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Radioimmunoassay of the antidiarrhoeal loperamide
Life Sciences, Vol . 21, pp . 451-460, 1977 Printed In The U.S .A .
Pergamon Press
RADIOIMMUNOASSAY OF THE ANTIDIARRHOEAL LOPERAMIDE M . Michiels, R. Hendrike and J . Heykants Department of Drug Metabolism Janssen Pharmaceutics, Research Laboratories B-2340 Beerse, Belgium (Received in final form July 5, 1977) Summa ry Production of antibodies against loperamide was induced in rabbits that were repeatedly injected with a loperamide-protein conjugate. By using the antiserum, a sensitive and specific radioimmunoassay procedure for loperamide wa.s developed. The drug could be assayed directly in human plasma in amounts as low as 50 picogram . None of the known metabolites of loperamide interfered with the radioimmunologic determination of loperamide in biological fluids . The disposition of loperamide was studied in man. Following a single oral dose of 4 mg in a tablet formulation, peak plasma levels, corresponding to about 0. 75 ng~ml, were reached 4 hours after drug intake and drug plasma concentrations could be measured up to 24 hours after administration . Loperamide, 4-(4-chloropheayl)-4-hydroxy-N, N-dimethyl-a , a -dipheayl1-piperidinebutanamide hydrochloride (R 18 553) is a very potent, orally long-acting and specific antidiarrhoeal (1) . It specifically inhibits peristaltic activity by a direct effect on the gaetro-intestinal wall, interacting locally with cholinergic as well as with non-cholinergic neuronal mechanisms involved in the peristaltic reflex (2) . Due to the lack of a sensitive and convenient analytical method for the quantification of the drug, the only information on the pharmacokinetics of loperamide in man thus far available is based on experiments with the tritium-labelled compound in which the unaltered drug was estimated by the inverse isotope dilution method (3) . In this study, the development of a sensitive and specific radioimmunoassay for loperamide is described. The applicability of the assay for pharmacokinetic studies of loperamide in man is also demonstrated .
Materials and Methode Preparation of the loperamide-protei n çonjuRate
Loperamide was converted to 4-{4-(4-chlorophenyl)-1-~4-(dimethylamino) -4-oxo-3, 3-diphenylbutylJ-4-piperidinyloxy}-4-oxobutanoic acid as shown in figure 1 . One hundred milligram of the hapten was dissolved in 1 ml of 451
Radioi~unoassay of Loperamide
452
Vol . 21, No . 3, 1977
Hi Ov Nv ~C CH3 C-CH?-CHZ-N,
CIfZ -C
CI
0
~0 CHZ-C+ 0 Pyridin~ Drrn :m
1 . DMA/MCI Wrbddnimidrr i.BSA
0
v eW
~7 n N 0H7
0 0 O-C-CHZ-CHZ-C-NH-BSA
C-CHZ-CHq-N
CI
FI G. 1 Synthesis of the loperamide-protein conjugate used for the immunization of the rabbit e.
dimethylacetamide, acidified with 2 drops of concentrated hydrochloric acid and this solution was added dropwiee to 400 mg of 1-ethyl-3-(3-dimethylaminopropyl)-carbodümide hydrochloride dissolved in 5 ml of water. The pH was adjusted at 5. 0 with 0 . 1 N sodium hydroxide . To this mixture 200 mg of bovine serum albumin (BSA), dissolved in 20 ml of 0 . 05 M phosphate buffer (pH 6. 0) was added dropwise and this solution was stirred overnight at 4° C. The reaction mixture was dialyzed against distilled water for 12 hours and lyophilized afterwards . Immunization
Two female New Zealand albino rabbits were injected intradermally in multiple sites on the back with 0 . 5 mg of the loperamide conjugate in a 50 °f° emulsion of complete Freund's adjuvant . Beginning one month after the initial dose, seven booster injections were given subcutaneouely with inter~ra.le of three weeks. The rabbits were sacrificed 9 days after the last booster and the collected serum was pooled and stored at -20° C.
Vol . 21, No . 3, 1977
Radioimmunoassay of Loperamide
453
Ra dioimmunoasea~of loperamide Antiserum-titers were determined by adding 0 . 2 ml of various antiserum dilutione to 0 . 5 ml of phosphate buffer (pH 7. 4) and 0. 5 ng of tritiated loperamide (spec . act. 9 Ci~mM, corresponding to about 22, 000 dpm) in 0 . 05 ml of 30 P/° methanol-water, Incubations were carried out in 1 . 3 ml plastic tubes with continuous rotation (25 rev. min) for 2 hours at room temperature, al though it was found that this incubation time was not critical at all. Sound and free loperamide were separated with dextran-coated charcoal . Aliquots of 0, 2 ml of a suepeneion, containing 500 mg activated charcoal (Norit A-supra), 50 mg dextran and 100 mg sodium azide in 100 ml of phosphate buffer (pH 7 . 4), were added to the incubation mixture and allowed to equilibrate for 1 hour at room temperature with continuous rotation . Thereafter the charcoal was removed by centrifugation at 10, 000 rev. min for 10 minutes . The supernatant was pipetted into a counting vial, mixed with 10 ml of a scintillation cocktail (Riafluor-NEN) and the radioactivity was determined in a liquid scintillation spectrometer (Packard Tri-Garb, Model 3380, 544 AAA) . Standard curves of loperamide, added to control plasma, were generated by the incubation of 0 . 2 ml of an appropriate antiserum dilution with a fixed amount of tritium-labelled and varying quantities of unlabelled loperamide in 0 . 5 ml of blank plasma . The amount of charcoal required for the separation of free and antibody-bound loperamide and for keeping blank values within acceptable limits was raised to 2 °j° ww . Antibody-specificity was determined in buffer by measuring the inhibition of the antibody-loperamide complex formation caused by increasing amounts of the known metabolites or various structurally related compounds incubated with 0 . 2 ml of the appropriate antiserum dilution, in the presence of 1 ng of 3H-loperamide . Human stud
Three healthy male volunteers participated in this study, They fasted overnight prior to drug intake . A blank blood sample was taken on heparin just before drug administration, The morning urine served as cont 1 urine . Each subject swallowed two 2-mg capsules of loperamide (Irrodium ~) . Only mineral water was taken during a further fasting period of two hour e. Venous blood was taken oa heparin (200 EU~10 ml of blood) at intervals up to 72 hours after administration . After centrifuging at 2, 500 rev. min, the collected plasma was stored at -20° C until assayed, Urine and faeces were collected for up to three days after drug intake and the samples were stored at -20° C until analysis . Unaltered loperamide was determined directly in 0 . 5 ml aliquots of the subject's plasma ae described. The drug was also assayed directly in urine by interpolation on a simultaneously obtained standard curve of the drug added to control urine, The pH of all urine samples wa.e adjusted at 7 . 4 before incubation . Separation of bound and free loperamide was effected with an 0 . 5 °J° charcoal suepeneion . Loperamide conjugated with glucuronic acid was assayed after hydrolysis with ß -glucuronidase . Faecal homogenates were prepared in methanol (1 :10 w :v) using an Ultraturrax TP 182 and centrifuged afterwards . Aliquots of 0 . 02 ml of the super natant were assayed in phosphate buffer (pH 7. 4) as described. Concentrations were calculated from a standard curve generated with a control faecal extract.
454
Radioi~uaoassay of Loperamide
Vol. 21, No . 3, 1977
Results The data obtained from the antiserum titrations demonstrated that the rabbit serum contained antibodies capable of binding 3H-loperamide . Antieerum diluted 1130 bound nearly 50 °fo of the added tracer under the assay conditions described. p,
~i~ ~0 ô x
~ m ~n
~
np of bp~ramid~ ~dMd
,.o
FI G. 2 Logit-log plot for the displacement of 0 . 5 ng of tritium-labelled loperamide, effected by various amounts of unlabelled loperamide added to control human plasma. . Sensitivity of the aesa.y Figure 2 represents a typical standard curve obtained with 1130 antiserum dilution and 0 . 5 ng of the labelled tracer, for loperamide added to control human plasma . The detection limit of the assay, when applied in this way, is about 50 picogram and it may be stated that loperamide can be accurately assayed in a range from 100 picogram up to 10 nanogram contained in 0. 5 ml of plasma . The remainder of the radioactivity in the supernatant, due to unepecific adsorption to plasma constituents, never exceeded 2 °fo and this value was always subtracted for each assay. Specificity of the antiserum The cross-reactivity of various test compounds with the loperamide-antibody is illustrated in figure 3 and table 1 . Data are expressed as the molar ratio required for 50 % inhibition of antibody-binding of 1 ng 3H-loperamide . Under the assay conditions described, 50 °fo inhibition of 3H-loperamide binding was achieved with equimolar amounts of the labelled and unlabelled drug .
Vol. 21, No . 3, 1977
Radioi~unoassay of Loperamide
455
99
95
a .~ . ôn 0 - so v c c ao Ë
.
> vo
m Y
5
~ww~~T~a.
a X00
ng of compound added FIG. 3
Immunological cross-reactivity with loperamide antiserum: displacement of 1 ng 3H-loperamide from antibody-binding sites, effected by various amounts of unlabelled loperamide or related compounds . Fluperamide (compound 2), differing from loperamide by a trifluoromethyl eubstituent in the 3-position of the 4-phenyl, was the only compound tested that was able to displace to a considerable extent the parent drug from the antibody-binding sites. The antibody did not bind to any degree the structurally related drugs haloperidol, diphenoxylate and difenoxin (compounds 3-5) . The main biliary metabolites of loperamide, i . e . 4-(4-chlorophenyl)-4-hydroxy_N-methyl-a , a -diphenyl-l-piperidinebutanamide and 4-(4-chlorophenyl)-4hydroxy-a , a -diphenyl-l-piperidinebutanamide (4) (compounds b-7), interfered only very slightly in the competition for antibody-binding with 3H-loperamide . A molar excess of about 750 and 4, 800 respectively was required to inhibit by 50 °fo the antibody-binding of 3H-loperamide . No cross-reaction was observed with the possible metabolite 4-(4-chlorophenyl)-4-hydroxy-a , a diphenyl-l-piperidinebutanoic acid (not identified) or with 4-(4-chlorophenyl)4-hydroxy-piperidine, formed by oxydative N-dealkylation of loperamide (4), (compounds 8-9) even at 5, 000-fold molar excess . Disposition of lope ramide in man
Figure 4 represents the loperamide plasma levels after oral administration of 4 mg loperamide . Peak levels of unchanged drug were reached 4 hours after drug intake, corresponding to about 0 . 75 ng~ml . The cumulative percent of loperamide absorbed was estimated from the mean C-t plasma curve for subjects 1 and 2, using the Wagner-Nelson method (5) . About 26 °fo of the administered drug had entered the systemic circulation within 30 minutes after
456
Radioi3l3mt3noassay of Lopera3nide
Vol. 21, No . 3, 1977
TABLE 1 Antibody-Specificity : Competition between various Teat Compounds and 3H-Loperamide for Antibody-Binding Compound
1
~ ~
pnstary nsma
`
C CH3 /~~OH C-CMZ-CNZ-N J( \ ~,
I
b~ndingol 1 ny loperamide ~H by 50'i a
1
RtlS53 tppa " am~aa
CH3 ~\~OH O'C~ N'CH3 ~ ~ C-CHZ-CHT-N X
®I
,~~Cß
-
3
Molar rat~o to mhiDi7
ÇH3 N
0~
i
~Rataranea numbarornon-pro~
5tructura
numDer
~~
R tB9t0 fluPnsmae
7
R7l75 H"lopandol
> 5,000
R1137 ]vphanoeylate
>5,000
CI
0
/^~,~OH P+ ® " C-CHZ-CHZ-CHZ-N %
CI CN G
5
'~~----~~v~~~ - 0-CH1-CH3
C-CH ? -CHZ -N \
~ ~
,~
,
0 CN ^, C-OH ~ ~ C-CH?-CH T-N X ~ i \
H ' //~~~'ON ~ 'CH3 C-CHZ-CH7-N~
CN
R15G03 Dvlenoam
>5,000
0, 6
~ ~
R20905
750
Rt13GS
G,CSO
Rt9S3i
>S,ODO
R tStS
> S,DDO
\~I
CI ,H N H OH ;~®CMZ-CH?-N~, 0~
7
1 0, ,OM
!
~
~
Ç
/~~OH
C-CH=-CHZ-N
J(
I !
H-N
OH
CI
(!)
Cross-reaction is axprassed as the molar ratio of loperamide and the teat compounds to Inhibit bt~ 50 the complex formation between 3H-loperamide and the antiserum,
(~)
r
Croee-reaction ie expressed ae the molar ratio of loperamide and the test compounds to inhibit by 50 yp the complex formation between 3H-loperamide and the anti serum.
Radioi~unoassay of Loperamide
Vol . 21, No . 3, 1977
457
FI G. 4
Plasma levels of unaltered loperamide plotted against time in three subjects orally treated with 4 mg of the drug . drug intake, 67 °fo after 2 hours and the absorption phase was almost completely terminated 4 hours after administration . From that time, loperamide disappeared from the plasma with a half-life of about 15 hours and 48 hours after administration drug concentrations were lower than 0 . 1 ng~ml, i. e. the detec tion limit of the assay method . Since plasma. concentrations of loperamide, assayed after hydrolysis with ß -glucuronidase did not increase significantly, it can be stated that no or only negligible amounts of loperamide-glucuronide were present in the plasma (data not shown) . TABLE 2
Excretion of Unchanged Loperamide with Urine and Faeces of Man up to three Days after an Oral Dose of 4 mg (Medians of three Subjects) K ofadministered dose Time interval
Urine Free loperamide
0-8 h 8-24 h 24-32 h 32-48 h 48-56 h 56-72 h
o. 297
G~mulathe
p, q24
0. 0. 0. 0. 0.
402 085 085 030 025
Glucuronic acid conjugate 0. 0. 0. 0. 0. 0.
Total
110 100 040 060 015 040
0 . 407 0 . 502 0 . 125 0 . 145 0 . 045 0 . 065
0 . 365
1 . 289
Faeces
8 . 99 12 .23 2 . 95 24 . 17
458
Radioimmunoassay of Loperamide
Vol . 21, No . 3, 1977
Urinary and faecal excretion of loperamide are summarized in table 2. From 0 . 63 to 1 . 4 °fo of the dose (median 0. 92 °fo) was excreted with the urine ae unaltered drug, whereas total urinary excretion of free and conjugated drug averaged 1 . 35 °Jo (median 1 . 29 °fo) . Drug excretion occurred mainly with the first day urine and was not correlated with the urinary pH . Faecal excretion was the main route and amounted to about 25 °~o of the administered dose .
Di scue sinn Antibodies with high loperamide-binding capacity were obtained by repeated injections of rabbits with a loperamide-BSA conjugate . By using the antiserum, loperamide could be assayed with accuracy, directly in plasma in a range from 0 . 1 up to 20 nanogram . Although the incubation volumes of 0 . 5 ml of plasma were rather large, blank values were less than 2 °fo, indicating that normal plasma constituents did not interfere . The affinity of the antibody for loperamide is evidenced by the results of the cross-reactivity experiments . On the one hand, a high degree of cross-reaction was observed between fluperamide and 3H-loperamide, but on the other hand, the antibody sharply differentiated between the parent drug and its structurally very closely related metabolites . This indicates that in practice these N-dealkylated metabolites will not interfere with the radioimmunoaseay of loperamide . From these results and the data obtained with the structurally related drugs, ae there are the neuroleptic haloperidol and the antidiarrhoeale diphenoxylate and difenoxin, it can be concluded that the antibody sharply recog nized not only the entire dimethylamide-moiety of the loperamide molecule, but also that the intact loperamide structure was required for strong binding . So, ae the amide-function was the principal distinctive mark of loperamide, the hapten synthesis via substitution of the tertiary alcohol was preferred, just to leave intact this important functional group. Moreover, by coupling the hapten in this way, a real possibility was created for the antiserum to be used not only for the determination of loperamide but also for the quantification of the closely related antidiarrhoeal fluperamide (6) . Based on the high degree of cross-reaction (table 1), the antiserum can presumably also be used for the assay of fluperamide . It can be concluded that the method presented in this paper, is very specific for the determination of loperamide, without interferences of its _N-dealkylated metabolites. The results of the preliminary pharrnacokinetic analysis of loperamide in man demonstrate that the accuracy, feasibility and sensitivity àre sufficient to enable this radioimmunoaesay to be used for routine determination of loperamide concentrations in clinical and pharmacokinetic studies. AcknowledAments The authors wish to thank Mr . A . Knaeps and Mr . J. Thijesen for the synthesis of the loperamide hapten .
Vol . 21, No . 3, 1977
Radioia~unoassay of Loperamide
459
References e 1 . C . J . E. NIEMEGEERS, F. M. LENAERTS and P. A, J. JANSSEN, Arzrieim . -Forsch. (Dru¢ Res . ) 24, 1633-1636 (1974) . 2. J . M. VAN NUETEN, P. A, J . JANSSEN and J. FONTAINE, Arzneiur . Forsch . (Drug Res. ) 24, 1641-1645 (1974) . 3. J . HEYKANTS, M . MICHIELS, A. KNAEPS and J . BRUGMANS, Arzneiur . Forsch . (Drug Res. ) 24, 1649-1653 (1974) . 4. J. HEYKANTS, W . MEULDERMANS, A. KNAEPS and M . MICHIELS, Europ. J . Drug Metabolism and Pharmacokinetics , submitted . 5. J . G. WAGNER, in Biopharmaceutice and relevant pharmacokiaetics , p. 278-280, Drug Intelligence Publications, Hamilton, Ill, (1971), b. C. NIEMEGEERS, F. LENAERTS and F. AWOUTERS, in Synthetic antidiarrheal drums, Van Bever and Lal (Eds), p. 65-114, Marcel Dekker, Inc . , New York (1976) .
Pergamon Press
RADIOIMMUNOASSAY OF THE ANTIDIARRHOEAL LOPERAMIDE M . Michiels, R. Hendrike and J . Heykants Department of Drug Metabolism Janssen Pharmaceutics, Research Laboratories B-2340 Beerse, Belgium (Received in final form July 5, 1977) Summa ry Production of antibodies against loperamide was induced in rabbits that were repeatedly injected with a loperamide-protein conjugate. By using the antiserum, a sensitive and specific radioimmunoassay procedure for loperamide wa.s developed. The drug could be assayed directly in human plasma in amounts as low as 50 picogram . None of the known metabolites of loperamide interfered with the radioimmunologic determination of loperamide in biological fluids . The disposition of loperamide was studied in man. Following a single oral dose of 4 mg in a tablet formulation, peak plasma levels, corresponding to about 0. 75 ng~ml, were reached 4 hours after drug intake and drug plasma concentrations could be measured up to 24 hours after administration . Loperamide, 4-(4-chloropheayl)-4-hydroxy-N, N-dimethyl-a , a -dipheayl1-piperidinebutanamide hydrochloride (R 18 553) is a very potent, orally long-acting and specific antidiarrhoeal (1) . It specifically inhibits peristaltic activity by a direct effect on the gaetro-intestinal wall, interacting locally with cholinergic as well as with non-cholinergic neuronal mechanisms involved in the peristaltic reflex (2) . Due to the lack of a sensitive and convenient analytical method for the quantification of the drug, the only information on the pharmacokinetics of loperamide in man thus far available is based on experiments with the tritium-labelled compound in which the unaltered drug was estimated by the inverse isotope dilution method (3) . In this study, the development of a sensitive and specific radioimmunoassay for loperamide is described. The applicability of the assay for pharmacokinetic studies of loperamide in man is also demonstrated .
Materials and Methode Preparation of the loperamide-protei n çonjuRate
Loperamide was converted to 4-{4-(4-chlorophenyl)-1-~4-(dimethylamino) -4-oxo-3, 3-diphenylbutylJ-4-piperidinyloxy}-4-oxobutanoic acid as shown in figure 1 . One hundred milligram of the hapten was dissolved in 1 ml of 451
Radioi~unoassay of Loperamide
452
Vol . 21, No . 3, 1977
Hi Ov Nv ~C CH3 C-CH?-CHZ-N,
CIfZ -C
CI
0
~0 CHZ-C+ 0 Pyridin~ Drrn :m
1 . DMA/MCI Wrbddnimidrr i.BSA
0
v eW
~7 n N 0H7
0 0 O-C-CHZ-CHZ-C-NH-BSA
C-CHZ-CHq-N
CI
FI G. 1 Synthesis of the loperamide-protein conjugate used for the immunization of the rabbit e.
dimethylacetamide, acidified with 2 drops of concentrated hydrochloric acid and this solution was added dropwiee to 400 mg of 1-ethyl-3-(3-dimethylaminopropyl)-carbodümide hydrochloride dissolved in 5 ml of water. The pH was adjusted at 5. 0 with 0 . 1 N sodium hydroxide . To this mixture 200 mg of bovine serum albumin (BSA), dissolved in 20 ml of 0 . 05 M phosphate buffer (pH 6. 0) was added dropwise and this solution was stirred overnight at 4° C. The reaction mixture was dialyzed against distilled water for 12 hours and lyophilized afterwards . Immunization
Two female New Zealand albino rabbits were injected intradermally in multiple sites on the back with 0 . 5 mg of the loperamide conjugate in a 50 °f° emulsion of complete Freund's adjuvant . Beginning one month after the initial dose, seven booster injections were given subcutaneouely with inter~ra.le of three weeks. The rabbits were sacrificed 9 days after the last booster and the collected serum was pooled and stored at -20° C.
Vol . 21, No . 3, 1977
Radioimmunoassay of Loperamide
453
Ra dioimmunoasea~of loperamide Antiserum-titers were determined by adding 0 . 2 ml of various antiserum dilutione to 0 . 5 ml of phosphate buffer (pH 7. 4) and 0. 5 ng of tritiated loperamide (spec . act. 9 Ci~mM, corresponding to about 22, 000 dpm) in 0 . 05 ml of 30 P/° methanol-water, Incubations were carried out in 1 . 3 ml plastic tubes with continuous rotation (25 rev. min) for 2 hours at room temperature, al though it was found that this incubation time was not critical at all. Sound and free loperamide were separated with dextran-coated charcoal . Aliquots of 0, 2 ml of a suepeneion, containing 500 mg activated charcoal (Norit A-supra), 50 mg dextran and 100 mg sodium azide in 100 ml of phosphate buffer (pH 7 . 4), were added to the incubation mixture and allowed to equilibrate for 1 hour at room temperature with continuous rotation . Thereafter the charcoal was removed by centrifugation at 10, 000 rev. min for 10 minutes . The supernatant was pipetted into a counting vial, mixed with 10 ml of a scintillation cocktail (Riafluor-NEN) and the radioactivity was determined in a liquid scintillation spectrometer (Packard Tri-Garb, Model 3380, 544 AAA) . Standard curves of loperamide, added to control plasma, were generated by the incubation of 0 . 2 ml of an appropriate antiserum dilution with a fixed amount of tritium-labelled and varying quantities of unlabelled loperamide in 0 . 5 ml of blank plasma . The amount of charcoal required for the separation of free and antibody-bound loperamide and for keeping blank values within acceptable limits was raised to 2 °j° ww . Antibody-specificity was determined in buffer by measuring the inhibition of the antibody-loperamide complex formation caused by increasing amounts of the known metabolites or various structurally related compounds incubated with 0 . 2 ml of the appropriate antiserum dilution, in the presence of 1 ng of 3H-loperamide . Human stud
Three healthy male volunteers participated in this study, They fasted overnight prior to drug intake . A blank blood sample was taken on heparin just before drug administration, The morning urine served as cont 1 urine . Each subject swallowed two 2-mg capsules of loperamide (Irrodium ~) . Only mineral water was taken during a further fasting period of two hour e. Venous blood was taken oa heparin (200 EU~10 ml of blood) at intervals up to 72 hours after administration . After centrifuging at 2, 500 rev. min, the collected plasma was stored at -20° C until assayed, Urine and faeces were collected for up to three days after drug intake and the samples were stored at -20° C until analysis . Unaltered loperamide was determined directly in 0 . 5 ml aliquots of the subject's plasma ae described. The drug was also assayed directly in urine by interpolation on a simultaneously obtained standard curve of the drug added to control urine, The pH of all urine samples wa.e adjusted at 7 . 4 before incubation . Separation of bound and free loperamide was effected with an 0 . 5 °J° charcoal suepeneion . Loperamide conjugated with glucuronic acid was assayed after hydrolysis with ß -glucuronidase . Faecal homogenates were prepared in methanol (1 :10 w :v) using an Ultraturrax TP 182 and centrifuged afterwards . Aliquots of 0 . 02 ml of the super natant were assayed in phosphate buffer (pH 7. 4) as described. Concentrations were calculated from a standard curve generated with a control faecal extract.
454
Radioi~uaoassay of Loperamide
Vol. 21, No . 3, 1977
Results The data obtained from the antiserum titrations demonstrated that the rabbit serum contained antibodies capable of binding 3H-loperamide . Antieerum diluted 1130 bound nearly 50 °fo of the added tracer under the assay conditions described. p,
~i~ ~0 ô x
~ m ~n
~
np of bp~ramid~ ~dMd
,.o
FI G. 2 Logit-log plot for the displacement of 0 . 5 ng of tritium-labelled loperamide, effected by various amounts of unlabelled loperamide added to control human plasma. . Sensitivity of the aesa.y Figure 2 represents a typical standard curve obtained with 1130 antiserum dilution and 0 . 5 ng of the labelled tracer, for loperamide added to control human plasma . The detection limit of the assay, when applied in this way, is about 50 picogram and it may be stated that loperamide can be accurately assayed in a range from 100 picogram up to 10 nanogram contained in 0. 5 ml of plasma . The remainder of the radioactivity in the supernatant, due to unepecific adsorption to plasma constituents, never exceeded 2 °fo and this value was always subtracted for each assay. Specificity of the antiserum The cross-reactivity of various test compounds with the loperamide-antibody is illustrated in figure 3 and table 1 . Data are expressed as the molar ratio required for 50 % inhibition of antibody-binding of 1 ng 3H-loperamide . Under the assay conditions described, 50 °fo inhibition of 3H-loperamide binding was achieved with equimolar amounts of the labelled and unlabelled drug .
Vol. 21, No . 3, 1977
Radioi~unoassay of Loperamide
455
99
95
a .~ . ôn 0 - so v c c ao Ë
.
> vo
m Y
5
~ww~~T~a.
a X00
ng of compound added FIG. 3
Immunological cross-reactivity with loperamide antiserum: displacement of 1 ng 3H-loperamide from antibody-binding sites, effected by various amounts of unlabelled loperamide or related compounds . Fluperamide (compound 2), differing from loperamide by a trifluoromethyl eubstituent in the 3-position of the 4-phenyl, was the only compound tested that was able to displace to a considerable extent the parent drug from the antibody-binding sites. The antibody did not bind to any degree the structurally related drugs haloperidol, diphenoxylate and difenoxin (compounds 3-5) . The main biliary metabolites of loperamide, i . e . 4-(4-chlorophenyl)-4-hydroxy_N-methyl-a , a -diphenyl-l-piperidinebutanamide and 4-(4-chlorophenyl)-4hydroxy-a , a -diphenyl-l-piperidinebutanamide (4) (compounds b-7), interfered only very slightly in the competition for antibody-binding with 3H-loperamide . A molar excess of about 750 and 4, 800 respectively was required to inhibit by 50 °fo the antibody-binding of 3H-loperamide . No cross-reaction was observed with the possible metabolite 4-(4-chlorophenyl)-4-hydroxy-a , a diphenyl-l-piperidinebutanoic acid (not identified) or with 4-(4-chlorophenyl)4-hydroxy-piperidine, formed by oxydative N-dealkylation of loperamide (4), (compounds 8-9) even at 5, 000-fold molar excess . Disposition of lope ramide in man
Figure 4 represents the loperamide plasma levels after oral administration of 4 mg loperamide . Peak levels of unchanged drug were reached 4 hours after drug intake, corresponding to about 0 . 75 ng~ml . The cumulative percent of loperamide absorbed was estimated from the mean C-t plasma curve for subjects 1 and 2, using the Wagner-Nelson method (5) . About 26 °fo of the administered drug had entered the systemic circulation within 30 minutes after
456
Radioi3l3mt3noassay of Lopera3nide
Vol. 21, No . 3, 1977
TABLE 1 Antibody-Specificity : Competition between various Teat Compounds and 3H-Loperamide for Antibody-Binding Compound
1
~ ~
pnstary nsma
`
C CH3 /~~OH C-CMZ-CNZ-N J( \ ~,
I
b~ndingol 1 ny loperamide ~H by 50'i a
1
RtlS53 tppa " am~aa
CH3 ~\~OH O'C~ N'CH3 ~ ~ C-CHZ-CHT-N X
®I
,~~Cß
-
3
Molar rat~o to mhiDi7
ÇH3 N
0~
i
~Rataranea numbarornon-pro~
5tructura
numDer
~~
R tB9t0 fluPnsmae
7
R7l75 H"lopandol
> 5,000
R1137 ]vphanoeylate
>5,000
CI
0
/^~,~OH P+ ® " C-CHZ-CHZ-CHZ-N %
CI CN G
5
'~~----~~v~~~ - 0-CH1-CH3
C-CH ? -CHZ -N \
~ ~
,~
,
0 CN ^, C-OH ~ ~ C-CH?-CH T-N X ~ i \
H ' //~~~'ON ~ 'CH3 C-CHZ-CH7-N~
CN
R15G03 Dvlenoam
>5,000
0, 6
~ ~
R20905
750
Rt13GS
G,CSO
Rt9S3i
>S,ODO
R tStS
> S,DDO
\~I
CI ,H N H OH ;~®CMZ-CH?-N~, 0~
7
1 0, ,OM
!
~
~
Ç
/~~OH
C-CH=-CHZ-N
J(
I !
H-N
OH
CI
(!)
Cross-reaction is axprassed as the molar ratio of loperamide and the teat compounds to Inhibit bt~ 50 the complex formation between 3H-loperamide and the antiserum,
(~)
r
Croee-reaction ie expressed ae the molar ratio of loperamide and the test compounds to inhibit by 50 yp the complex formation between 3H-loperamide and the anti serum.
Radioi~unoassay of Loperamide
Vol . 21, No . 3, 1977
457
FI G. 4
Plasma levels of unaltered loperamide plotted against time in three subjects orally treated with 4 mg of the drug . drug intake, 67 °fo after 2 hours and the absorption phase was almost completely terminated 4 hours after administration . From that time, loperamide disappeared from the plasma with a half-life of about 15 hours and 48 hours after administration drug concentrations were lower than 0 . 1 ng~ml, i. e. the detec tion limit of the assay method . Since plasma. concentrations of loperamide, assayed after hydrolysis with ß -glucuronidase did not increase significantly, it can be stated that no or only negligible amounts of loperamide-glucuronide were present in the plasma (data not shown) . TABLE 2
Excretion of Unchanged Loperamide with Urine and Faeces of Man up to three Days after an Oral Dose of 4 mg (Medians of three Subjects) K ofadministered dose Time interval
Urine Free loperamide
0-8 h 8-24 h 24-32 h 32-48 h 48-56 h 56-72 h
o. 297
G~mulathe
p, q24
0. 0. 0. 0. 0.
402 085 085 030 025
Glucuronic acid conjugate 0. 0. 0. 0. 0. 0.
Total
110 100 040 060 015 040
0 . 407 0 . 502 0 . 125 0 . 145 0 . 045 0 . 065
0 . 365
1 . 289
Faeces
8 . 99 12 .23 2 . 95 24 . 17
458
Radioimmunoassay of Loperamide
Vol . 21, No . 3, 1977
Urinary and faecal excretion of loperamide are summarized in table 2. From 0 . 63 to 1 . 4 °fo of the dose (median 0. 92 °fo) was excreted with the urine ae unaltered drug, whereas total urinary excretion of free and conjugated drug averaged 1 . 35 °Jo (median 1 . 29 °fo) . Drug excretion occurred mainly with the first day urine and was not correlated with the urinary pH . Faecal excretion was the main route and amounted to about 25 °~o of the administered dose .
Di scue sinn Antibodies with high loperamide-binding capacity were obtained by repeated injections of rabbits with a loperamide-BSA conjugate . By using the antiserum, loperamide could be assayed with accuracy, directly in plasma in a range from 0 . 1 up to 20 nanogram . Although the incubation volumes of 0 . 5 ml of plasma were rather large, blank values were less than 2 °fo, indicating that normal plasma constituents did not interfere . The affinity of the antibody for loperamide is evidenced by the results of the cross-reactivity experiments . On the one hand, a high degree of cross-reaction was observed between fluperamide and 3H-loperamide, but on the other hand, the antibody sharply differentiated between the parent drug and its structurally very closely related metabolites . This indicates that in practice these N-dealkylated metabolites will not interfere with the radioimmunoaseay of loperamide . From these results and the data obtained with the structurally related drugs, ae there are the neuroleptic haloperidol and the antidiarrhoeale diphenoxylate and difenoxin, it can be concluded that the antibody sharply recog nized not only the entire dimethylamide-moiety of the loperamide molecule, but also that the intact loperamide structure was required for strong binding . So, ae the amide-function was the principal distinctive mark of loperamide, the hapten synthesis via substitution of the tertiary alcohol was preferred, just to leave intact this important functional group. Moreover, by coupling the hapten in this way, a real possibility was created for the antiserum to be used not only for the determination of loperamide but also for the quantification of the closely related antidiarrhoeal fluperamide (6) . Based on the high degree of cross-reaction (table 1), the antiserum can presumably also be used for the assay of fluperamide . It can be concluded that the method presented in this paper, is very specific for the determination of loperamide, without interferences of its _N-dealkylated metabolites. The results of the preliminary pharrnacokinetic analysis of loperamide in man demonstrate that the accuracy, feasibility and sensitivity àre sufficient to enable this radioimmunoaesay to be used for routine determination of loperamide concentrations in clinical and pharmacokinetic studies. AcknowledAments The authors wish to thank Mr . A . Knaeps and Mr . J. Thijesen for the synthesis of the loperamide hapten .
Vol . 21, No . 3, 1977
Radioia~unoassay of Loperamide
459
References e 1 . C . J . E. NIEMEGEERS, F. M. LENAERTS and P. A, J. JANSSEN, Arzrieim . -Forsch. (Dru¢ Res . ) 24, 1633-1636 (1974) . 2. J . M. VAN NUETEN, P. A, J . JANSSEN and J. FONTAINE, Arzneiur . Forsch . (Drug Res. ) 24, 1641-1645 (1974) . 3. J . HEYKANTS, M . MICHIELS, A. KNAEPS and J . BRUGMANS, Arzneiur . Forsch . (Drug Res. ) 24, 1649-1653 (1974) . 4. J. HEYKANTS, W . MEULDERMANS, A. KNAEPS and M . MICHIELS, Europ. J . Drug Metabolism and Pharmacokinetics , submitted . 5. J . G. WAGNER, in Biopharmaceutice and relevant pharmacokiaetics , p. 278-280, Drug Intelligence Publications, Hamilton, Ill, (1971), b. C. NIEMEGEERS, F. LENAERTS and F. AWOUTERS, in Synthetic antidiarrheal drums, Van Bever and Lal (Eds), p. 65-114, Marcel Dekker, Inc . , New York (1976) .