European Journal of Clinical Pharmacology
Eur. J. Clin. Pharmacol. 16, 341-344 (1979)
© by Springer-Verlag 1979
The Binding of Gitoxin to Human Plasma Proteins N. Verbeke 1, P. Pellegrin2., Anne Vienne 1, and M. Lesne 2 tD6partement de Pharmacologie, A. Christiaens S. A., and 2Laboratoire de Pharmacodynamie G6n6rale UCL, Bruxelles, Belgium
Summary. The binding of gitoxin, digitoxin and digoxin to human plasma proteins was measured by ultracentrifugation and equilibrium dialysis. At concentrations in the range of therapeutic plasma levels, protein binding amounted, respectively, to 85, 92 and 20%, the last two values being consistent with data reported in the literature. The affinity of purified human albumin was not significantly different for the three cardiac glycosides tested. No other protein than albumin was found to bind gitoxin in human plasma.
Key words: gitoxin, digitoxin, digoxin, protein binding
Although the cardiotonic properties of gitoxin have been well known for almost half a century, this digitalis glycoside has never been used in cardiac therapy, due to its apparent lack of absorption after administration by the oral route (Lesne et al., 1978). Recent studies have demonstrated that fully bioavailable oral dosage forms can be developed (Hupin et al., 1979). Favourable pharmacokinetic parameters of gitoxin have been advanced, especially in renal patients (Lesne, 1978; Lesne et al., 1977). The aim of the present work was to study the binding of gitoxin to human plasma proteins in comparison with digoxin and digitoxin. The protein binding of cardiac glycosides has been claimed by several authors to be an important factor in evaluation of the pharmacokinetic parameters of these drugs, such as blood level, renal excretion, tissue uptake and etc. (Kuschinsky, 1969; Lukas and De Martino, 1969; Lukas, 1971; Evered, 1972; * P. Pellegrin is Aspirant du Fonds National de la Recherche Scientifique
Ohnhaus et al., 1972; Wallace and Whiting, 1974; Storstein, 1976). However, divergent results have been obtained on use of different methods, as has recently been reviewed by Jusko and Gretch (1976). The plasma protein to which binding occurs has been assumed to be albumin, but no incontrovertible demonstration has been reported (Kuschinsky, 1969; Lukas and De Martino, 1969; Evered, 1972). A variety of methodological approaches and experimental conditions have given rise to divergent affinity constants of human plasma proteins for digoxin and digitoxin (Lukas and De Martino, 1969; Evered, 1972; Ohnhaus et al., 1972; Sholtan et al., 1966; Brock, 1974, 1975; Okita, 1967). We have compared the protein binding of gitoxin, digoxin and digitoxin, determined by equilibrium dialysis and ultracentrifugation, using plasma concentrations of the drugs in the range of therapeutic levels. Plasma proteins have been fractionated by isoelectric focussing, in order to recognise the nature of the binding proteins of gitoxin. The equilibrium constant of the association of gitoxin, digoxin and digitoxin with purified human albumin has been studied by ultracentrifugation over a hundredfold range of concentrations.
Materials and Methods
Equilibrium Dialysis Dialysis was performed at room temperature through premoistened Cuprophan 930225-01 dialysis membranes (Technicon, USA) in i ml Diapack cells (Kontron, Switzerland). Plasma containing gitoxin (10ng/ml), digoxin (1 ng/ml) or digitoxin (10ng/ ml), labelled with tritium (+ 0.15 gCi/ml), was dialysed against an ultrafiltrate of plasma containing NaN 3 (150ng/ml), obtained by filtration through 0031-6970/79/0016/0341/$01.00
342
N. Verbeke et al.: Plasma Protein Binding of Gitoxin
Table 1. Binding of gitoxin, digitoxin and digoxin to human plasma proteins. Protein binding was determined by equilibrium dialysis and by ultracentrifugation as described under 'Methods'. Data are the average of several determinations _+ SEM; the number of experiments is given in parentheses Cardiac glycosides
Plasma level (ng/ml)
Percent protein Nnding Equilibrium Ultracentfidialysis fugation
Gitoxin
10
87.2 ,+ 0.5
Digitoxin Digoxin
10 1
85.8 .+ 0.7
(7)
(4)
93.4 _+ 0.3 (7) 12.1 _+ 1.7
92.1 _+ 0.5 (4) 20.2 _+ 2
(4)
(4)
Table 2. Association constants of gitoxin, digitoxin and digoxin
with human serum albumin calculated from the slopes of Scatchard plots s h o ~ in Figure 1. Values between brackets represent 95% confidence intervals Cardiac Glycosides
Association constants (M -1)
Gitoxin Digitoxin Digoxin
2.38 × 105 6.02 x 105 3.26 × 105
[1.26 × 105-3.5 × 105] [2.27 x 105-9.77 x 105] [0.25 x 105-6.27 x 105]
Pellicon PTGC 02510 membranes (Millipore, USA). After the 16 h required to reach equilibrium, 50 gl aliquots from both compartments of the dialysis cell were sampled and assayed for radioactivity by liquid scintillation counting. The percentage binding was calculated as follows: % protein binding =
% Cp -of.
lOO
Cp and Cf refer to concentrations of cardiac glycosides respectively in the plasma and in the ultrafiltrate chambers after dialysis.
Ultracentrifugation Triplicate samples of tritiated gitoxin (10 ng/ml), digoxin (1 ng/ml) or digitoxin (10 ng/ml) in human plasma (+ 0.15 ~tCi/ml) were spun at 300000 g, for 15 h, at 25 ° C in an Omega II centrifuge with rotor 9792 (Christ, GFR). Radioactivity was measured before and after centrifugation, in 50 and 500 ~tl aliquots, respectively. Protein binding was calculated as follows: % protein binding _-
Cp
• 100
Cp and C~ refer to concentrations of cardiac glycosides in plasma before centrifugation and in the supernatant after centrifugation. Sedimentation of
albumin, a 4 S protein, was complete after the run. In pilot studies using solutions of 3H-glycosides in plasma ultrafiltrate, prepared as described above, no adsorption was found when solutions were spun in polyallomer tubes (Christ, GFR). Determination of the equlibrium constant for the binding of gitoxin, digoxin and digitoxin to human serum albumin was performed by ultracentrifugation at 20 ° C. Solutions of tritium - labelled cardiac glycosides (gitoxin 0.775mCi/mg; digoxin 0.833mCi/mg; digitoxin 0.879mCi/mg), at concentrations of 10, 30, 100, 300, 600 and 1000 ng/ml, were made in a 4% solution of purified human albumin in buffer (phosphate buffer 0.01 M, pH 7.4 containing NaC1 0.9%). Experimental data were plotted according to Scatchard (1949), in order to calculate the association constants.
Isoelectric Focussing Fractionation of plasma containing gitoxin (10 ng/ml - 0.16 ~Ci/ml) was carried out by isoelectric focussing on a LKB 8100-2 column (LKB, Sweden). Plasma 5 ml was incorporated in a 0-46% sucrose gradient decreasing between anode and cathode, and containing 0.25% ampholines pH 3.5-10 to achieve a linear pH gradient. The column was run at 4 ° C. The potential was gradually increased to 1200 V and was maintained for 48 h. 2.5 ml fractions were collected. Optical density was recorded at 280 nm. Radioactivity was assayed as described previously. pH was quickly measured on alternating fifth fractions.
Reagents 3H-digoxin (12.8 mCi/mg) and 3H-digitoxin (9.7 mCi/mg) were purchased from the Radiochemical Centre, Amersham (UK). 3H-gitoxin (15 mCi/ mg) was purchased from the Institut des Radio616ments (Belgium). Slightly outdated lyophilised plasma from normal human donors was used (total proteins 46.5 g/l; total lipids 4.6 g/l; urea 0.216 g/l). Human serum albumin was issued from Poviet (Holland). Ampholines were obtained from LKB (Sweden). All other reagents were analytical grade and were purchased from Merck (GFR).
Results
The percentage protein binding of gitoxin, digitoxin and digoxin, at concentrations in the range of therapeutic plasma levels, are listed in Table 1.
N. V e r b e k e et al.: PIasma Protein Binding of Gitoxin
343
i /
BIFi y=o+bx 6.8 ~ " ~
r
Digitoxin
ab == -6.02.105 6,8 M- t r ~: 0,92
2.o ~0000
6.A
• ~,
b=-2.38.10
M-1
1,5 -30 000 !
01
I
0,5
..........................
| 1 1 II
I
1.0 B x t 0 6 ( M ~)
BIF~ Digoxin
1.0 70000
o = 0,155 5 M-I b = -3.26" 10 r = 0.872 0,15
%
0.11 0
~
I 0.5
05 I0 000
_ I 1,0 13x 107 (M-~)
Fig. L Scartchard plots of gitoxin, digitoxin and digoxin binding to human serum albumin. Bound and free fractions of the cardiac
glycosides were determined by ultracentrifugation, as specified under 'Methods'. Vertical bars represent SE of B/F values
Table 3. Maximum number of binding sites of gitoxin, digitoxin and digoxin per milligram human serum albumin calculated from the intercepts of Scatchard plots with the abscissa. Values between brackets represent 95% confidence intervals Cardiac Glycosides
Maximum number of binding sites per mg wotein
Gitoxin Digitoxin Digoxin
1.82 x 1014 [1.26 × 1014-3.38 × 1014] 1.70 × 10 ~4 [1.08 × 1014-4.37 × 10 TM] 7.39 X 10 t2 [4.32 × 101a-8.43 × 1013]
0
O - -
pH 10
ill [ 50
I
100
I
150
Nr. Fraction Fig, 2, Fractionation of human plasma containing 3H-gltoxin by isoelectric focussing. (0) Optical density at 280 n m ; (ll) pH; (*) Radioactivity
(p > 0.05) for digoxin than for gitoxin and digitoxin (Table 3). Protein fractionation of plasma containing 3Hgitoxin is illustrated in Figure 2. The pH gradient permitted good separation of plasma proteins (Bours, 1973; Scheil, 1973; Spieker-Polet and Polet, 1976). Radioactivity was found only in the albumin peak, ,which indicates that this protein is the sole binding site of gitoxin in human plasma. Discussion
Experimental data obtained by ultracentrifugation and equilibrium dialysis for digitoxin and gitoxin were equivalent (p < 0.05), whereas different results and a notable scatter were found for digoxin. Scatchard plots of gitoxin, digitoxin and digoxin binding to human serum albumin, obtained from ultracentrifugation data, are represented in Figure 1. The association constants of the three cardiac glycosides are reported in Table 2. No significant difference between the equilibrium constants was found (p < 0.05). In fact, the maximum number of binding sites calculated from the same data is twenty fold lower
Gitoxin was found to be extensively protein-bound, as has previously been shown for 16 alpha-gitoxin (Richter and Haustein, 1977). The present experimental values are in good agreement with previous reports that have shown a considerable difference between the percentage protein binding of digoxin and digitoxin (Baggot and Davis, 1973; Sholtan et al., 1966; Hempelman and Heinz, 1978; Storstein, 1976). The association constant found for digoxin was close to that reported by Evered (1972) and Ohnhaus et al. (1972), and different from those of
344
Scholtan et al. (1966) and Brock (1974). For digitoxin affinity constant obtained was tenfold higher than the values of Brock (1975) and Lukas and De Martino (1969). Recent studies have suggested that gitoxin should be of therapeutic interest, especially in cases of renal insufficiency, since urinary excretion amounts to only 15% of a given dose (Lesne et al., 1977). The small proportion of gitoxin lost via the kidney may be explained by the fact that the major fraction of the drug is bound to plasma proteins. In the case of digoxin, its low protein binding appears to be responsible for its predominant renal excretion, and consequently the marked increase in its biological half-life in renal patients (Doherty et al., 1969). Nevertheless, despite a percentage protein binding close to that of digitoxin, gitoxin has a biological half-life of approximately 24 h, even shorter than digoxin. This could be due to rapid sulpho - or glucurono - conjugation on the 16-beta-hydroxyl position. Work is in progress to elucidate this hypothesis.
Acknowledgements. We are greatly indebted to Prof. T. Godfraind for use of his ultracentrifuge, to Dr. H. Grosjean for use of his Diapack dialysis apparatus and to Prof. J. Urbain for use of his isoelectric focussing system. We wish to thank Mrs. H. MabytQuisthoudt for her expert technical assistance. References Baggot, J.D., Davis, L. E.: Plasma protein binding of digitoxin and digoxin in several mammalian species. Res. Vet. Sci. 15, 81-87 (1973) Bouts, J.: The use of protein markers in thin-layer isoelectric focusing. Sci. Tools 20, 29-31 (1973) Brock, A.: Binding of digoxin to human serum proteins: influence of pH on the binding of digoxin to human albumin. Acta Pharmacol. Toxicot. 34, 260-266 (1974) Brock, A.: Binding of digitoxin to human serum proteins: influence of pH on the binding of digoxin to human albumin. Acta Pharmacol. Toxicol. 36, 13-24 (1975) Doherty, J.E., Ferrel, C.B., Towbin, E.J.: Localization of the renal excretion of the tritiated digoxin. Am. J. Med. Sci. 258, 181-189 (1969) Evered, D. C.: The binding of digoxin by the serum proteins. Eur. J. Pharmacol. 18, 236-244 (1972) Hempelmann, F.W., Heinz, N.: Liphophilie und Proteinbindung bei Cardenotiden. Arzneim. Forsch. 28, 2185-2187 (1978) Hupin, C., de Suray, J.M., Versluys, J., Lorent, M., Dodion, L.,
N. Verbeke et al.: Plasma Protein Binding of Gitoxin Lesne, M.: Bioavailability study of gitoxin in a solid dosage form. Int. J. Clin. Pharmacol. Biopharm. 17, 197-204 (1979) Jusko, W.J., Gretch, M.: Plasma and tissue protein binding of drugs in pharmacokinetics. Drug Metab. Rev. 5, 43-140 (1976) Kuschinksy, K.: l ~ e r die Bindungseigenschaften von Plasma-proteinen fiir Herzglykoside. Natmyn Sehmiedebergs Arch. Pharmakol. Exp. Pathol. 262, 388-398 (1969) Lesne, M.: Pharmacological reevaluation of gitoxin in man. Int. J. Clin. PharmacoI. 16, 456--459 (1978) Lesne, M., Van Ypersele, C., Vandenbroecke, J.M.: Etude pr6liminaire de la pharmacocin6tique de la gitoxine dans l'insuffisance r6nale. J. Pharmacol. 8, 572 (1977) Lesne, M., de Suray, J.M., Hupin, C., Verbeke, N.: Favorable pharmacokinetic parameters of gitoxin in man. Proceedings of the 7th International Congress of Pharmacology, Paris, 1977, p. 1403. Paris: Pergamon Press 1978 Lukas, D.S.: Some aspects of the distribution and disposition of digitoxin in man. Ann. NY Acad. Sci. 179, 338-361 (1971) Lukas, D. S., De Martino, A.G.: Binding of digitoxin and some related cardenolides to htmaan plasma proteins. J. Clin. Invest. 48, 1041-I053 (1969) Ohnhaus, E. E., Spring, P., Dettli, L.: Protein bindingof digoxin in human serum. Eur. J. Clin. Pharmacol. 5, 34-76 (1972) Okita, G.T.: Binding of cardiac glycosides. In: Myocardial contractility. Tanz, R.O. (ed.), pp. 549-561. New York: Academic Press 1967 Richter, M., Haustein, K. O.: Binding of 16 alpha-gitoxin and its 16-acetate to human serum albumin. Eur. J. Clin. Pharmacol. 11, 459-461 (1977) Scatehard, G.: The attractions of proteins for small molecules and ions. Ann. NY Acad. Sci. 51, 660--664 (1949) ShoRan, W., Slossmann, K., Rosenkranz, H.: Bestimmung tier EiweiBbindung von Digitalis mittels Ultrazentrifuge. Arzneim. Forsch. 16, 109-118 (1966) Scheil, H. G.: Protein-mapping von menschlichen Seren (Isoelektrisches Fokusieren mit anschliel3enderGelelektrophorese). Z. Klin. Chem. Klin. Biochem. 11, 456-468 (1973) Spieker-Polet, H., Polet, H.: Identification of albumin as the serum factor essential for the growth of activated human lymphocytes. J. Biol. Chem. 251, 987-992 (1976) Storstein, L.: Studies on digitalis. V. The influence of imparied renal function, hemodialysis, and drug interaction on serum protein binding of digitoxin and digoxin. Clin. Pharmacot. Ther. 20, 6-14 (1976) Wallace, S., Whiting, B.: Some clinical implications of the protein binding of digoxin. Br. J. Clin. Pharmacot. 1,325-328 (1974) Received: December 7, 1977 in first revised form: March 6, 1979 in second revised form: July 3, 1979 accepted: July 5, 1979 Dr. N. Verbeke A. Christiaens S. A. rue de l'Etuve, 60 B-1000 Bruxelles, Belgium
Eur. J. Clin. Pharmacol. 16, 341-344 (1979)
© by Springer-Verlag 1979
The Binding of Gitoxin to Human Plasma Proteins N. Verbeke 1, P. Pellegrin2., Anne Vienne 1, and M. Lesne 2 tD6partement de Pharmacologie, A. Christiaens S. A., and 2Laboratoire de Pharmacodynamie G6n6rale UCL, Bruxelles, Belgium
Summary. The binding of gitoxin, digitoxin and digoxin to human plasma proteins was measured by ultracentrifugation and equilibrium dialysis. At concentrations in the range of therapeutic plasma levels, protein binding amounted, respectively, to 85, 92 and 20%, the last two values being consistent with data reported in the literature. The affinity of purified human albumin was not significantly different for the three cardiac glycosides tested. No other protein than albumin was found to bind gitoxin in human plasma.
Key words: gitoxin, digitoxin, digoxin, protein binding
Although the cardiotonic properties of gitoxin have been well known for almost half a century, this digitalis glycoside has never been used in cardiac therapy, due to its apparent lack of absorption after administration by the oral route (Lesne et al., 1978). Recent studies have demonstrated that fully bioavailable oral dosage forms can be developed (Hupin et al., 1979). Favourable pharmacokinetic parameters of gitoxin have been advanced, especially in renal patients (Lesne, 1978; Lesne et al., 1977). The aim of the present work was to study the binding of gitoxin to human plasma proteins in comparison with digoxin and digitoxin. The protein binding of cardiac glycosides has been claimed by several authors to be an important factor in evaluation of the pharmacokinetic parameters of these drugs, such as blood level, renal excretion, tissue uptake and etc. (Kuschinsky, 1969; Lukas and De Martino, 1969; Lukas, 1971; Evered, 1972; * P. Pellegrin is Aspirant du Fonds National de la Recherche Scientifique
Ohnhaus et al., 1972; Wallace and Whiting, 1974; Storstein, 1976). However, divergent results have been obtained on use of different methods, as has recently been reviewed by Jusko and Gretch (1976). The plasma protein to which binding occurs has been assumed to be albumin, but no incontrovertible demonstration has been reported (Kuschinsky, 1969; Lukas and De Martino, 1969; Evered, 1972). A variety of methodological approaches and experimental conditions have given rise to divergent affinity constants of human plasma proteins for digoxin and digitoxin (Lukas and De Martino, 1969; Evered, 1972; Ohnhaus et al., 1972; Sholtan et al., 1966; Brock, 1974, 1975; Okita, 1967). We have compared the protein binding of gitoxin, digoxin and digitoxin, determined by equilibrium dialysis and ultracentrifugation, using plasma concentrations of the drugs in the range of therapeutic levels. Plasma proteins have been fractionated by isoelectric focussing, in order to recognise the nature of the binding proteins of gitoxin. The equilibrium constant of the association of gitoxin, digoxin and digitoxin with purified human albumin has been studied by ultracentrifugation over a hundredfold range of concentrations.
Materials and Methods
Equilibrium Dialysis Dialysis was performed at room temperature through premoistened Cuprophan 930225-01 dialysis membranes (Technicon, USA) in i ml Diapack cells (Kontron, Switzerland). Plasma containing gitoxin (10ng/ml), digoxin (1 ng/ml) or digitoxin (10ng/ ml), labelled with tritium (+ 0.15 gCi/ml), was dialysed against an ultrafiltrate of plasma containing NaN 3 (150ng/ml), obtained by filtration through 0031-6970/79/0016/0341/$01.00
342
N. Verbeke et al.: Plasma Protein Binding of Gitoxin
Table 1. Binding of gitoxin, digitoxin and digoxin to human plasma proteins. Protein binding was determined by equilibrium dialysis and by ultracentrifugation as described under 'Methods'. Data are the average of several determinations _+ SEM; the number of experiments is given in parentheses Cardiac glycosides
Plasma level (ng/ml)
Percent protein Nnding Equilibrium Ultracentfidialysis fugation
Gitoxin
10
87.2 ,+ 0.5
Digitoxin Digoxin
10 1
85.8 .+ 0.7
(7)
(4)
93.4 _+ 0.3 (7) 12.1 _+ 1.7
92.1 _+ 0.5 (4) 20.2 _+ 2
(4)
(4)
Table 2. Association constants of gitoxin, digitoxin and digoxin
with human serum albumin calculated from the slopes of Scatchard plots s h o ~ in Figure 1. Values between brackets represent 95% confidence intervals Cardiac Glycosides
Association constants (M -1)
Gitoxin Digitoxin Digoxin
2.38 × 105 6.02 x 105 3.26 × 105
[1.26 × 105-3.5 × 105] [2.27 x 105-9.77 x 105] [0.25 x 105-6.27 x 105]
Pellicon PTGC 02510 membranes (Millipore, USA). After the 16 h required to reach equilibrium, 50 gl aliquots from both compartments of the dialysis cell were sampled and assayed for radioactivity by liquid scintillation counting. The percentage binding was calculated as follows: % protein binding =
% Cp -of.
lOO
Cp and Cf refer to concentrations of cardiac glycosides respectively in the plasma and in the ultrafiltrate chambers after dialysis.
Ultracentrifugation Triplicate samples of tritiated gitoxin (10 ng/ml), digoxin (1 ng/ml) or digitoxin (10 ng/ml) in human plasma (+ 0.15 ~tCi/ml) were spun at 300000 g, for 15 h, at 25 ° C in an Omega II centrifuge with rotor 9792 (Christ, GFR). Radioactivity was measured before and after centrifugation, in 50 and 500 ~tl aliquots, respectively. Protein binding was calculated as follows: % protein binding _-
Cp
• 100
Cp and C~ refer to concentrations of cardiac glycosides in plasma before centrifugation and in the supernatant after centrifugation. Sedimentation of
albumin, a 4 S protein, was complete after the run. In pilot studies using solutions of 3H-glycosides in plasma ultrafiltrate, prepared as described above, no adsorption was found when solutions were spun in polyallomer tubes (Christ, GFR). Determination of the equlibrium constant for the binding of gitoxin, digoxin and digitoxin to human serum albumin was performed by ultracentrifugation at 20 ° C. Solutions of tritium - labelled cardiac glycosides (gitoxin 0.775mCi/mg; digoxin 0.833mCi/mg; digitoxin 0.879mCi/mg), at concentrations of 10, 30, 100, 300, 600 and 1000 ng/ml, were made in a 4% solution of purified human albumin in buffer (phosphate buffer 0.01 M, pH 7.4 containing NaC1 0.9%). Experimental data were plotted according to Scatchard (1949), in order to calculate the association constants.
Isoelectric Focussing Fractionation of plasma containing gitoxin (10 ng/ml - 0.16 ~Ci/ml) was carried out by isoelectric focussing on a LKB 8100-2 column (LKB, Sweden). Plasma 5 ml was incorporated in a 0-46% sucrose gradient decreasing between anode and cathode, and containing 0.25% ampholines pH 3.5-10 to achieve a linear pH gradient. The column was run at 4 ° C. The potential was gradually increased to 1200 V and was maintained for 48 h. 2.5 ml fractions were collected. Optical density was recorded at 280 nm. Radioactivity was assayed as described previously. pH was quickly measured on alternating fifth fractions.
Reagents 3H-digoxin (12.8 mCi/mg) and 3H-digitoxin (9.7 mCi/mg) were purchased from the Radiochemical Centre, Amersham (UK). 3H-gitoxin (15 mCi/ mg) was purchased from the Institut des Radio616ments (Belgium). Slightly outdated lyophilised plasma from normal human donors was used (total proteins 46.5 g/l; total lipids 4.6 g/l; urea 0.216 g/l). Human serum albumin was issued from Poviet (Holland). Ampholines were obtained from LKB (Sweden). All other reagents were analytical grade and were purchased from Merck (GFR).
Results
The percentage protein binding of gitoxin, digitoxin and digoxin, at concentrations in the range of therapeutic plasma levels, are listed in Table 1.
N. V e r b e k e et al.: PIasma Protein Binding of Gitoxin
343
i /
BIFi y=o+bx 6.8 ~ " ~
r
Digitoxin
ab == -6.02.105 6,8 M- t r ~: 0,92
2.o ~0000
6.A
• ~,
b=-2.38.10
M-1
1,5 -30 000 !
01
I
0,5
..........................
| 1 1 II
I
1.0 B x t 0 6 ( M ~)
BIF~ Digoxin
1.0 70000
o = 0,155 5 M-I b = -3.26" 10 r = 0.872 0,15
%
0.11 0
~
I 0.5
05 I0 000
_ I 1,0 13x 107 (M-~)
Fig. L Scartchard plots of gitoxin, digitoxin and digoxin binding to human serum albumin. Bound and free fractions of the cardiac
glycosides were determined by ultracentrifugation, as specified under 'Methods'. Vertical bars represent SE of B/F values
Table 3. Maximum number of binding sites of gitoxin, digitoxin and digoxin per milligram human serum albumin calculated from the intercepts of Scatchard plots with the abscissa. Values between brackets represent 95% confidence intervals Cardiac Glycosides
Maximum number of binding sites per mg wotein
Gitoxin Digitoxin Digoxin
1.82 x 1014 [1.26 × 1014-3.38 × 1014] 1.70 × 10 ~4 [1.08 × 1014-4.37 × 10 TM] 7.39 X 10 t2 [4.32 × 101a-8.43 × 1013]
0
O - -
pH 10
ill [ 50
I
100
I
150
Nr. Fraction Fig, 2, Fractionation of human plasma containing 3H-gltoxin by isoelectric focussing. (0) Optical density at 280 n m ; (ll) pH; (*) Radioactivity
(p > 0.05) for digoxin than for gitoxin and digitoxin (Table 3). Protein fractionation of plasma containing 3Hgitoxin is illustrated in Figure 2. The pH gradient permitted good separation of plasma proteins (Bours, 1973; Scheil, 1973; Spieker-Polet and Polet, 1976). Radioactivity was found only in the albumin peak, ,which indicates that this protein is the sole binding site of gitoxin in human plasma. Discussion
Experimental data obtained by ultracentrifugation and equilibrium dialysis for digitoxin and gitoxin were equivalent (p < 0.05), whereas different results and a notable scatter were found for digoxin. Scatchard plots of gitoxin, digitoxin and digoxin binding to human serum albumin, obtained from ultracentrifugation data, are represented in Figure 1. The association constants of the three cardiac glycosides are reported in Table 2. No significant difference between the equilibrium constants was found (p < 0.05). In fact, the maximum number of binding sites calculated from the same data is twenty fold lower
Gitoxin was found to be extensively protein-bound, as has previously been shown for 16 alpha-gitoxin (Richter and Haustein, 1977). The present experimental values are in good agreement with previous reports that have shown a considerable difference between the percentage protein binding of digoxin and digitoxin (Baggot and Davis, 1973; Sholtan et al., 1966; Hempelman and Heinz, 1978; Storstein, 1976). The association constant found for digoxin was close to that reported by Evered (1972) and Ohnhaus et al. (1972), and different from those of
344
Scholtan et al. (1966) and Brock (1974). For digitoxin affinity constant obtained was tenfold higher than the values of Brock (1975) and Lukas and De Martino (1969). Recent studies have suggested that gitoxin should be of therapeutic interest, especially in cases of renal insufficiency, since urinary excretion amounts to only 15% of a given dose (Lesne et al., 1977). The small proportion of gitoxin lost via the kidney may be explained by the fact that the major fraction of the drug is bound to plasma proteins. In the case of digoxin, its low protein binding appears to be responsible for its predominant renal excretion, and consequently the marked increase in its biological half-life in renal patients (Doherty et al., 1969). Nevertheless, despite a percentage protein binding close to that of digitoxin, gitoxin has a biological half-life of approximately 24 h, even shorter than digoxin. This could be due to rapid sulpho - or glucurono - conjugation on the 16-beta-hydroxyl position. Work is in progress to elucidate this hypothesis.
Acknowledgements. We are greatly indebted to Prof. T. Godfraind for use of his ultracentrifuge, to Dr. H. Grosjean for use of his Diapack dialysis apparatus and to Prof. J. Urbain for use of his isoelectric focussing system. We wish to thank Mrs. H. MabytQuisthoudt for her expert technical assistance. References Baggot, J.D., Davis, L. E.: Plasma protein binding of digitoxin and digoxin in several mammalian species. Res. Vet. Sci. 15, 81-87 (1973) Bouts, J.: The use of protein markers in thin-layer isoelectric focusing. Sci. Tools 20, 29-31 (1973) Brock, A.: Binding of digoxin to human serum proteins: influence of pH on the binding of digoxin to human albumin. Acta Pharmacol. Toxicot. 34, 260-266 (1974) Brock, A.: Binding of digitoxin to human serum proteins: influence of pH on the binding of digoxin to human albumin. Acta Pharmacol. Toxicol. 36, 13-24 (1975) Doherty, J.E., Ferrel, C.B., Towbin, E.J.: Localization of the renal excretion of the tritiated digoxin. Am. J. Med. Sci. 258, 181-189 (1969) Evered, D. C.: The binding of digoxin by the serum proteins. Eur. J. Pharmacol. 18, 236-244 (1972) Hempelmann, F.W., Heinz, N.: Liphophilie und Proteinbindung bei Cardenotiden. Arzneim. Forsch. 28, 2185-2187 (1978) Hupin, C., de Suray, J.M., Versluys, J., Lorent, M., Dodion, L.,
N. Verbeke et al.: Plasma Protein Binding of Gitoxin Lesne, M.: Bioavailability study of gitoxin in a solid dosage form. Int. J. Clin. Pharmacol. Biopharm. 17, 197-204 (1979) Jusko, W.J., Gretch, M.: Plasma and tissue protein binding of drugs in pharmacokinetics. Drug Metab. Rev. 5, 43-140 (1976) Kuschinksy, K.: l ~ e r die Bindungseigenschaften von Plasma-proteinen fiir Herzglykoside. Natmyn Sehmiedebergs Arch. Pharmakol. Exp. Pathol. 262, 388-398 (1969) Lesne, M.: Pharmacological reevaluation of gitoxin in man. Int. J. Clin. PharmacoI. 16, 456--459 (1978) Lesne, M., Van Ypersele, C., Vandenbroecke, J.M.: Etude pr6liminaire de la pharmacocin6tique de la gitoxine dans l'insuffisance r6nale. J. Pharmacol. 8, 572 (1977) Lesne, M., de Suray, J.M., Hupin, C., Verbeke, N.: Favorable pharmacokinetic parameters of gitoxin in man. Proceedings of the 7th International Congress of Pharmacology, Paris, 1977, p. 1403. Paris: Pergamon Press 1978 Lukas, D.S.: Some aspects of the distribution and disposition of digitoxin in man. Ann. NY Acad. Sci. 179, 338-361 (1971) Lukas, D. S., De Martino, A.G.: Binding of digitoxin and some related cardenolides to htmaan plasma proteins. J. Clin. Invest. 48, 1041-I053 (1969) Ohnhaus, E. E., Spring, P., Dettli, L.: Protein bindingof digoxin in human serum. Eur. J. Clin. Pharmacol. 5, 34-76 (1972) Okita, G.T.: Binding of cardiac glycosides. In: Myocardial contractility. Tanz, R.O. (ed.), pp. 549-561. New York: Academic Press 1967 Richter, M., Haustein, K. O.: Binding of 16 alpha-gitoxin and its 16-acetate to human serum albumin. Eur. J. Clin. Pharmacol. 11, 459-461 (1977) Scatehard, G.: The attractions of proteins for small molecules and ions. Ann. NY Acad. Sci. 51, 660--664 (1949) ShoRan, W., Slossmann, K., Rosenkranz, H.: Bestimmung tier EiweiBbindung von Digitalis mittels Ultrazentrifuge. Arzneim. Forsch. 16, 109-118 (1966) Scheil, H. G.: Protein-mapping von menschlichen Seren (Isoelektrisches Fokusieren mit anschliel3enderGelelektrophorese). Z. Klin. Chem. Klin. Biochem. 11, 456-468 (1973) Spieker-Polet, H., Polet, H.: Identification of albumin as the serum factor essential for the growth of activated human lymphocytes. J. Biol. Chem. 251, 987-992 (1976) Storstein, L.: Studies on digitalis. V. The influence of imparied renal function, hemodialysis, and drug interaction on serum protein binding of digitoxin and digoxin. Clin. Pharmacot. Ther. 20, 6-14 (1976) Wallace, S., Whiting, B.: Some clinical implications of the protein binding of digoxin. Br. J. Clin. Pharmacot. 1,325-328 (1974) Received: December 7, 1977 in first revised form: March 6, 1979 in second revised form: July 3, 1979 accepted: July 5, 1979 Dr. N. Verbeke A. Christiaens S. A. rue de l'Etuve, 60 B-1000 Bruxelles, Belgium
