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Development of a radioimmunoassay for aprindine
Journal of Immunological Me thods, 17 (1977 ) 189--198
189
© Elsevier/North-Holland Biomedical Press
D E V E L O P M E N T O F A RADIOIMMUNOASSAY FO R APRINDINE
MICHEL LESNE and Rl~GIS DOLPHEN * Service de Biopharmacie Clinique, Laboratoire de Pharmacodynamie Gdndrale, UCL 7350, Avenue Mounier 73, 1200 Bruxelles, Belgium
(Received 7 March 1977, accepted 24 April 1977)
We have developed a radioimmunoassay for aprindine, a new antiarrhythmic drug used in the treatment of ventricular disorders. The antibodies were produced by immunization of New-Zealand rabbits with aprindine coupled to human serum albumin. Their biochemical characteristics have been determined. Tritiated aprindine was used as radioactive competitor. The cross-reactivity with several metabolites of aprindine was studied too. Finally, the results obtained by RIA in plasma and tissues of dogs were compared to those obtained by gas-chromatography.
INTRODUCTION Aprindine is a new antiarrhythmic drug especially indicated in the disorders o f the ventricular excitability (Van Durme et al., 1974a; Georges et al., 1973). Its structure is illustrated in fig. 1. Although pharmacologically related to lidocaine, its pharmacokinetics is however very different. In fact, in contrast with lidocaine which is n o t used orally due to its very high hepatic clearance, aprindine, given orally, reaches plasma levels identical t o those observed after intravenous administration. T h e r e f o r e , it m ay be used n o t only in intravenous perfusion in the treatm e n t o f acute cases but also orally in the chronic t r e a t m e n t of ventricular arrhythmias. As the therapeutic index of this drug is rather low (Van Durme et al., 1974b), it seems o f interest to control its therapeutic use by means o f a rapid and precise m e t h o d of d e t e r m i n a t i o n in plasma, in order to avoid a t o o high f r e q u e n c y o f iatrogenic intoxications t h a t are manifested especially through cerebellar disorders. T h e r e f o r e , its seemed t o us interesting to develop a radioimmunological m e t h o d t o det erm i ne aprindine in biological samples instead o f the long and tedious gas-chromatography m e t h o d . ( R u t h e r f o r d et al., 1976).
* Assistant F.R.S.M.
190 SYNTHESIS OF ANTIGEN
CH2)3-N/c2H5
/CzH~" CI" CH2)3-N \C2H5
~~~[~
NC2H 5 SYNTHESIS
OF
HAPTEN APRINDINE
coupling
N=N* ~
HAPTEN
hapten - albumin
~
7C2H5
CH213-N\
C2H5
N=N-AH ANT IGEN
Fig. 1. Synthesis of the antigen using aprindine (N-(3-(diethylamino)propyl)-N-phenyl-2indanamine) and human serum albumin.
MATERIALS AND METHODS
The radioimmunoassay of aprindine requires the use of three compounds: unlabelled aprindine as standard, tritiated aprindine as radioactive competitor and specific antiaprindine antibodies. The tritiated aprindine used has been prepared in the laboratories of The Radioelements Institute (I.R.E.) in Fleurus, Belgium. The molecule was specifically labelled in the fourth position of the phenyl group (fig. 1); the specific activity obtained was 1.6 Ci/mmole. The purity of the preparation was controlled by means of thin-layer-chromatography and U.V. spectrophotometry. Due to the fact that aprindine has no immunogenic properties by itself, the production of these antibodies required, first, the synthesis of an antigen, obtained by coupling the hapten, in the form of aprindine diazotated in the fourth position of the phenyl group, to human serum albumin (fig. 1). The analysis has shown that eleven aprindine molecules are bound to each molecule of serum albumin. The antigen was then administrated to five NewZealand rabbits (A, B, C, D and E) by the multiple intradermic injection
191
technique on both sides of the vertebral column (Vaitukaitis et al., 1971). The production of antibodies in the serum of the rabbits has been followed in the different samples of blood taken from week to week. For each serum, we have measured the binding of a limited a m o u n t of tritiated aprindine to increasing dilutions of the antiserum in phosphate saline buffer.
Composition of the phosphate saline buffer 1.392 g Potassium phosphate, dibasic, K2HPO4; 0.276 g sodium phosphate, monobasic, m o n o h y d r a t e , Na H2PO4" H20; 8.76 g sodium chloride, NaC1, 1 liter distilled water. By expressing the percentage of binding of the tritiated aprindine versus the logarithm of antiserum dilution, it appears a sigmoidal shaped curve that, for the same rabbit, shifts to the right when the antibody level in serum becomes higher (fig. 2). Moreover these binding curves make it possible to define the antiserum with the highest titer. The next step was the determination of the selected antiserum characteristics, namely the concentration of antibodies and their affinity for aprindine. In order to do that, an appropriate dilution of the antiserum was incubated at 4 ° C in presence of increasing concentrations of tritiated aprindine. For each of the different concentrations used, after the equilibrium has been reached, one part of the solution was taken in order to measure the radioactivity corresponding to the total concentration of radioactive aprindine.
loo.
o"
-"-o
i T
o ~ c
50.
2_222
O. O.S
I
1.5
Log. dilution antiserum
Fig. 2. Binding of [3H]aprindine (4 ng) to increA.sing dilutions of antiserum in phosphate saline buffer.
192
T h e o t h e r p a r t was t h e n p l a c e d in p r e s e n c e o f d e x t r a n c o a t e d c h a r c o a l t o a d s o r b a p r i n d i n e selectively. A f t e r c e n t r i f u g a t i o n a t 3 0 0 0 r p m f o r 10 m i n , t h e m e a s u r e o f the radioactivity in the s u p e r n a t a n t a l l o w e d the d e t e r m i n a t i o n o f the a p r i n d i n e c o n c e n t r a t i o n b o u n d to the a n t i b o d i e s . T h e s t a n d a r d curve f o r t h e r a d i o i m m u n o a s s a y o f a p r i n d i n e was e s t a b l i s h e d b y m e a s u r i n g the i n h i b i t i o n o f b i n d i n g o f a small a m o u n t o f t r i t i a t e d a p r i n d i n e (4 ng) t o an a p p r o p r i a t e d i l u t i o n o f the a n t i s e r u m b y increasing c o n c e n t r a t i o n s o f u n l a b e U e d a p r i n d i n e ; these were c h o s e n in the range o f
NH--CH2-CH~--CHg-N./'CzHs .~Czpis
II
I"
Ddsindanyl-aprindine (AC 1817)
[••H
z-CH 2"-CH2-N HCzH s
Dg h~nyl- oprindine P (AC zzz6)
\
Ddsgfhyl-aprindine (AO g187)
2 5
Aprindine (AC 180;')
\
/ C2H6
Hydroxy- aprindine
OH
Hydroxy-aprindine
(typeII)
(type 1)
Fig. 3. Metabolism of aprindine in man. Two main ways: N-desethylation of the lateral chain and aromatic hydroxylation, third way, less frequent: loss of phenyl or indanyl cycles (after Dodion et al., 1974).
193
therapeutic concentrations (0.75 to 2 pg/ml) previously determined by G.C. in plasma samples of patients treated with this drug (Van Durme et al., 1974). The antibody specificity for aprindine was also determined by measuring the inhibition obtained in the same conditions and at the same concentrations for 4 metabolites of aprindine previously identified in man (Dodion et al., 1974): dephenylaprindine, desindanylaprindine, parahydroxyaprindine and desethylaprindine (fig. 3). Finally, the levels of aprindine reached in plasma and tissues of dogs treated by this antiarrhythmic drug were measured by both G.C. and R.I.A. The determination of plasma levels of aprindine by G.C. requires a preliminary double extraction; on the other hand, the R.I.A. was performed directly on plasma. As far as the tissues are concerned, the determination by G.C. was performed in the chloroformic solution obtained after a double extraction of aprindine from the tissue homogenate. In order to perform the R.I.A., aprindine was extracted from the tissue homogenate by means of diethyl ether; the organic phase was then evaporated to dryness and the residue was redissolved in phosphate buffer containing serum albumin. This aqueous extract was used for R.I.A. Composition o f the buffer: 0.75 g Potassium phosphate, monobasic, KH2POa; 7.8 g sodium phosphate, dibasic, dihydrate, Na2HPO4" 2H20; 0.5 g sodium azide, NaNa; 5 g bovine serum albumin; 1 liter distilled water. RESULTS
The antiserum studied was taken from rabbit D on february 2, 1976. It is the result of three immunizations: the first, made ten months earlier with 1 mg of antigen in Freund's adjuvant, the second, six months earlier by means of 100 #g of antigen dissolved in physiological solution and the third, one month before. The antibody level and their affinity constants were determined at the equilibrium of the reaction between hapten (tritiated aprindine) and antibodies, according to the following equation: Antibody + hapten ~ Antibody--hapten complex N--R C R At that time, R represents the concentration of antibody sites bound to the hapten, C, the concentration of free hapten and N--R, the concentration of binding sites available in the dilution of the antiserum used. The affinity constant (K0) corresponds to the following equation: R Ko = ( N - - R ) C
194 Antiserum Apr~ndine Rabbit D 2FEB 1976 1 . 123x106 M o Dilution : 1:10 N o
75.
o/
"6 E 50.
,~ 25.
1 N=
J
j 50
100 C -1 (107 )
150 (retire-mole)
200
250
Fig. 4. Relationship b e t w e e n the reciprocal of b o u n d sites c o n c e n t r a t i o n ( l / R ) and the reciprocal of free h a p t e n c o n c e n t r a t i o n (1/C) at equilibrium for the antiserum taken on F e b r u a r y 2, 1976 f r o m rabbit D. The straight line was d e t e r m i n e d by linear regression analysis o f the results, r = 0.98.
The measured parameters R and C were introduced in the following equation: , used in dialysis equilibrium where 'a' is the heterogeneity constant of the population of antibodies. (Werblin et al., 1972, 1973) The reciprocal of the concentration of b o u n d sites ( l / R ) plotted versus the reciprocal of the concentration of free hapten (1/C), yields a straight line of which y-intercept is 1IN (fig. 4.). The value of N allows to determine the a m o u n t of antibodies in the antiserum by reference to the molecular weight of the immunoglobulin G (around 160,000) and by assuming that there are two binding sites present in each antibody molecule (Butler et al., 1973). The concentration of antibodies in the antiserum taken on February 2, 1976 from rabbit D was estimated at 0.6 mg/ml. The affinity constant was calculated from fig. 5 that shows the relationship between the values of the logarithm of the ratio R/(N--R) and the logarithm of the concentration C of free competitor according to the following equation : R log N - - R = a log K0 + a log C The plot issues from the linear regression analysis of these values crosses the abscissa at a concentration value that represents the reciprical of the affinity constant. For that antiserum, the affinity constant was estimated at 1.58 • 108/M.
o:l
195
Antiserum Aprindine Rabbit D 2 FEB 1976 Dilution:l:10 Ko=1.S8x108 M "t
/®
=i=,_o.s_ °= .J
-is
,ool" o
o
oSS
/
o.,~[°
?J
-~.0.
oo
°
O
r = 0.98
o
-2.0 - 9.s
-9
- is
-8
-~?s
Log C
Fig. 5. Determination of the affinity constant (K0) of the a n t i b o d y for [3H]aprindine. S l o p e o f the straight line: 'a' ~ 1.
100. ,~--v
o
v.
o
~,~.,~
CROSS-REACTIVITY : ANTISERUtl APPRINDINE RABBII D :' FEB 1976 0 aprindine A para-hydroxy-apri ndine
I-I ~. ~ ~
o e,, e,.
~
°~
,,r!
I"I desethyl-aprindine ~ dephen)~l-aprindine
0~. =~.~
~
desindanyl-aprindi ne
50.
o C ,~ C
lla
0
'
'
'
0.25 0.50 0.75 Concentration of unlabelled drug
II
1 5 10 15 (10-6M)
Fig. 6. Standard curve o b t a i n e d in the R I A o f aprindine ( o ) and cross-reactivity curves w i t h 4 m e t a b o l i t e s (~, ~, v , 0 ) (The binding o f [ 3 H ] a p r i n d i n e t o the a n t i b o d i e s is e x p r e s s e d in p e r c e n t o f the value measured w i t h o u t unlabelled drug.)
196 TABLE 1 D e t e r m i n a t i o n of aprinidine in plasma of dogs treated with this drug. Dog
G.C. (pg/ml)
APR APR APR APR APR APR APR APR APR APR APR APR
15 16 17 18 19 20 21 22 23 24 25 30
0.19 2.72 1.98 ( 0.1 1.11 0.87 0.51 ( 0.1 1.08 0.76 0.15 ( 0.1
R I A (pg/ml) 0.15 2.65 2.06 0.07 1.17 0.74 0.52 0.03 1.17 0.99 0.16 0.06
The value of the slope a, corresponding to the heterogeneity coefficient of the population of antibodies, is n o t significantly different from 1, so that the population may be considered as homogeneous. The standard curve obtained in the R.I.A. for aprindine is shown in fig 6. The cross-reactivity curves shown on the same figure point o u t that parahydroxyaprindine and to a lesser extent desethylaprindine are able to displace the tritiated aprindine from its binding sites on the antibody. On the other hand, dephenylaprindine and desindanylaprindine do n o t show any affinity for the antiaprindine antibody. The determination of aprindine in plasma and tissues of dogs by G.C. and by R.I.A. allows to compare the results obtained by both methods (tables 1 and 2). The correlation between these values was determined both for plasma and tissues. The statistical analysis of these results shows that, for plasma assays as well as tissue assays, the value of Y-intercept b of linear regression analysis is n o t significantly different from zero (P < 0.05) and the slope is n o t significantly different from 1 (P < 0.05) (figs. 7 and 8). TABLE 2 D e t e r m i n a t i o n of aprindine in tissues of dogs treated with this drug. Tissue LG GL LG LG LG LG
53 53 53 53 53 55
heart, heart, heart, heart, heart, lung
infarcted left ventricle right ventricle left auricle right auricle left ventricle
G.C. (pg/g)
R I A (pg/g)
21.3 22.6 14.0 17.0 14.8 52.0
19.8 24.1 17.1 19.2 13.6 50.0
197
50,
/
E c. w
t9
=ax+b 25.
a : 0.99/.
!
b=.-O.021 r = 0.992
o O
0 0
0,/(5
G
/ ,
i R.I.A. [jug/ml )
~
o -z,3
7°
b=-2.13/. r = 0.991
'
,
25
50
R.I.A. ( / J g / g )
Fig. 7. C o r r e l a t i o n b e t w e e n t h e results o f t h e d e t e r m i n a t i o n o f a p r i n d i n e b y G.C. a n d R I A in p l a s m a o f dogs. b --~ 0; a --~ 1 ( S t u d e n t ' s test, P ~ 0.05). Fig. 8. C o r r e l a t i o n b e t w e e n t h e results o f t h e d e t e r m i n a t i o n o f a p r i n d i n e b y G.C. a n d R I A in tissues of dogs. b -~ 0, a ~-- 1 ( S t u d e n t ' s t e s t , P < 0.05).
As far as the sensitivity is concerned, the values taken from table 1 point out that G.C. does not allow plasma levels lower than 0.1 pg/ml to be measured while R.I.A. allows to determine, with sufficient precision, concentrations as low as 0.01 pg/ml. DISCUSSION
The value of the affinity constant shows that the antibody has a high affinity for aprindine. Moreover, the cross-reactivity curves reveal that the affinity of the antibody for aprindine is higher than for its metabolites. In fact, parahydroxyaprindine does not interfere with plasma assay of aprindine since it exists only in traces in plasma (Dodion et al., unpublished). The determination of aprindine in urine is not interesting because it contains only very small amounts of unchanged aprindine; on the other hand, urine contains phenyl or indanyl hydroxylated derivatives in the form of conjugates, and in lower amounts, desethylaprindine. Dephenylaprindine and desindanylaprindine, however essentially free in biological fluids, do not interfere with aprindine assay since their affinity for the antibody is negligible. In conclusion the production of antibodies with a high affinity for aprindine makes it possible to perform R.I.A. of this antiarrhythmic drug in plasma with very little interference, and to consider not only the therapeutic monitoring of this drug but also its application to the study of its pharmacokinetics in animals and humans.
198 REFERENCES Butler, V., J. Watson, D. Sehmidt, J. Gardner, W. Mandel and C. Skelton, 1973, Pharmacol. Rev. 25,239. Dodion, L., J.M. de Suray, M. Deblecker and A. Georges, 1974, Th4rapie 29,221. Georges, A., A. Hosslet, G. Duvernay, 1973, Acta Cardiol. 28,166. Rutherford, B.S. and R.H. Bishara, 1976, J. Pharm. Sci. 65, 1322. Vaitukaitis, J., J.B. Robbins, E. Nieschlag and G.T. Ross, 1971, J. Clin. Endoerinol. Metab., 33,988. Van Durme, J.P., M.G. Bogaert and M.T. Rossel, 1974a, Brit. J. Clin. Pharmacol. 1,461. Van Durme, J.P., M.G. Bogaert and M.T. Rossel, 1974b, Brit. J. Clin. Pharmacol. 7,343. Werblin, Th_P. and G.W. Siskind, 1972, Immunochemistry 9,987. Werblin, Th., Y. Tai Kim, Ch. Smith and G.W. Siskind, 1973, Immunoehemistry 10, 3.
189
© Elsevier/North-Holland Biomedical Press
D E V E L O P M E N T O F A RADIOIMMUNOASSAY FO R APRINDINE
MICHEL LESNE and Rl~GIS DOLPHEN * Service de Biopharmacie Clinique, Laboratoire de Pharmacodynamie Gdndrale, UCL 7350, Avenue Mounier 73, 1200 Bruxelles, Belgium
(Received 7 March 1977, accepted 24 April 1977)
We have developed a radioimmunoassay for aprindine, a new antiarrhythmic drug used in the treatment of ventricular disorders. The antibodies were produced by immunization of New-Zealand rabbits with aprindine coupled to human serum albumin. Their biochemical characteristics have been determined. Tritiated aprindine was used as radioactive competitor. The cross-reactivity with several metabolites of aprindine was studied too. Finally, the results obtained by RIA in plasma and tissues of dogs were compared to those obtained by gas-chromatography.
INTRODUCTION Aprindine is a new antiarrhythmic drug especially indicated in the disorders o f the ventricular excitability (Van Durme et al., 1974a; Georges et al., 1973). Its structure is illustrated in fig. 1. Although pharmacologically related to lidocaine, its pharmacokinetics is however very different. In fact, in contrast with lidocaine which is n o t used orally due to its very high hepatic clearance, aprindine, given orally, reaches plasma levels identical t o those observed after intravenous administration. T h e r e f o r e , it m ay be used n o t only in intravenous perfusion in the treatm e n t o f acute cases but also orally in the chronic t r e a t m e n t of ventricular arrhythmias. As the therapeutic index of this drug is rather low (Van Durme et al., 1974b), it seems o f interest to control its therapeutic use by means o f a rapid and precise m e t h o d of d e t e r m i n a t i o n in plasma, in order to avoid a t o o high f r e q u e n c y o f iatrogenic intoxications t h a t are manifested especially through cerebellar disorders. T h e r e f o r e , its seemed t o us interesting to develop a radioimmunological m e t h o d t o det erm i ne aprindine in biological samples instead o f the long and tedious gas-chromatography m e t h o d . ( R u t h e r f o r d et al., 1976).
* Assistant F.R.S.M.
190 SYNTHESIS OF ANTIGEN
CH2)3-N/c2H5
/CzH~" CI" CH2)3-N \C2H5
~~~[~
NC2H 5 SYNTHESIS
OF
HAPTEN APRINDINE
coupling
N=N* ~
HAPTEN
hapten - albumin
~
7C2H5
CH213-N\
C2H5
N=N-AH ANT IGEN
Fig. 1. Synthesis of the antigen using aprindine (N-(3-(diethylamino)propyl)-N-phenyl-2indanamine) and human serum albumin.
MATERIALS AND METHODS
The radioimmunoassay of aprindine requires the use of three compounds: unlabelled aprindine as standard, tritiated aprindine as radioactive competitor and specific antiaprindine antibodies. The tritiated aprindine used has been prepared in the laboratories of The Radioelements Institute (I.R.E.) in Fleurus, Belgium. The molecule was specifically labelled in the fourth position of the phenyl group (fig. 1); the specific activity obtained was 1.6 Ci/mmole. The purity of the preparation was controlled by means of thin-layer-chromatography and U.V. spectrophotometry. Due to the fact that aprindine has no immunogenic properties by itself, the production of these antibodies required, first, the synthesis of an antigen, obtained by coupling the hapten, in the form of aprindine diazotated in the fourth position of the phenyl group, to human serum albumin (fig. 1). The analysis has shown that eleven aprindine molecules are bound to each molecule of serum albumin. The antigen was then administrated to five NewZealand rabbits (A, B, C, D and E) by the multiple intradermic injection
191
technique on both sides of the vertebral column (Vaitukaitis et al., 1971). The production of antibodies in the serum of the rabbits has been followed in the different samples of blood taken from week to week. For each serum, we have measured the binding of a limited a m o u n t of tritiated aprindine to increasing dilutions of the antiserum in phosphate saline buffer.
Composition of the phosphate saline buffer 1.392 g Potassium phosphate, dibasic, K2HPO4; 0.276 g sodium phosphate, monobasic, m o n o h y d r a t e , Na H2PO4" H20; 8.76 g sodium chloride, NaC1, 1 liter distilled water. By expressing the percentage of binding of the tritiated aprindine versus the logarithm of antiserum dilution, it appears a sigmoidal shaped curve that, for the same rabbit, shifts to the right when the antibody level in serum becomes higher (fig. 2). Moreover these binding curves make it possible to define the antiserum with the highest titer. The next step was the determination of the selected antiserum characteristics, namely the concentration of antibodies and their affinity for aprindine. In order to do that, an appropriate dilution of the antiserum was incubated at 4 ° C in presence of increasing concentrations of tritiated aprindine. For each of the different concentrations used, after the equilibrium has been reached, one part of the solution was taken in order to measure the radioactivity corresponding to the total concentration of radioactive aprindine.
loo.
o"
-"-o
i T
o ~ c
50.
2_222
O. O.S
I
1.5
Log. dilution antiserum
Fig. 2. Binding of [3H]aprindine (4 ng) to increA.sing dilutions of antiserum in phosphate saline buffer.
192
T h e o t h e r p a r t was t h e n p l a c e d in p r e s e n c e o f d e x t r a n c o a t e d c h a r c o a l t o a d s o r b a p r i n d i n e selectively. A f t e r c e n t r i f u g a t i o n a t 3 0 0 0 r p m f o r 10 m i n , t h e m e a s u r e o f the radioactivity in the s u p e r n a t a n t a l l o w e d the d e t e r m i n a t i o n o f the a p r i n d i n e c o n c e n t r a t i o n b o u n d to the a n t i b o d i e s . T h e s t a n d a r d curve f o r t h e r a d i o i m m u n o a s s a y o f a p r i n d i n e was e s t a b l i s h e d b y m e a s u r i n g the i n h i b i t i o n o f b i n d i n g o f a small a m o u n t o f t r i t i a t e d a p r i n d i n e (4 ng) t o an a p p r o p r i a t e d i l u t i o n o f the a n t i s e r u m b y increasing c o n c e n t r a t i o n s o f u n l a b e U e d a p r i n d i n e ; these were c h o s e n in the range o f
NH--CH2-CH~--CHg-N./'CzHs .~Czpis
II
I"
Ddsindanyl-aprindine (AC 1817)
[••H
z-CH 2"-CH2-N HCzH s
Dg h~nyl- oprindine P (AC zzz6)
\
Ddsgfhyl-aprindine (AO g187)
2 5
Aprindine (AC 180;')
\
/ C2H6
Hydroxy- aprindine
OH
Hydroxy-aprindine
(typeII)
(type 1)
Fig. 3. Metabolism of aprindine in man. Two main ways: N-desethylation of the lateral chain and aromatic hydroxylation, third way, less frequent: loss of phenyl or indanyl cycles (after Dodion et al., 1974).
193
therapeutic concentrations (0.75 to 2 pg/ml) previously determined by G.C. in plasma samples of patients treated with this drug (Van Durme et al., 1974). The antibody specificity for aprindine was also determined by measuring the inhibition obtained in the same conditions and at the same concentrations for 4 metabolites of aprindine previously identified in man (Dodion et al., 1974): dephenylaprindine, desindanylaprindine, parahydroxyaprindine and desethylaprindine (fig. 3). Finally, the levels of aprindine reached in plasma and tissues of dogs treated by this antiarrhythmic drug were measured by both G.C. and R.I.A. The determination of plasma levels of aprindine by G.C. requires a preliminary double extraction; on the other hand, the R.I.A. was performed directly on plasma. As far as the tissues are concerned, the determination by G.C. was performed in the chloroformic solution obtained after a double extraction of aprindine from the tissue homogenate. In order to perform the R.I.A., aprindine was extracted from the tissue homogenate by means of diethyl ether; the organic phase was then evaporated to dryness and the residue was redissolved in phosphate buffer containing serum albumin. This aqueous extract was used for R.I.A. Composition o f the buffer: 0.75 g Potassium phosphate, monobasic, KH2POa; 7.8 g sodium phosphate, dibasic, dihydrate, Na2HPO4" 2H20; 0.5 g sodium azide, NaNa; 5 g bovine serum albumin; 1 liter distilled water. RESULTS
The antiserum studied was taken from rabbit D on february 2, 1976. It is the result of three immunizations: the first, made ten months earlier with 1 mg of antigen in Freund's adjuvant, the second, six months earlier by means of 100 #g of antigen dissolved in physiological solution and the third, one month before. The antibody level and their affinity constants were determined at the equilibrium of the reaction between hapten (tritiated aprindine) and antibodies, according to the following equation: Antibody + hapten ~ Antibody--hapten complex N--R C R At that time, R represents the concentration of antibody sites bound to the hapten, C, the concentration of free hapten and N--R, the concentration of binding sites available in the dilution of the antiserum used. The affinity constant (K0) corresponds to the following equation: R Ko = ( N - - R ) C
194 Antiserum Apr~ndine Rabbit D 2FEB 1976 1 . 123x106 M o Dilution : 1:10 N o
75.
o/
"6 E 50.
,~ 25.
1 N=
J
j 50
100 C -1 (107 )
150 (retire-mole)
200
250
Fig. 4. Relationship b e t w e e n the reciprocal of b o u n d sites c o n c e n t r a t i o n ( l / R ) and the reciprocal of free h a p t e n c o n c e n t r a t i o n (1/C) at equilibrium for the antiserum taken on F e b r u a r y 2, 1976 f r o m rabbit D. The straight line was d e t e r m i n e d by linear regression analysis o f the results, r = 0.98.
The measured parameters R and C were introduced in the following equation: , used in dialysis equilibrium where 'a' is the heterogeneity constant of the population of antibodies. (Werblin et al., 1972, 1973) The reciprocal of the concentration of b o u n d sites ( l / R ) plotted versus the reciprocal of the concentration of free hapten (1/C), yields a straight line of which y-intercept is 1IN (fig. 4.). The value of N allows to determine the a m o u n t of antibodies in the antiserum by reference to the molecular weight of the immunoglobulin G (around 160,000) and by assuming that there are two binding sites present in each antibody molecule (Butler et al., 1973). The concentration of antibodies in the antiserum taken on February 2, 1976 from rabbit D was estimated at 0.6 mg/ml. The affinity constant was calculated from fig. 5 that shows the relationship between the values of the logarithm of the ratio R/(N--R) and the logarithm of the concentration C of free competitor according to the following equation : R log N - - R = a log K0 + a log C The plot issues from the linear regression analysis of these values crosses the abscissa at a concentration value that represents the reciprical of the affinity constant. For that antiserum, the affinity constant was estimated at 1.58 • 108/M.
o:l
195
Antiserum Aprindine Rabbit D 2 FEB 1976 Dilution:l:10 Ko=1.S8x108 M "t
/®
=i=,_o.s_ °= .J
-is
,ool" o
o
oSS
/
o.,~[°
?J
-~.0.
oo
°
O
r = 0.98
o
-2.0 - 9.s
-9
- is
-8
-~?s
Log C
Fig. 5. Determination of the affinity constant (K0) of the a n t i b o d y for [3H]aprindine. S l o p e o f the straight line: 'a' ~ 1.
100. ,~--v
o
v.
o
~,~.,~
CROSS-REACTIVITY : ANTISERUtl APPRINDINE RABBII D :' FEB 1976 0 aprindine A para-hydroxy-apri ndine
I-I ~. ~ ~
o e,, e,.
~
°~
,,r!
I"I desethyl-aprindine ~ dephen)~l-aprindine
0~. =~.~
~
desindanyl-aprindi ne
50.
o C ,~ C
lla
0
'
'
'
0.25 0.50 0.75 Concentration of unlabelled drug
II
1 5 10 15 (10-6M)
Fig. 6. Standard curve o b t a i n e d in the R I A o f aprindine ( o ) and cross-reactivity curves w i t h 4 m e t a b o l i t e s (~, ~, v , 0 ) (The binding o f [ 3 H ] a p r i n d i n e t o the a n t i b o d i e s is e x p r e s s e d in p e r c e n t o f the value measured w i t h o u t unlabelled drug.)
196 TABLE 1 D e t e r m i n a t i o n of aprinidine in plasma of dogs treated with this drug. Dog
G.C. (pg/ml)
APR APR APR APR APR APR APR APR APR APR APR APR
15 16 17 18 19 20 21 22 23 24 25 30
0.19 2.72 1.98 ( 0.1 1.11 0.87 0.51 ( 0.1 1.08 0.76 0.15 ( 0.1
R I A (pg/ml) 0.15 2.65 2.06 0.07 1.17 0.74 0.52 0.03 1.17 0.99 0.16 0.06
The value of the slope a, corresponding to the heterogeneity coefficient of the population of antibodies, is n o t significantly different from 1, so that the population may be considered as homogeneous. The standard curve obtained in the R.I.A. for aprindine is shown in fig 6. The cross-reactivity curves shown on the same figure point o u t that parahydroxyaprindine and to a lesser extent desethylaprindine are able to displace the tritiated aprindine from its binding sites on the antibody. On the other hand, dephenylaprindine and desindanylaprindine do n o t show any affinity for the antiaprindine antibody. The determination of aprindine in plasma and tissues of dogs by G.C. and by R.I.A. allows to compare the results obtained by both methods (tables 1 and 2). The correlation between these values was determined both for plasma and tissues. The statistical analysis of these results shows that, for plasma assays as well as tissue assays, the value of Y-intercept b of linear regression analysis is n o t significantly different from zero (P < 0.05) and the slope is n o t significantly different from 1 (P < 0.05) (figs. 7 and 8). TABLE 2 D e t e r m i n a t i o n of aprindine in tissues of dogs treated with this drug. Tissue LG GL LG LG LG LG
53 53 53 53 53 55
heart, heart, heart, heart, heart, lung
infarcted left ventricle right ventricle left auricle right auricle left ventricle
G.C. (pg/g)
R I A (pg/g)
21.3 22.6 14.0 17.0 14.8 52.0
19.8 24.1 17.1 19.2 13.6 50.0
197
50,
/
E c. w
t9
=ax+b 25.
a : 0.99/.
!
b=.-O.021 r = 0.992
o O
0 0
0,/(5
G
/ ,
i R.I.A. [jug/ml )
~
o -z,3
7°
b=-2.13/. r = 0.991
'
,
25
50
R.I.A. ( / J g / g )
Fig. 7. C o r r e l a t i o n b e t w e e n t h e results o f t h e d e t e r m i n a t i o n o f a p r i n d i n e b y G.C. a n d R I A in p l a s m a o f dogs. b --~ 0; a --~ 1 ( S t u d e n t ' s test, P ~ 0.05). Fig. 8. C o r r e l a t i o n b e t w e e n t h e results o f t h e d e t e r m i n a t i o n o f a p r i n d i n e b y G.C. a n d R I A in tissues of dogs. b -~ 0, a ~-- 1 ( S t u d e n t ' s t e s t , P < 0.05).
As far as the sensitivity is concerned, the values taken from table 1 point out that G.C. does not allow plasma levels lower than 0.1 pg/ml to be measured while R.I.A. allows to determine, with sufficient precision, concentrations as low as 0.01 pg/ml. DISCUSSION
The value of the affinity constant shows that the antibody has a high affinity for aprindine. Moreover, the cross-reactivity curves reveal that the affinity of the antibody for aprindine is higher than for its metabolites. In fact, parahydroxyaprindine does not interfere with plasma assay of aprindine since it exists only in traces in plasma (Dodion et al., unpublished). The determination of aprindine in urine is not interesting because it contains only very small amounts of unchanged aprindine; on the other hand, urine contains phenyl or indanyl hydroxylated derivatives in the form of conjugates, and in lower amounts, desethylaprindine. Dephenylaprindine and desindanylaprindine, however essentially free in biological fluids, do not interfere with aprindine assay since their affinity for the antibody is negligible. In conclusion the production of antibodies with a high affinity for aprindine makes it possible to perform R.I.A. of this antiarrhythmic drug in plasma with very little interference, and to consider not only the therapeutic monitoring of this drug but also its application to the study of its pharmacokinetics in animals and humans.
198 REFERENCES Butler, V., J. Watson, D. Sehmidt, J. Gardner, W. Mandel and C. Skelton, 1973, Pharmacol. Rev. 25,239. Dodion, L., J.M. de Suray, M. Deblecker and A. Georges, 1974, Th4rapie 29,221. Georges, A., A. Hosslet, G. Duvernay, 1973, Acta Cardiol. 28,166. Rutherford, B.S. and R.H. Bishara, 1976, J. Pharm. Sci. 65, 1322. Vaitukaitis, J., J.B. Robbins, E. Nieschlag and G.T. Ross, 1971, J. Clin. Endoerinol. Metab., 33,988. Van Durme, J.P., M.G. Bogaert and M.T. Rossel, 1974a, Brit. J. Clin. Pharmacol. 1,461. Van Durme, J.P., M.G. Bogaert and M.T. Rossel, 1974b, Brit. J. Clin. Pharmacol. 7,343. Werblin, Th_P. and G.W. Siskind, 1972, Immunochemistry 9,987. Werblin, Th., Y. Tai Kim, Ch. Smith and G.W. Siskind, 1973, Immunoehemistry 10, 3.