CA2109644A1 - Agent and method for the immunological determination of amiodarone and its metabolites - Google Patents
Agent and method for the immunological determination of amiodarone and its metabolitesInfo
- Publication number
- CA2109644A1 CA2109644A1 CA002109644A CA2109644A CA2109644A1 CA 2109644 A1 CA2109644 A1 CA 2109644A1 CA 002109644 A CA002109644 A CA 002109644A CA 2109644 A CA2109644 A CA 2109644A CA 2109644 A1 CA2109644 A1 CA 2109644A1
- Authority
- CA
- Canada
- Prior art keywords
- amiodarone
- general formula
- derivative
- antibodies
- crosslinker
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/9453—Cardioregulators, e.g. antihypotensives, antiarrhythmics
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
- C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D307/78—Benzo [b] furans; Hydrogenated benzo [b] furans
- C07D307/79—Benzo [b] furans; Hydrogenated benzo [b] furans with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to carbon atoms of the hetero ring
- C07D307/80—Radicals substituted by oxygen atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
Abstract
Abstract The invention relates to an agent and method for the immunological determination of amiodarone and its metabolites in body fluids, as well as to the tracers, immunogens and antibodies which are required for a determination, and their preparation.
Description
- 1- 210964~
Merck Patent Gesellschaft mit beschrankter Haftung 6100 D a r m s t a d t Agent and method for the immunological determination of amiodarone and it8 metabolite~
The invention relates to an agent and method for the immunological determination of amiodarone and its metabolites in body fluids or in pretreated body fluids, as well a~ to the tracers, immunogens and antibodies which are required for a determination, and their preparation.
Underdoses or overdoses of drugs are f requently administered. This experience calls to an increasing extent for the measurement of the serum concentration of the drugs. This is all the more important when these drugs have a narrow therapeutic range and complications increase greatly when this range is exceeded. Immuno-assays, in particular fluorescence-polarization immuno-assays or enzyme immunoassays, are particularly suitable and rapid determination methods. Amiodarone (2-butyl-3-[3,5-diiodo-4-~-diethylaminoethoxy)-benzoyl]benzofuran) possesse~ anti-anginal and anti-arrhythmic properties. It is frequently employed in ventricular and supra-ventricular arrhythmias. Amiodarone is metabolized in part to the therapeutically active desethylamiodarone.
Taking this drug not infrequently leads, in a dose-dependent manner, to side effects such as photo-sensitivity, discolouration of the skin, microdepositions on the cornea, and disturbance of thyroid gland function.
For this reason, it is recommended in the literature that a serum level of 1-2 mg/l should not be exceeded. The serum levels correlate with the dose administered, with the morphological alterations in some tissues, and with the toxic side effects. Serum levels of amiodarone have hitherto been determined using various HPLC methods.
However, these methods require pretreatment of the sample (protein precipitation) and elaborate analytical equip-ment; in addition, they are very time-consuming.
, - -,: . . . . .
- ~ - 2 - 2109644 As is known, tracers, immunogens and specific antibodies are required for preparing an immunoassay.
According to the methods described in the state of the art, a tracer is prepared by coupling an analyte which is to be determined, or its metabolites, to a label`
(chromogen, fluorogen, enzyme or radioactive label). An immunogen is prepared by coupling a hapten or hapten derivative to a protein. If desired, the coupling reac-tions are effected with the aid of a crosslinker or spacer. Immunogens are prepared in an aqueous system, preferably in a buffer system, and subsequently purified.
Owing to the very poor solubility of amiodarone and its derivatives in aqueous solution, it is not possible to prepare the corresponding immunogens using the known methods.
The invention is based on the object of providing an agent and method which permit amiodarone and its metabolites to be determined rapidly and reproducibly without using highly elaborate analytical procedures.
The invention relates to compounds of the general formula ~I) 21 n W ~ Z ) ~~Q l I ) in which X = O, S, or NRl W = CO, NR', S, or O, Z = a crosslinker or spacer, Q = a label or a carrier protein, R' = H or alkyl having 1-6 C atoms, n = 1-10 m = 0 or l.
The invention further relates to anti~odies which can be obtained by immunizing with an Lmmunogen of the general formula (I), in which Q is a carrier protein.
The invention also relates to an agent for the - 3 ~ 210964~
immunological determination of amiodarone and its meta-bolites in body fluids, which agent is characterized in that it contain~
a) a labelled amiodarone derivative of the general formula (I), in which Q is a label, b) antibodies which can bind amiodarone, its meta-bolites and the labelled amiodarone derivative, and c) where appropriate, an enzyme substrate.
The invention further relates to a method for the immunological determination of amiodarone and its metabo-lites in body fluids by incubating the sample to be examined with a labelled amiodarone derivative of the general formula (I), in which Q is a label, and anti-bodies which can bind amiodarone, its metabolite~ and the labelled amiodarone derivative, as well as, where appro-priate, with an enzyme substrate, and measuring the fluorescence or the enzyme activity.
In the case of a fluorescence-polarization immunoassay, the tracer i9 labelled with a fluorescing compound, and in the case of an enzyme immunoassay with an enzyme. The tracer consists of an amiodarone deriva-tive of the general formula II, ~ ~ X~(CH2)n~Y (II) in which X = O, S, or NR~, .
Y = N~R1, COR2, SH, OH, halogen or C~, Rl = H or-alkyl having 1-6 C atoms, R2 = OH, halogen, N~Rl or OR1, n = 1-10, which is bound, if desired with the aid of a crosslinker or spacer, to a fluorescein derivative or to an enzyme.
- Preparation of the compounds of the general formula (II) involve~ starting with, for example, 2-butyl-3-(4-hydroxyphenyl-car~onyl)-benzofuran and '.~' '' ~
'" ' :.
,:.:
..~ ' _ 4 _ 21 09 6 ~ 4 iodinating the phenyl ring in the 3 and 5 positions and subsequently alkylating in the 4 position with bromo-carboxylic acid ethyl ester. Alkaline hydrolysis leads to 2-butyl-3-[3,5-diiodo-4-(carboxyalkyloxy)-benzoyl]benzo-furan. The corresponding derivatives can be prepared fromthe free carboxylic acid by known methods, which deriva-tives can be reacted with a crosslinker or spacer, or else directly with a label or carrier protein.
Alkyl is methyl, ethyl, propyl, butyl, pentyl or hexyl, and their isomers, preferably ethyl, propyl, isopropyl, n-butyl, sec-butyl or tert-butyl. ~alogen is preferably chlorine and bromine. The compounds of the general formula (II) which are preferred according to the invention are the carboxylic acid esters having 2 to 6 C
atoms in the alkyl group, in particular the propylene and butylene compounds.
Suitable fluorescein derivatives are, for example, all aminofluoresceins, carboxyfluorescein, fluoresceinisothiocyanate,2,4-dichloro-1,3,5-triazin-2-ylamino-~luorescein, 4-chloro-6-methoxy-1,3,5-triazin-2-ylamino-fluorescein,2-iodoacetamidofluorescein,2-bromo-acetamidofluorescein, fluorescein thiocarbamylethylene-diamine and 5-aminomethylfluorescein, preferably 4-aminofluorescein.
Suitable crosslinker~ or spacers for coupling the fluorescein derivative to the amiodarone derivative of the general formula (II~ are known from the literature.
Preferred crosslinkers for the method according to the invention are, for example, 1-ethyl-3-dimethylamino-propylcarbodiimide hydrochloride (EDC), N hydroxysuccin-imide (NHS), N-(3-dimethylaminopropyl)-N'-ethylcarbo-diimide hydrochloride (ECD) and dicyclohexylcarbodiimide (DCC)-The tracers are~prepared by ~nown methods in organic sQlvents, such as dimethylformamide, dimethyl sulfoxide, acetonitrile, methanol, pyridine, etc., and at room temperature.
The specific antibodies which are necessary for the determination, according to the invention, of ~,, .... ., .,, ..... ....... ,~.. , . ,~ .
.. ^.~.:. ' . ~ . : , ~ .
~:.. ",, . :: ~: :
~'~c.' amiodarone are obtained by immunizing with an immunogen consisting of an amiodarone derivative of the general formula (II) which is covalently coupled to a carrier protein, where appropriate via a crosslinker or spacer.
The compounds mentioned above in association with the preparation of tracers are suitable as crosslinkers.
Keyhole limpet hemocyanin (KL~), bovine serum albumin (BSA), IgG, ovalbumin, thyroglobulin and lactalbumin are examples of proteins which are used as carrier proteins.
As already mentioned, it is not possible, owing to the poor solubility of amiodarone and its derivatives, to prepare the requisite immunogens in a customary manner in aqueous solution. They principally dissolve in the customary organic solvent~, such as dimethyl sulfoxide, dimethylformamide, ethanol, etc., leading, however, to the irreversible denaturation of the carrier protein.
Surprisingly, it was found that amiodarone and its derivatives dissolve very well in diethylene glycol monoalkyl ether, diethylene glycol dialkyl ether, 1,3-dimethyl-2-imidazolidinone, 3-butyrolactone, glycerol formal and 2,2-dimethyl-4-hydroxymethyl-1,3-dioxolane. In this way, an immunogen consisting of an amiodarone of the general formula (II) and a carrier protein has been successfully prepared for the first time. Synthesis of the immunogen otherwise proceeds in accordance with methods described in the literature (Ann. Clin. Bioahem.
23, 37 (1986)).
To prepare the necessary antibodies, the purified immunogen is injected in a customary manner into parti-cular animals (sheep, goats, mice or rabbits) at inter-vals of two to three weeks. The desired antibodies have formed after 3 to 6 immunizations. Processing and purifi-cation of the antibodies takes place in accordance with known methods.
The tracer for the enzyme immunoassay is prepared from a derivative of the general formula (II) and a labeiling enzyme, for example alkaline phosphatase, glucosidase, galactosidase or horseradish peroxidase. The synthesis is effected in the presence of the solvents ....
.
:,.; ., .
:, ,:~ . . .
:, :., , . :
. ;:: ..
,................................................. .
. :-.
- 6 - 21096~4 which have been mentioned as being used for the immunogen synthesis, it being possible to dispense with the use of the crosslinker in this instance.
The method according to the invention for deter-mining amiodarone and its metabolites, in particulardesethylamiodarone~ is carried out by mixing the ~ample solution, which has been diluted with buffer, with the tracer and the antibody. After a given incubation period, the concentration of amiodarone and its metabolites is determined directly in the reaction solution.
In the case of the fluorescence-polarization immunoas~ay, the tracer is excited with blue polarized light (488 nm), for example; the polarization of the green emitted light (532 nm) depends reciprocally on the size, the shape and the movement of the tracer. The polarization of the emitted light increases when the tracer is bound to the specific antibody. Fluorescence-polarization immunoa~says are competitive Lmmunoa~says, i.e. tracer and analyte compete for the binding site on the antibody. The fluorescence-polarization signal depends, in an inversely proportional manner, on the concentration of the analyte in the sample. If, for example, the concentration of the analyte in the sample is high, only a small quantity of the tracer binds to the antibody and the fluorescence-polarization signal is low.
In the case of the competitive enzyme immuno-assay, the specific antibody i8 immobilized on a solid support, for example on microtitre plates, in small tubes or on magnetic particles, and incubated with the sample solution and the enzyme conjugate. Subsequently, the support is washed and the corresponding enzyme substrate is added, and the concentration of the analyte i8 deter-mined photometrically.
~xample 1 35 Preparation of 2-butyl-3-~3,5-diiodo-4-(3-carboxypropyl-oxy)benzoyllbenzofuran 32.8 g (60 mmol) of 2-butyl-3-[3,5-diiodo-4-hydroxybenzoyl]benzofuran and 13.8 g (100 mmol~ of anhydrou~ pota~sium carbonate are initially introduced ~:- ;- :
i j ,~ '' , ~
, ~."
:: .
_ 7 _ 21 09 64 4 into 200 ml of acetonitrile. 17.6 g (90 mmol) of ethyl 4-bromobutyrate are added dropwise at room temperature within the space of 30 minutes, and the suspension i8 subsequently heated under reflux for 14 h. 200 ml of distilled water are added to the reaction solution once the latter has cooled down to room temperature, and the mixture is then extracted 3 times with 100 ml of MTB
ether. The combined organic extracts are dried and concentrated to dryness. The residue, which consists of 2-butyl-3-[3,5-diiodo-4-(3-ethoxycarbonylpropyloxy)-benzoyl]benzofuran, is introduced into 100 ml of isopro-panol and a solution of 39.6 g (600 mmol) of 85~
potassium hydroxide in 400 ml of distilled water is added. The suspension is heated at about 60C until a clear solution is formed. 100 ml of lN hydrochloric acid are added to the solution, once it has been cooled down to room temperature, and the mixture i8 then adjusted to a pH of 3 with 32% hydrochloric acid, while cooling, and then extracted 3 times with 200 ml of MTB ether on each occasion. The combined organic, dried extracts are taken up in 50 ml of dichloromethane and left at 0C for several hours. The pale brown crystals which have pre-cipitated out are stirred up with a further S0 ml of dichloromethane. A~ter filtering with suction, washing and drying, 5.7 g (20%) of almost colourless crystal~ of 2-butyl-3- r 3,5-diiodo-4-(3-carboxypropyloxy)benzoyl]-benzofuran are obtained which, if desired, can be further purified on a silica gel column (MTB ether/methanol =
9:1) .
~xample 2 Preparation of an immunogen using XLH
230 mg (0.3b mmol) of the amiodarone derivative prepared according to Example 1 are dissolved in 1.5 ml of diethylene glycol monoethyl ether. 50 mg (0.72 ~mol) of KL~ are likewise dissolved in 25 ml of diethylene glycol monoethyl ether. The two solutions are mixed together and stirred at room temperature for 3 hours.
After the reaction i8 complete, the mixture i8 dialysed against demineralized water at 4C. After the dialysis, , ,.: , : ..
:;:
,, :. :
21096~4 the solution, which contains the conjugate consisting of the amiodarone derivative and KLH, is used for the immunization.
Example 3 Preparation of an immunogen using BSA
500 ~l of demineralized water, in which 57 mg ~0.3 mmol) of EDC and 38 mg (0.33 mmol) of NHS have been dissolved, are added to the solution of the amiodarone derivative according to Example 1. This solution is added to a solution of 50 mg of BSA in 25 ml of diethylene glycol monoethyl ether. The subsequent procedure is as given in Example 2. An immunoconjugate is obtained consisting of the amiodarone derivative according to Example 1, the said crosslinkers and BSA.
Example 4 Preparation of a fluorescence-labelled tracer 11 mg (5.6 mmol) of the amiodarone derivative according to Example 1 are dis~olved in 1 ml of dimethyl-formamide together with 15 mg (19.6 mmol) of EDC and 3 mg (6.5 mmol) of N~S, and the mixture is stirred at room temperature for 30 minutes.
10 mg (5.1 mmol) of fluorescein thiocarbamyl-ethylenediamine are dissolved in 3 ml of dimethylform-amide and the solution is stirred at room temperature for 10 minutes. Subsequently, this solution is added dropwise to the first solution and the whole mixture is stirred at room temperature overn~ght. Isolation and purification of the tracer takes place using thin layer chromatography.
Eluent: chloroform + methanol 8+1.
~xample 5 Preparation of an enzyme-labelled tracer 57 mg of EDC and 38 mg of NHS are dissolved in 500 ~l of demineralized water and a solution of the amiodarone derivative according to Example 1 (230 mg) in 1.5 ml of diethylene glycol monoethyl ether is added.
50 mg of horseradi~h peroxidase are dissolved in 25 ml of diethylene glycol monoethyl ether. The two solutions are mixed together ànd stirred at room temperature for 3 hours. Once the reaction is complete, the whole mixture .;, i ~
:.
21096~4 g is dialysed against phosphate buffer at 4C.
Example 6 Preparation of the antibody against amiodarone and its metabolites 0.8 mg of the immunogen according to Example 3 are emulsified with an adjuvant (Freund's complete or incomplete adjuvant, aluminium hydroxide, water-oil emul-sion or muramyl dipeptide). The animal is immunized twice with this emulsion at an interval of 21 days. Subse-quently it is immunized 4-12 times with an emulsion consisting of 0.8 mg of amiodarone-BSA, and containing Freund's incomplete adjuvant, at intervals of 21 days.
Example 7 Fluorescence-polarization Lmmunoassay ~uman serum is deproteinized with a customary precipitating reagent and the precipitation supernatant, or the diluted precipitation supernatant, i8 employed in the test. Depending on their titre, the sheep antibodies are diluted between 1:5 and 1:1000 with Tris buffer, pH
7.5. The test is carried out according to the following pipetting schedule:
1000 ~l of system buffer (Tris buffer, pH 7.5), 50 ~l of antibody solution, 150 ~l of precipitation supernatant or diluted preci-pitation supernatant or calibrator.
After mea~uring the sample blank value, 50 ~l of tracer, which i8 adjusted to a concentration of 100 nmol/l, are added. After incubating at 37C for 6 minutes, the concentration of amiodarone is measured. The system is calibrated with 6 standards; these standards, which are based on human serum, contain amiodarone in concen-trations from 0-6 mg/l.
If appropriate, the test can also be carried out without prior deproteinization.
Example 8 Enzyme immunoassay The determination takes piace at room temperature in accordance with the principle of the competitive enzyme Lmmunoassay. The same standards are used as in - , ~ .
;.:- .
.~.. ~ .; . .. . :
2109~44 Example 7. The antibody is immobilized either directly or indirectly (via anti-sheep IgG) on microtitre plates or in small tubes. Using microtitre plates, the pipetting schedule is:
50 ~1 of diluted precipitated supernatant or diluted calibrator (diluted 1:50), 50 ~l of enzyme conjugate (horseradish peroxidase) from Example 5.
Incubation takes place at room temperature for 1 hour.
After the incubation vessels have been washed, 100 ~l of substrate (2,2'-azinobis(3-ethylbenzothia-zoline-6-sulphonic acid) diammonium salt) are added.
Incubation is continued for a further 15 minutes.
Subsequently the optical density is measured at 405 nm.
Merck Patent Gesellschaft mit beschrankter Haftung 6100 D a r m s t a d t Agent and method for the immunological determination of amiodarone and it8 metabolite~
The invention relates to an agent and method for the immunological determination of amiodarone and its metabolites in body fluids or in pretreated body fluids, as well a~ to the tracers, immunogens and antibodies which are required for a determination, and their preparation.
Underdoses or overdoses of drugs are f requently administered. This experience calls to an increasing extent for the measurement of the serum concentration of the drugs. This is all the more important when these drugs have a narrow therapeutic range and complications increase greatly when this range is exceeded. Immuno-assays, in particular fluorescence-polarization immuno-assays or enzyme immunoassays, are particularly suitable and rapid determination methods. Amiodarone (2-butyl-3-[3,5-diiodo-4-~-diethylaminoethoxy)-benzoyl]benzofuran) possesse~ anti-anginal and anti-arrhythmic properties. It is frequently employed in ventricular and supra-ventricular arrhythmias. Amiodarone is metabolized in part to the therapeutically active desethylamiodarone.
Taking this drug not infrequently leads, in a dose-dependent manner, to side effects such as photo-sensitivity, discolouration of the skin, microdepositions on the cornea, and disturbance of thyroid gland function.
For this reason, it is recommended in the literature that a serum level of 1-2 mg/l should not be exceeded. The serum levels correlate with the dose administered, with the morphological alterations in some tissues, and with the toxic side effects. Serum levels of amiodarone have hitherto been determined using various HPLC methods.
However, these methods require pretreatment of the sample (protein precipitation) and elaborate analytical equip-ment; in addition, they are very time-consuming.
, - -,: . . . . .
- ~ - 2 - 2109644 As is known, tracers, immunogens and specific antibodies are required for preparing an immunoassay.
According to the methods described in the state of the art, a tracer is prepared by coupling an analyte which is to be determined, or its metabolites, to a label`
(chromogen, fluorogen, enzyme or radioactive label). An immunogen is prepared by coupling a hapten or hapten derivative to a protein. If desired, the coupling reac-tions are effected with the aid of a crosslinker or spacer. Immunogens are prepared in an aqueous system, preferably in a buffer system, and subsequently purified.
Owing to the very poor solubility of amiodarone and its derivatives in aqueous solution, it is not possible to prepare the corresponding immunogens using the known methods.
The invention is based on the object of providing an agent and method which permit amiodarone and its metabolites to be determined rapidly and reproducibly without using highly elaborate analytical procedures.
The invention relates to compounds of the general formula ~I) 21 n W ~ Z ) ~~Q l I ) in which X = O, S, or NRl W = CO, NR', S, or O, Z = a crosslinker or spacer, Q = a label or a carrier protein, R' = H or alkyl having 1-6 C atoms, n = 1-10 m = 0 or l.
The invention further relates to anti~odies which can be obtained by immunizing with an Lmmunogen of the general formula (I), in which Q is a carrier protein.
The invention also relates to an agent for the - 3 ~ 210964~
immunological determination of amiodarone and its meta-bolites in body fluids, which agent is characterized in that it contain~
a) a labelled amiodarone derivative of the general formula (I), in which Q is a label, b) antibodies which can bind amiodarone, its meta-bolites and the labelled amiodarone derivative, and c) where appropriate, an enzyme substrate.
The invention further relates to a method for the immunological determination of amiodarone and its metabo-lites in body fluids by incubating the sample to be examined with a labelled amiodarone derivative of the general formula (I), in which Q is a label, and anti-bodies which can bind amiodarone, its metabolite~ and the labelled amiodarone derivative, as well as, where appro-priate, with an enzyme substrate, and measuring the fluorescence or the enzyme activity.
In the case of a fluorescence-polarization immunoassay, the tracer i9 labelled with a fluorescing compound, and in the case of an enzyme immunoassay with an enzyme. The tracer consists of an amiodarone deriva-tive of the general formula II, ~ ~ X~(CH2)n~Y (II) in which X = O, S, or NR~, .
Y = N~R1, COR2, SH, OH, halogen or C~, Rl = H or-alkyl having 1-6 C atoms, R2 = OH, halogen, N~Rl or OR1, n = 1-10, which is bound, if desired with the aid of a crosslinker or spacer, to a fluorescein derivative or to an enzyme.
- Preparation of the compounds of the general formula (II) involve~ starting with, for example, 2-butyl-3-(4-hydroxyphenyl-car~onyl)-benzofuran and '.~' '' ~
'" ' :.
,:.:
..~ ' _ 4 _ 21 09 6 ~ 4 iodinating the phenyl ring in the 3 and 5 positions and subsequently alkylating in the 4 position with bromo-carboxylic acid ethyl ester. Alkaline hydrolysis leads to 2-butyl-3-[3,5-diiodo-4-(carboxyalkyloxy)-benzoyl]benzo-furan. The corresponding derivatives can be prepared fromthe free carboxylic acid by known methods, which deriva-tives can be reacted with a crosslinker or spacer, or else directly with a label or carrier protein.
Alkyl is methyl, ethyl, propyl, butyl, pentyl or hexyl, and their isomers, preferably ethyl, propyl, isopropyl, n-butyl, sec-butyl or tert-butyl. ~alogen is preferably chlorine and bromine. The compounds of the general formula (II) which are preferred according to the invention are the carboxylic acid esters having 2 to 6 C
atoms in the alkyl group, in particular the propylene and butylene compounds.
Suitable fluorescein derivatives are, for example, all aminofluoresceins, carboxyfluorescein, fluoresceinisothiocyanate,2,4-dichloro-1,3,5-triazin-2-ylamino-~luorescein, 4-chloro-6-methoxy-1,3,5-triazin-2-ylamino-fluorescein,2-iodoacetamidofluorescein,2-bromo-acetamidofluorescein, fluorescein thiocarbamylethylene-diamine and 5-aminomethylfluorescein, preferably 4-aminofluorescein.
Suitable crosslinker~ or spacers for coupling the fluorescein derivative to the amiodarone derivative of the general formula (II~ are known from the literature.
Preferred crosslinkers for the method according to the invention are, for example, 1-ethyl-3-dimethylamino-propylcarbodiimide hydrochloride (EDC), N hydroxysuccin-imide (NHS), N-(3-dimethylaminopropyl)-N'-ethylcarbo-diimide hydrochloride (ECD) and dicyclohexylcarbodiimide (DCC)-The tracers are~prepared by ~nown methods in organic sQlvents, such as dimethylformamide, dimethyl sulfoxide, acetonitrile, methanol, pyridine, etc., and at room temperature.
The specific antibodies which are necessary for the determination, according to the invention, of ~,, .... ., .,, ..... ....... ,~.. , . ,~ .
.. ^.~.:. ' . ~ . : , ~ .
~:.. ",, . :: ~: :
~'~c.' amiodarone are obtained by immunizing with an immunogen consisting of an amiodarone derivative of the general formula (II) which is covalently coupled to a carrier protein, where appropriate via a crosslinker or spacer.
The compounds mentioned above in association with the preparation of tracers are suitable as crosslinkers.
Keyhole limpet hemocyanin (KL~), bovine serum albumin (BSA), IgG, ovalbumin, thyroglobulin and lactalbumin are examples of proteins which are used as carrier proteins.
As already mentioned, it is not possible, owing to the poor solubility of amiodarone and its derivatives, to prepare the requisite immunogens in a customary manner in aqueous solution. They principally dissolve in the customary organic solvent~, such as dimethyl sulfoxide, dimethylformamide, ethanol, etc., leading, however, to the irreversible denaturation of the carrier protein.
Surprisingly, it was found that amiodarone and its derivatives dissolve very well in diethylene glycol monoalkyl ether, diethylene glycol dialkyl ether, 1,3-dimethyl-2-imidazolidinone, 3-butyrolactone, glycerol formal and 2,2-dimethyl-4-hydroxymethyl-1,3-dioxolane. In this way, an immunogen consisting of an amiodarone of the general formula (II) and a carrier protein has been successfully prepared for the first time. Synthesis of the immunogen otherwise proceeds in accordance with methods described in the literature (Ann. Clin. Bioahem.
23, 37 (1986)).
To prepare the necessary antibodies, the purified immunogen is injected in a customary manner into parti-cular animals (sheep, goats, mice or rabbits) at inter-vals of two to three weeks. The desired antibodies have formed after 3 to 6 immunizations. Processing and purifi-cation of the antibodies takes place in accordance with known methods.
The tracer for the enzyme immunoassay is prepared from a derivative of the general formula (II) and a labeiling enzyme, for example alkaline phosphatase, glucosidase, galactosidase or horseradish peroxidase. The synthesis is effected in the presence of the solvents ....
.
:,.; ., .
:, ,:~ . . .
:, :., , . :
. ;:: ..
,................................................. .
. :-.
- 6 - 21096~4 which have been mentioned as being used for the immunogen synthesis, it being possible to dispense with the use of the crosslinker in this instance.
The method according to the invention for deter-mining amiodarone and its metabolites, in particulardesethylamiodarone~ is carried out by mixing the ~ample solution, which has been diluted with buffer, with the tracer and the antibody. After a given incubation period, the concentration of amiodarone and its metabolites is determined directly in the reaction solution.
In the case of the fluorescence-polarization immunoas~ay, the tracer is excited with blue polarized light (488 nm), for example; the polarization of the green emitted light (532 nm) depends reciprocally on the size, the shape and the movement of the tracer. The polarization of the emitted light increases when the tracer is bound to the specific antibody. Fluorescence-polarization immunoa~says are competitive Lmmunoa~says, i.e. tracer and analyte compete for the binding site on the antibody. The fluorescence-polarization signal depends, in an inversely proportional manner, on the concentration of the analyte in the sample. If, for example, the concentration of the analyte in the sample is high, only a small quantity of the tracer binds to the antibody and the fluorescence-polarization signal is low.
In the case of the competitive enzyme immuno-assay, the specific antibody i8 immobilized on a solid support, for example on microtitre plates, in small tubes or on magnetic particles, and incubated with the sample solution and the enzyme conjugate. Subsequently, the support is washed and the corresponding enzyme substrate is added, and the concentration of the analyte i8 deter-mined photometrically.
~xample 1 35 Preparation of 2-butyl-3-~3,5-diiodo-4-(3-carboxypropyl-oxy)benzoyllbenzofuran 32.8 g (60 mmol) of 2-butyl-3-[3,5-diiodo-4-hydroxybenzoyl]benzofuran and 13.8 g (100 mmol~ of anhydrou~ pota~sium carbonate are initially introduced ~:- ;- :
i j ,~ '' , ~
, ~."
:: .
_ 7 _ 21 09 64 4 into 200 ml of acetonitrile. 17.6 g (90 mmol) of ethyl 4-bromobutyrate are added dropwise at room temperature within the space of 30 minutes, and the suspension i8 subsequently heated under reflux for 14 h. 200 ml of distilled water are added to the reaction solution once the latter has cooled down to room temperature, and the mixture is then extracted 3 times with 100 ml of MTB
ether. The combined organic extracts are dried and concentrated to dryness. The residue, which consists of 2-butyl-3-[3,5-diiodo-4-(3-ethoxycarbonylpropyloxy)-benzoyl]benzofuran, is introduced into 100 ml of isopro-panol and a solution of 39.6 g (600 mmol) of 85~
potassium hydroxide in 400 ml of distilled water is added. The suspension is heated at about 60C until a clear solution is formed. 100 ml of lN hydrochloric acid are added to the solution, once it has been cooled down to room temperature, and the mixture i8 then adjusted to a pH of 3 with 32% hydrochloric acid, while cooling, and then extracted 3 times with 200 ml of MTB ether on each occasion. The combined organic, dried extracts are taken up in 50 ml of dichloromethane and left at 0C for several hours. The pale brown crystals which have pre-cipitated out are stirred up with a further S0 ml of dichloromethane. A~ter filtering with suction, washing and drying, 5.7 g (20%) of almost colourless crystal~ of 2-butyl-3- r 3,5-diiodo-4-(3-carboxypropyloxy)benzoyl]-benzofuran are obtained which, if desired, can be further purified on a silica gel column (MTB ether/methanol =
9:1) .
~xample 2 Preparation of an immunogen using XLH
230 mg (0.3b mmol) of the amiodarone derivative prepared according to Example 1 are dissolved in 1.5 ml of diethylene glycol monoethyl ether. 50 mg (0.72 ~mol) of KL~ are likewise dissolved in 25 ml of diethylene glycol monoethyl ether. The two solutions are mixed together and stirred at room temperature for 3 hours.
After the reaction i8 complete, the mixture i8 dialysed against demineralized water at 4C. After the dialysis, , ,.: , : ..
:;:
,, :. :
21096~4 the solution, which contains the conjugate consisting of the amiodarone derivative and KLH, is used for the immunization.
Example 3 Preparation of an immunogen using BSA
500 ~l of demineralized water, in which 57 mg ~0.3 mmol) of EDC and 38 mg (0.33 mmol) of NHS have been dissolved, are added to the solution of the amiodarone derivative according to Example 1. This solution is added to a solution of 50 mg of BSA in 25 ml of diethylene glycol monoethyl ether. The subsequent procedure is as given in Example 2. An immunoconjugate is obtained consisting of the amiodarone derivative according to Example 1, the said crosslinkers and BSA.
Example 4 Preparation of a fluorescence-labelled tracer 11 mg (5.6 mmol) of the amiodarone derivative according to Example 1 are dis~olved in 1 ml of dimethyl-formamide together with 15 mg (19.6 mmol) of EDC and 3 mg (6.5 mmol) of N~S, and the mixture is stirred at room temperature for 30 minutes.
10 mg (5.1 mmol) of fluorescein thiocarbamyl-ethylenediamine are dissolved in 3 ml of dimethylform-amide and the solution is stirred at room temperature for 10 minutes. Subsequently, this solution is added dropwise to the first solution and the whole mixture is stirred at room temperature overn~ght. Isolation and purification of the tracer takes place using thin layer chromatography.
Eluent: chloroform + methanol 8+1.
~xample 5 Preparation of an enzyme-labelled tracer 57 mg of EDC and 38 mg of NHS are dissolved in 500 ~l of demineralized water and a solution of the amiodarone derivative according to Example 1 (230 mg) in 1.5 ml of diethylene glycol monoethyl ether is added.
50 mg of horseradi~h peroxidase are dissolved in 25 ml of diethylene glycol monoethyl ether. The two solutions are mixed together ànd stirred at room temperature for 3 hours. Once the reaction is complete, the whole mixture .;, i ~
:.
21096~4 g is dialysed against phosphate buffer at 4C.
Example 6 Preparation of the antibody against amiodarone and its metabolites 0.8 mg of the immunogen according to Example 3 are emulsified with an adjuvant (Freund's complete or incomplete adjuvant, aluminium hydroxide, water-oil emul-sion or muramyl dipeptide). The animal is immunized twice with this emulsion at an interval of 21 days. Subse-quently it is immunized 4-12 times with an emulsion consisting of 0.8 mg of amiodarone-BSA, and containing Freund's incomplete adjuvant, at intervals of 21 days.
Example 7 Fluorescence-polarization Lmmunoassay ~uman serum is deproteinized with a customary precipitating reagent and the precipitation supernatant, or the diluted precipitation supernatant, i8 employed in the test. Depending on their titre, the sheep antibodies are diluted between 1:5 and 1:1000 with Tris buffer, pH
7.5. The test is carried out according to the following pipetting schedule:
1000 ~l of system buffer (Tris buffer, pH 7.5), 50 ~l of antibody solution, 150 ~l of precipitation supernatant or diluted preci-pitation supernatant or calibrator.
After mea~uring the sample blank value, 50 ~l of tracer, which i8 adjusted to a concentration of 100 nmol/l, are added. After incubating at 37C for 6 minutes, the concentration of amiodarone is measured. The system is calibrated with 6 standards; these standards, which are based on human serum, contain amiodarone in concen-trations from 0-6 mg/l.
If appropriate, the test can also be carried out without prior deproteinization.
Example 8 Enzyme immunoassay The determination takes piace at room temperature in accordance with the principle of the competitive enzyme Lmmunoassay. The same standards are used as in - , ~ .
;.:- .
.~.. ~ .; . .. . :
2109~44 Example 7. The antibody is immobilized either directly or indirectly (via anti-sheep IgG) on microtitre plates or in small tubes. Using microtitre plates, the pipetting schedule is:
50 ~1 of diluted precipitated supernatant or diluted calibrator (diluted 1:50), 50 ~l of enzyme conjugate (horseradish peroxidase) from Example 5.
Incubation takes place at room temperature for 1 hour.
After the incubation vessels have been washed, 100 ~l of substrate (2,2'-azinobis(3-ethylbenzothia-zoline-6-sulphonic acid) diammonium salt) are added.
Incubation is continued for a further 15 minutes.
Subsequently the optical density is measured at 405 nm.
Claims (10)
1. Compounds of the general formula (I) (I) in which X = O, S, or NR1 W = CO, NR1, S, or O, Z = a crosslinker or spacer, Q = a label or a carrier protein, R1 = H or alkyl having 1-6 C atoms, n = 1-10 m = 0 or 1.
2. Antibodies obtainable by immunizing with an immunogen of the general formula (I), in which X, Z, R1, n and m have the given meaning and Q is a carrier protein.
3. Agent for the immunological determination of amiodarone and its metabolites in body fluids, which agent is characterized in that it contains a) a labelled amiodarone derivative of the general formula (I), in which Q is a label, b) antibodies which can bind amiodarone, its meta-bolites and the labelled amiodarone derivative, and c) where appropriate, an enzyme substrate.
4. Agent according to Claim 3, characterized in that the labelled amiodarone derivative consists of a compound of the general formula (I) which is bound to a label via a crosslinker or spacer.
5. Agent according to Claim 3, characterized in that the immunogen for preparing the antibodies consists of a compound of the general formula (I) which is bound to a carrier protein via a crosslinker or spacer.
6. Method for the immunological determination of amiodarone and its metabolites in body fluids by incubat-ing the sample to be examined with a labelled amiodarone derivative of the general formula (I), in which Q is a label, and antibodies which can bind amiodarone, its metabolites and the labelled amiodarone derivative, as well as, where appropriate, with an enzyme substrate, and measuring the fluorescence or the enzyme activity.
7. Method according to Claim 6, characterized in that a compound of the general formula (II), (II) in which X = O, S, or NR1, Y = NHR1, COR2, SH, OH, halogen or CN, R1 = H or alkyl having 1-6 C atoms, R2 = OH, halogen, NHR1 or OR1, n = 1-10, which is bound to a label via a crosslinker or spacer, is employed as the amiodarone derivative.
8. Method according to Claim 6, chracterized in that the antibodies are prepared by using as the immunogen a compound of the general formula (II) which is bound to a carrier protein via a crosslinker or spacer.
9. Method according to Claim 8, characterized in that the preparation of the immunogen is carried out in the presence of diethylene glycol monoalkyl ether, diethylene glycol dialkyl ether, 1,3-dimethyl-2-imid-azolidinone, 3-butyrolactone, glycerol formal or 2,2-dimethyl-4-hydroxymethyl-1,3-dioxolane.
10. The compounds of the general formula (II) (II) in which X, Y and n have the abovementioned meanings.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE4239430A DE4239430A1 (en) | 1992-11-24 | 1992-11-24 | Means and method for the immunological determination of amiodarone and its metabolites |
| DEP4239430.9 | 1992-11-24 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2109644A1 true CA2109644A1 (en) | 1994-05-25 |
Family
ID=6473500
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002109644A Abandoned CA2109644A1 (en) | 1992-11-24 | 1993-11-22 | Agent and method for the immunological determination of amiodarone and its metabolites |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP0599141A2 (en) |
| JP (1) | JPH06228118A (en) |
| CA (1) | CA2109644A1 (en) |
| CZ (1) | CZ251493A3 (en) |
| DE (1) | DE4239430A1 (en) |
| IL (1) | IL107697A0 (en) |
| ZA (1) | ZA938744B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6376669B1 (en) * | 1999-11-05 | 2002-04-23 | 3M Innovative Properties Company | Dye labeled imidazoquinoline compounds |
| DE19963407A1 (en) * | 1999-12-28 | 2001-07-12 | Giesecke & Devrient Gmbh | Portable data carrier with access protection through message alienation |
-
1992
- 1992-11-24 DE DE4239430A patent/DE4239430A1/en not_active Withdrawn
-
1993
- 1993-11-11 EP EP93118265A patent/EP0599141A2/en not_active Withdrawn
- 1993-11-19 JP JP5290443A patent/JPH06228118A/en active Pending
- 1993-11-22 CA CA002109644A patent/CA2109644A1/en not_active Abandoned
- 1993-11-22 IL IL10769793A patent/IL107697A0/en unknown
- 1993-11-23 ZA ZA938744A patent/ZA938744B/en unknown
- 1993-11-23 CZ CZ932514A patent/CZ251493A3/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| ZA938744B (en) | 1994-06-30 |
| IL107697A0 (en) | 1994-02-27 |
| EP0599141A2 (en) | 1994-06-01 |
| CZ251493A3 (en) | 1994-06-15 |
| JPH06228118A (en) | 1994-08-16 |
| DE4239430A1 (en) | 1994-05-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US5359093A (en) | Reagents and methods for the detection and quantification of thyroxine in fluid samples | |
| US5593896A (en) | Reagents and methods for the detection and quantification of thyroxine in fluid samples | |
| EP0876385B1 (en) | Topiramate immunoassay, as well as analogs and antibodies | |
| US6946547B2 (en) | Ecstasy-class analogs and use of same in detection of ecstasy-class compounds | |
| US6632624B1 (en) | Human chromogranin A (CgA) immunologic assay, antibodies, reagents and kits for said assay | |
| US6262265B1 (en) | Non-hydrolyzable analogs of heroin metabolites suitable for use in immunoassay | |
| US6201109B1 (en) | Assay for bone alkaline phosphatase | |
| US5741715A (en) | Quinidine immunoassay and reagents | |
| CA2109644A1 (en) | Agent and method for the immunological determination of amiodarone and its metabolites | |
| US5221611A (en) | Ddi immunoassays, derivatives, conjugates and antibodies | |
| JP4035754B2 (en) | Bisphenol A hapten compound, hybridoma, anti-bisphenol A antibody having resistance to organic solvents, and method for measuring bisphenol A using them | |
| JP3419084B2 (en) | Antibody Recognizing Active MAP Kinase | |
| EP0597034B1 (en) | Haptens, tracers, immunogens and antibodies for immunoassays for propoxyphene | |
| CN105440128A (en) | Antibody, method and kit for detecting and determining phillyrin | |
| JP2004264024A (en) | Hair growth activity evaluating method | |
| JPH0346562A (en) | Immunization and immunoassay | |
| WO2011007180A9 (en) | Immunodetection and quantification of pyrazolopyrimidine sedatives | |
| JP2000065829A (en) | Antibody against stanniocalcin | |
| JPH09278800A (en) | Monoclonal antibody specific to methoprene, hybridoma producing the same antibody and measurement of methoprene | |
| CN105440129A (en) | Antibody, method and kit for detecting and determining chenodeoxycholic acid | |
| MXPA98004368A (en) | Immunoassay of topiramato, as well as analogs and anticuer | |
| JPH08196294A (en) | Preparation of antibody and immunoassay |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Discontinued |