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Radioimmunoassay for determination of prazepam and its metabolites
Forensic Science International, 22 (1983) 243-248 Elsevier Scientific Publishers Ireland Ltd.
RADIOlMMUNOASSAY FOR AND ITS METABOLITES
H. K&-ILER-SCHMIDT Institut
fiir Rechtsmedizin
DETERMINATION
OF
PRAZEPAM
and G. BOHN der Westfiilischen,
Wilhelm+Universitiit,
Miinster
(F.R.G.)
(Received Octobw 20, 1982) (Revision received November 12, 1982) (Accepted December 29, 1982)
Summary The development of a radioimmunoassay for the analysis of prazepam and its metabolites is described. It includes the preparation of the immunogen, immunization and measurement of specificity and sensitivity. Key words:
RLA; Immunogen; Immunization; Prazepam; Specificity
Introduction Some years ago several radioimmunoassays were developed for the determination of several derivatives of benzodiazepines. For example, the diazepam-assay by Peskar and Spector in 1973 which is characterized by a high specificity of substrate Cl]. In addition, in 1979, Aderjan gave an account of a group-specific radioimmunoassay which covers several benzodiazepines 121. This paper describes the development of a radioimmunoassay for the measurement of prazepam and its metabolites. Prazepam is the active ingredient of Demetrin@ which gained some significance within the confines of our institute in connection with traffic cases. As for its structure as well as its metabolism, it has some resemblance to diazepam (Fig. 1). The main metabolic pathway is transformation into desmethyldiazepam by dealkylation in position 1; after this, into oxazepam by hydroxylation in position 3 [3,4]. Oxazepam as a glucuronide is excreted by the kidneys. In addition, prazepam can be hydroxylated to 3-OH-prazepam which is either excreted in the urine or further metabolized to oxazepam. After therapeutic dosage unchanged prazepam could not be detected in body fluids by chromatographic methods [51. 0379-0738/83/$03.00
0 1983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
244
7lH2
0
&$$CH2
-
C,~5H2 6
4 Prazepan
Desmethyidiazepum
I
I
Y C&&HO,
---c
C,J&$HOH
d0
b
3-OH-Prazepam
Oxazepan
METABOLISM
OF PRAZEPAM
Fig. 1. Metabolism of prazepam.
Experimental The immunogen was synthesized by the addition of equimolar portions of succinic anhydride to 3-hydrozyprazepam to give a hemisuccinate. The free carboxyl group was converted to a mixed anhydride by use of isobutyl chloroformate. The mixed anhydride then directly reacted with protein amino groups to form peptide bonds (Fig. 2). After suspension in Freund’ s adjuvant, each mg of immunogen was administered to New Zealand rabbits by intramuscular injection. The immunization was first repeated after a fortnight and then repeated every month. The process of immunization was observed by measuring at intervals the antibody titres in the various sera. The antibody titre is defined as that dilution of an antiserum that will bind
COUPLING OF THE HAPTEN TO PROTEIN CARRIER
Fig. 2. Coupling of the hapten to protein carrier.
245
-
dilution
factor
B
L loo 200 300 ANTISERUM Fig. 3. Antiserum
500 DILUTION
1000
750 CURVES
dilution curves.
50% of a fixed amount of labelled
antigen
in a fixed incubation
volume
(Fig.
3). As shown in Fig. 4, the antibody production of those rabbits which had developed a significant antibody production lessened after 12 and 15 weeks. Therefore the immunization was stopped at these times. The antiserum with the highest antibody titre was evaluated more closely. The specificity and sensitivity of the radioimmunoassay were measured in combination with prazepam, 3-hydroxyprazepam, desmethyldiazepam, oxazepam, and diazepam which was used as a tracer. For this purpose constant amounts of tracer and antiserum in buffer solution were added to aqueous solutions of the various benzodiazepines in ranges of concentrations from 0.1 ng/lOO F 1 to 100 ng/lOO ~1. After separating the free antigen fraction by adsorption to charcoal coated with dextran the radioactivity of the bound antigen fraction was measured by a scintillation counter (Fig. 5).
246
162.
lC0 Tag TIME SLOPE OF IMMUNIZATION
Fig. 4. Time slope of immunization.
ng/10Oul 0”
1
’
10
’
20
1
LO
60
60
100
STANDARD
CURVES
Fig. 5. Standard curves.
Results and discussion Contrasting the curves of prazepam respective to 3-hydroxyprazepam and desmethyldiazepam respective to oxazepam, we find a distinct difference in specificity. In combination with desmethyldiazepam respective to oxazepam, 1 ng/incubation volume (100 ~1) is sufficient to partially remove the tracer
247
cross
antigen
CROSS
reaction
REACTION
Fig. 6. Cross reactivity.
Cl
Hydroxyprozepam-succmoyi-BSA
Fig. 7. Immunogen.
from the antibodies, while concentrations of more than long/incubation volume are required to produce the same effect in combination with prazepam respective to 3-hydroxyprazepam. This difference in specificity is also illustrated by the determination of cross reactivity (Fig. 6). The antiserum therefore is not specific for prazepam and S-hydroxyprazepam but has some group specificity analogous to the antiserum prepared by Aderjan by means of immunization by oxazepam-3-succinoyl-bovine serum albumin conjugate [21. Small changes on position 1, as represented by the methyl group of diazepam in contrast to desmethyldiazepam, do not cause any change of cross reactivity. Conversely, the large methylcyclopropyl group of prazepam causes a significant decrease in cross reactivity. The basis for forming the immunogen seems to be the immediate dealkylation in position 1 (Fig. 7). Therefore the immune response is not induced by 3-hydroxyprazepam- 3-succinoyl-bovine serum albumin conjugate but by oxazepam-protein conjugate directly applied by Aderjan 121.
References 1 B. Peskar and S. Spector, Quantitative determination of Diazepam in blood by RIA. J. Pharmacol. I&p. Z’her., 166 (1973) 167-172. 2 R. Aderjan and Gg. Schmidt, Ein Screening-Radioimmunoaasay fiir 1,4Benzodiazepine in menschlichem Blut, Serum und Urin mit Antikiirpern gegen Oxazepamhemisuccinat. Z. Rechtsmed., 63 (1979) 191-200.
248 3 R.R. Brodie, L.F. Chassead and T. Taylor, Concentrations of N-Descyclopropylmethylprasepam in whole-blood, plasma, and milk after administration of prasepam to humans. Biopham. Drug. Llisp., (1981) 59-68. 4 E.U. Kolle, Zur Pharmakokinetik nach oraler Gabe von Prazepam. In Psychouegetatiue Allgemeinstirungen -Diagnose, Therupic und Grundlagen, Dr. L. Blaha, Herausgeber, 1981. 5 D.J. Greenblatt and R.I. Shader, Prazepam and Lorazepam, two new benzcdiasepines. New Engl. J. Med., 299 (1978) 1342-1344.
RADIOlMMUNOASSAY FOR AND ITS METABOLITES
H. K&-ILER-SCHMIDT Institut
fiir Rechtsmedizin
DETERMINATION
OF
PRAZEPAM
and G. BOHN der Westfiilischen,
Wilhelm+Universitiit,
Miinster
(F.R.G.)
(Received Octobw 20, 1982) (Revision received November 12, 1982) (Accepted December 29, 1982)
Summary The development of a radioimmunoassay for the analysis of prazepam and its metabolites is described. It includes the preparation of the immunogen, immunization and measurement of specificity and sensitivity. Key words:
RLA; Immunogen; Immunization; Prazepam; Specificity
Introduction Some years ago several radioimmunoassays were developed for the determination of several derivatives of benzodiazepines. For example, the diazepam-assay by Peskar and Spector in 1973 which is characterized by a high specificity of substrate Cl]. In addition, in 1979, Aderjan gave an account of a group-specific radioimmunoassay which covers several benzodiazepines 121. This paper describes the development of a radioimmunoassay for the measurement of prazepam and its metabolites. Prazepam is the active ingredient of Demetrin@ which gained some significance within the confines of our institute in connection with traffic cases. As for its structure as well as its metabolism, it has some resemblance to diazepam (Fig. 1). The main metabolic pathway is transformation into desmethyldiazepam by dealkylation in position 1; after this, into oxazepam by hydroxylation in position 3 [3,4]. Oxazepam as a glucuronide is excreted by the kidneys. In addition, prazepam can be hydroxylated to 3-OH-prazepam which is either excreted in the urine or further metabolized to oxazepam. After therapeutic dosage unchanged prazepam could not be detected in body fluids by chromatographic methods [51. 0379-0738/83/$03.00
0 1983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
244
7lH2
0
&$$CH2
-
C,~5H2 6
4 Prazepan
Desmethyidiazepum
I
I
Y C&&HO,
---c
C,J&$HOH
d0
b
3-OH-Prazepam
Oxazepan
METABOLISM
OF PRAZEPAM
Fig. 1. Metabolism of prazepam.
Experimental The immunogen was synthesized by the addition of equimolar portions of succinic anhydride to 3-hydrozyprazepam to give a hemisuccinate. The free carboxyl group was converted to a mixed anhydride by use of isobutyl chloroformate. The mixed anhydride then directly reacted with protein amino groups to form peptide bonds (Fig. 2). After suspension in Freund’ s adjuvant, each mg of immunogen was administered to New Zealand rabbits by intramuscular injection. The immunization was first repeated after a fortnight and then repeated every month. The process of immunization was observed by measuring at intervals the antibody titres in the various sera. The antibody titre is defined as that dilution of an antiserum that will bind
COUPLING OF THE HAPTEN TO PROTEIN CARRIER
Fig. 2. Coupling of the hapten to protein carrier.
245
-
dilution
factor
B
L loo 200 300 ANTISERUM Fig. 3. Antiserum
500 DILUTION
1000
750 CURVES
dilution curves.
50% of a fixed amount of labelled
antigen
in a fixed incubation
volume
(Fig.
3). As shown in Fig. 4, the antibody production of those rabbits which had developed a significant antibody production lessened after 12 and 15 weeks. Therefore the immunization was stopped at these times. The antiserum with the highest antibody titre was evaluated more closely. The specificity and sensitivity of the radioimmunoassay were measured in combination with prazepam, 3-hydroxyprazepam, desmethyldiazepam, oxazepam, and diazepam which was used as a tracer. For this purpose constant amounts of tracer and antiserum in buffer solution were added to aqueous solutions of the various benzodiazepines in ranges of concentrations from 0.1 ng/lOO F 1 to 100 ng/lOO ~1. After separating the free antigen fraction by adsorption to charcoal coated with dextran the radioactivity of the bound antigen fraction was measured by a scintillation counter (Fig. 5).
246
162.
lC0 Tag TIME SLOPE OF IMMUNIZATION
Fig. 4. Time slope of immunization.
ng/10Oul 0”
1
’
10
’
20
1
LO
60
60
100
STANDARD
CURVES
Fig. 5. Standard curves.
Results and discussion Contrasting the curves of prazepam respective to 3-hydroxyprazepam and desmethyldiazepam respective to oxazepam, we find a distinct difference in specificity. In combination with desmethyldiazepam respective to oxazepam, 1 ng/incubation volume (100 ~1) is sufficient to partially remove the tracer
247
cross
antigen
CROSS
reaction
REACTION
Fig. 6. Cross reactivity.
Cl
Hydroxyprozepam-succmoyi-BSA
Fig. 7. Immunogen.
from the antibodies, while concentrations of more than long/incubation volume are required to produce the same effect in combination with prazepam respective to 3-hydroxyprazepam. This difference in specificity is also illustrated by the determination of cross reactivity (Fig. 6). The antiserum therefore is not specific for prazepam and S-hydroxyprazepam but has some group specificity analogous to the antiserum prepared by Aderjan by means of immunization by oxazepam-3-succinoyl-bovine serum albumin conjugate [21. Small changes on position 1, as represented by the methyl group of diazepam in contrast to desmethyldiazepam, do not cause any change of cross reactivity. Conversely, the large methylcyclopropyl group of prazepam causes a significant decrease in cross reactivity. The basis for forming the immunogen seems to be the immediate dealkylation in position 1 (Fig. 7). Therefore the immune response is not induced by 3-hydroxyprazepam- 3-succinoyl-bovine serum albumin conjugate but by oxazepam-protein conjugate directly applied by Aderjan 121.
References 1 B. Peskar and S. Spector, Quantitative determination of Diazepam in blood by RIA. J. Pharmacol. I&p. Z’her., 166 (1973) 167-172. 2 R. Aderjan and Gg. Schmidt, Ein Screening-Radioimmunoaasay fiir 1,4Benzodiazepine in menschlichem Blut, Serum und Urin mit Antikiirpern gegen Oxazepamhemisuccinat. Z. Rechtsmed., 63 (1979) 191-200.
248 3 R.R. Brodie, L.F. Chassead and T. Taylor, Concentrations of N-Descyclopropylmethylprasepam in whole-blood, plasma, and milk after administration of prasepam to humans. Biopham. Drug. Llisp., (1981) 59-68. 4 E.U. Kolle, Zur Pharmakokinetik nach oraler Gabe von Prazepam. In Psychouegetatiue Allgemeinstirungen -Diagnose, Therupic und Grundlagen, Dr. L. Blaha, Herausgeber, 1981. 5 D.J. Greenblatt and R.I. Shader, Prazepam and Lorazepam, two new benzcdiasepines. New Engl. J. Med., 299 (1978) 1342-1344.