APPLICATIONS OF RADIOIMMUNOASSAY (RIA) IN PHARMACEUTICAL ANALYSIS
The scope of applicability of radioimmunoassay is rapidly expanding with the dawn of each day as RIA is being developed for newer pharmaceutical substances. It has attained wide recognition and application both in vitro and in vivo measurements of compounds of interest like insulin, gastrin, glucagon, and growth hormones on one hand ; whereas drugs like :
Morphine — Narcotic analgesic,
Hydromorphone and — Narcotic analgesic, antitussive and antipyretic, Hydrocodone on the other hand
Clonazepam — Sedative and anticonvulsant,
Flurazepam — Hypnotic and anticonvulsant,
Chlordiazepoxide — Sedative
Barbiturates — Hypnotic and anticonvulsant,
Flunisolide — A steroid having marked anti-inflammatory activity,
Neobentine — A novel antidysrhythmic and antifibrillatory agent,
Carteolol — B1-Adrenoreceptor blocker used in hypertension and
RIA of some of these drugs will be discussed in the sections that follows :
Synthesis of Immunogen : Morphine is first converted to 3-o-carboxymethymorphine by reacting the free base with sodium-p-chloroacetate in absolute ethanol
The product (II) is coupled to bovine-serum albumin by dissolving the former in distilled water contain-ing the latter, maintaining the pH of the resulting mixture to 5.5 and 1-ethyl-3-(3-dimethyl-aminopropyl) carbidiimide was added. The mixture is incubated overnight at room temperature and then dialyzed against distilled water to cause purification. The resulting purified product carboxy-methyl-bovine-serum conjugate is then labelled with tritium.
Antiserum Production : The immunogen, carboxymethylmorphine-bovine-serum-albumin, is emulsi-fied with equal volume of complete Freund’s adjuvant*. Initial immunization doses are injected into the New Zealand albino rabbits and later on this followed up with booster injections after a period of 6 weeks. The antiserum titer is determined with each booster dose injection and is duly harvested when the titre value is maximum. This is diluted suitably and employed in the radioimmunoassay**.
RIA-Procedure : The various steps followed are as stated below, namely :
1) Various dilutions of antiserums are incubated in the presence of fixed concentration of (3H)-dihydromorphine, and after incubation, a neutral saturated ammonium sulphate solution is added to all the tubes,
2) The complete precipitate, sedimented by centrifugation at 5000 rpm is washed twice with 50% ammonium sulphate solution,
3) The washed-precipitate, containing antibody-bound morphine, is dissolved in NCS-solubilizer, and the radioactivity is counted with the help of a Packard-Iri-card Liquid Scintillation Spectrometer, The tube which contained radioactive dihydromorphine and antiserum but no unlabelled mor-phine, served as a measure of maximum antibody-bound radioactivity,
4) The addition of increasing amount of unlabelled morphine to a fixed amount of (3H) dihydromorphine and antiserum results a competitive inhibition of the labelled dihydromorphine for the formation of the antibody-hapten complex, and
5) The assay sensitivity limit is found to be 100 pg of unlabelled-morhine per tube that caused 20% binding inhibition of labelled-dihydromorphine, (see Figure 32.3).
Hydromorphone (I) and hydrocodone (II) belong to the morphine group of drugs and are used invariably in combination with other ingredients in a number of proprietory antitussive and analgesic antipyretic mixtures. However, interest in the pharmacokinetics of hydromorphone and hydrocodone in human subjects required an adequate assay for drug levels in plasma.
RIA for hydromorphone*, ** and hydrocodone*** are fairly sensitive in the nanogram per millilitre range but essentially require the preparation of a specific antibody. The laid-out RIA method is quite capable of estimating the above drugs within a range of 2.5-20 ng ml–1 using standard 100 μ l plasma sample only.
RAI is carried out using morphine-6-antiserum and tritiated dihydromorphine (commercially avail-able). The free-drug is separated from bound drug using dextran coated charcoal and an aliquot of the supenate containing the antiserum-bound-drug is subsequently counted for radioactivity. However, the radioactivity measurements are normally ascertained in a Liquid Scintillation Counter provided with 20-ml glass scintilla-tion vials.
(i) Lyophilized morphine-6-antiserum : It is diluted 1 : 20 with phosphate buffer prior to use,
(ii) 3H-Dihydromorphine Solution : It is prepared by diluting 2 μ l of the radiolabelled compound in ethanol to 10 ml with phosphate buffer so that each 0.1 ml of solution contained 83 pg (0.5 mole),
(iii) Dextran-coated chrocoal suspension : It is prepared by mixing 2.5 g of charcoal in 100 ml of distilled water with 2.5 g of dextran in 100 ml of distilled water, and eliminating the fine particles by centrifugation, and
(i) Preparation of Saturated Solutions : Individual stock solutions containing the equivalent of 200 μ g of I or II base line are prepared using weighed quantities of the respective powders dissolved in distilled water. Dilutions of the drugs are made in individual 10 ml volumetric flasks to yield drug concentrations of 2.5, 5.0, 10.0 and 20 ng ml–1 for I and 10.0, 20.0, 40.0 and 80.0 ng ml–1 for II. The dilutions are made using blank plasma and phosphate buffer solutions
RIA-Procedure : The different steps to be followed are stated below, namely :
a) Various dilutions of unknown plasma, morphine-6-antiserum, 3H-dihydromorphone are prepared afresh,
b) The unknown plasma (0.1 ml) is incubated directly with morphine-6-antiserum (0.1 ml) and buffer (0.3 ml) for a duration of 50 minutes at room temperature (20 ± 2 °C) and immediately followed by 10 minutes at 4°C,
c) The ice-cold dextran-coated-charcoal suspension (0.1 ml) is added to all the above tubes, followed by immediate mixing and incubation for 10 minutes at 4°C,
d) All the tubes are then centrifuged for a period of 15 minutes at 3000 rpm,
e) A small portion (0.2 ml) of the supernate is removed and placed in a scintillation vial containing 0.5 ml of distilled water and 5 ml of scintilation fluid,
f) The contents of the scintillation vial are mixed thoroughly, and the radioactivity is measured in a Liquid Scintillation Counter for 10 minutes,
g) Duplicate hydromorphone 2.5, 5.0, 10.0 and 20.0 ng ml–1 or hydrocodone 10.0, 20.0, 40.0, and 80.0 μ g ml–1 standards are accurately assayed concurrently and the data is plotted in a graph, and
h) The regression equation, calculated from the standard solutions in each collection, is employed to determine quantitatively the drug concentration present in individual plasma samples.
Colonazepam belongs to the class of 1, 4-benzodiazepines that has been found to be therapeutically effective in controlling minor motor seizures i.e., petitmal epilepsy in humans*
Synthesis of Immunogen : The 3-hemisuccinyloxy derivative of clonazepam is covalently coupled to bovine serum albumin employing the mixed-anhydride method suggested by Erlanger and coworkers** (1959). After successive dialysis against dioxane-water borate buffer and water, the immunogen i.e., clonazepam-bovine-serum-albumin conjugate is isolated by lyophilization.
Preparation of 3H-Labelled Clonazepam : 3H-Clonazepam is prepared by tritium exchange employ-ing dimethyl formamide-titrated water having a specific activity*** of 100 ci g–1. The resulting product is subsequently purified by silica-gel-column-chromatography, thereby yielding a material which has a specific activity of 4.32 mci mg–1. This specific method of introducing 3H (tritium) probably provided exchange chiefly at C-3 position****.
Antibody Production : A thick emulsion of the immunogen (clonazepam-bovine-serum-albumin-con-jugate) is prepared employing complete Freund’s adjuvant and two New Zealand white female rabbits are immunized intradermally at multiple sites with the immunogen emulsion. The animals are then administered with booster doses intravenously with immunogen emulsion at monthly intervals, and serum is harvested 10-14 days after each administration. Both rabbits produced satisfactory titers of antibodies to clonazepam within a period of three months following the initial immunization. The resulting serum is pooled, diluted suitably and employed in the radioimmunoassay.
RIA-Procedure : The various steps involved in the RIA-procedure for clonazepam are enumerated below, namely :
1) A constant small (volume) portion of the control plasma is added to constant small (volume) portion of standard clonazepam in small test-tubes to generate a calibration (standard) curve,
2) Appropriate controls are included by adding the control plasma to small portion of buffer solutions,
3) Each unknown plasma sample is added to tubes containing buffer solution then the titrated (3H)-colnazepam solution followed by diluted antiserum is added,
4) The contents of each tube are mixed thoroughly and allowed to stand at room temperature for sometime,
5) Saturated ammonium sulphate solution is added to precipitate the globulin-bound-3H- clonazepam and after mixing, the tubes are allowed to stand for 15 minutes at 0 °C,
6) The tubes are subsequently centrifuged at 3000 rpm,
7) Each supernate, containing the unbound 3H-clonazepam, is decanted into a scintillation vial and toluene is added,
8) The samples are assayed for 3H-activity in a liquid scintillation counter, and
9) All samples including the standards, unknowns and controls are assayed in duplicate and the aver-age of the 3H-counts is employed for the percentage of binding.
Flurazepam belongs to the class of hypnotic agent used for the treatment of insomnia.
Synthesis of Immunogen (Hapten) : 3-Hydroxy flurazepam is refluxed with succinic anhydride in dichloromethane containing triethylamine to produce the desired hapten, 3-hemisuccinyloxy-flurazepam. It is coupled covalently to bovine-serum-albumin by the mixed-anhydride procedure developed by Erlanger et al (1959). The resulting conjugate is purified by dialysis against sodium bicarbonate solution followed by dialy-sis against distilled water and finally isolated by lyophilization.
Immunization and Antibody Production : The immunogen 3-hemisuccinyloxyflurazepam, is emulsi-fied with complete Freund’s adjuvant. It is injected intradermally into two female New Zealand albino (white) rabbits. Repeated doses are administered twice at interval of two weeks. Subsequently, booster injections of the thick-immunogen-emulsion-paste are administered after a span of 6-weeks. The antibody is harvested when its titer level is high enough, diluted to the suitable-level and employed in the RIA.
RIA-Procedure : The different steps followed in the RIA-procedure are as given below :
A calibration curve is generated by adding 3H-Flurazepam in 0.1 ml of buffer containing 0.03-0.2 ng range of flurazepam in buffer,
· Following preparation of the standards, duplicate portions of the reconstituted unknown flurazepam fractions are added to tubes containing 3H-Flurazepam,
· Diluted antiserum is added to all tubes except the non-specific-binding control specimen to which buffer is added,
· The contents of each tube is mixed gently on a Vortex Mixer and allowed to stand at room tempera-ture,
· Following incubation, the antibody-bound radio ligand is separated from the unbound fraction by precipitation with saturated ammonium sulphate,
· After the pellet is dissolved in water add 3 ml of scintillation fluid to produce a clear solution, and
· The radioactivity in each tube is quantified in a modified scintillation liquid counter.
RIA-Specificity* : The specificity of the antiserum initially is evaluated by cross-reactivity** studies involving all the flurazepam metabolites known to be present in plasma. The mono-as well as di-desethylmetabolites exhibited a cross-reactivity of 17 and 3.7% respectively, while other possible competitors cross-reacted less than 1% as shown in Table 32.1.
Evidently, due to the cross-reactivity of both mono- and di-desethyl metabolites, a specific assay of flurazepam could not be developed successfully without first separating it from its metabolites effectively by the help of column chromatography.
Chlordiazepoxide is the pioneer member of the 1, 4-benzodiazepines to be employed clinically as an antianxiety agent in humans***. A number of methods based on extraction processes are available for the assay of this drug, namely : spectrofluorometry, polarography and electron-capture GC-technique ; but RIA measures it directly in the blood without involving extraction and possesses very low sensitivity.
Synthesis of Immunogen : Chlordiazepoxide as suspension in N-methylformamide is treated with HCl in dioxane to yield a pale-yellow solution. The resulting mixture is cooled to -30 °C and isoamyl nitrite in dioxane is added. The solution is stirred at – 30° to – 40 °C and aqueous ammonium sulfamate is added with continuous stirring.
The chilled azide solution is added slowly, dropwise with constant vigorous stirring into a solution of bovine-serum albumin. The pH is maintained at 8.0 to 8.7 by the careful addition of NaOH solution. The resulting pale-yellow solution is kept at 4°C for a duration of 36 hours and then dialysed against trimethamine buffer. After further dialysis for two days against distilled water, the immunogen is isolated by lyophilization.
Immunization and Antibody Production : The lypphilized immunogen obtained above is dissolved in normal saline and emulsified with equal volumes of complete Freund’s adjuvant into a thick paste. Three New Zealand albino rabbits are immunized with the immunogen-paste through intradermal injections. The process is repeated twice at 2-weeks intervals followed by booster doses at monthly intervals. The antiserum is harvested when the plasma titer value is attained maximum.
RIA-Procedure : The various steps involved in the RIA procedure are enumerated below :
1) A constant volume of control human plasma is added to a constant volume of each standard of chlordiazepoxide to produce a calibration curve of 2 to 100 ng per tube,
2) The same volume of the unknown plasma samples is added to tubes containing constant volume of the solution of the labelled chlordiazepoxide and constant volume of the antiserum solution is now added to all the tubes,
3) The volumes in all the tubes are made upto 1 ml with buffer solution, mixed thoroughly on a Vortex Mixer, and each tube is immersed in an ice-water bath,
4) An equal volume of saturated ammonium sulphate solution is added to enable complete precipitation of globulin-bound chlordiazepoxide 14C,
5) After mixing the contents of the tubes thoroughly on a Vortex Mixer and allowing them to stand for a while at 4°C, the tubes are centrifuged at 3000 rpm,
6) The supernate thus obtained containing unbound chlordiazepoxide-14C is decanted into a count-ing vial and toluene is added, and
7) The radioactivity in the supernate and that in the precipitate are separately counted in a scintilla-tion counter.
Specificity of Antibody binding of Chlordiazepoxide : A good number of benzodiazepines are tested for their ability to complete with labelled chlordiazepoxide for the respective antibody binding site. The various competitors are adequately tested at a concentration of 200 ng i.e..,10-times the concentration of chlordiazepoxide required to produce a 50% inhibition of binding as shown in Table 32.2.
From table 32.2 it is evident that the highest cross-reaction is 5% with N-desmethylchlordiazepoxide while demoxepam, N-desmethyldiazepam, diazepam and clonazepam displayed less than 1% inhibition. How-ever, the RIA method appears to be reliable over a range of 2-100 ng per tube of chlordiazepoxide and, there-fore, the sensitivity limit stands at 20 ng ml–1 using a 1.0 ml sample of plasma.
Barbiturates represent a class of sedative and hypnotic drugs employed extensively in medicine. RIA provides a rapid, sensitive specific and reliable means for their determination in plasma levels upto 5 ng without indulging in any type of extraction, filtration or evaporation as required for other conventional analyti-cal methods**.
5-Allyl-5-(1-carboxyisopropyl) barbituric acid.
Synthesis of Immunogen (Hapten) : The barbiturate, 5-allyl-5-(1-carboxyisopropyl) barbituric acid (1) is first converted to 5-allyl-5-(1-p-nitrophenyloxycarbonylisopropyl) barbituric acid (II) by the interaction of the base with p-nitrophenol in N, N-dimethylformamide (DMF) as shown below :
The resulting product (II) is subsequently coupled to bovine-serum-albumin in a glycerol-water mixture in the presence of dicyclohexylcarbodiimide. The mixture is incubated overnight at 4°C, and the protein-hapten complex is dialysed against distilled water thereby causing its purification. Conjugation of the respective bar-biturate to the protein carrier, comparison of the barbiturate BGG-conjugate to control BGG-solution and preparation of 14C-pentobarbital sodium are carried out respectively.
Preparation of Antiserum : The barbiturate-bovine-serum-albumin conjugate is duly emulsified with an equal volume of complete Freund’s adjuvant and New Zealand albino rabbits are subsequently im munized with this particular emulsion. Six weeks after the initial does, booster doses are administered to the animals in each of their foot pads. Blood samples are collected 5-7 days after the booster injections and the serum is examined for antibodies to barbiturates. The antiserum is harvested when the serum antibody titer has attained its maximum level.
It has been observed that while normal, rabbit serum failed to bind labelled phenobarbital, the serum from immunized rabbits bound 75 to 80% of the added pentobarbital and there exists a linear relationship between 14C-phenobarbital and the concentration of added antibody. Besides, when variable quantities of 14C-pentobarbital are added to a constant quantity of antibody, there exists a linear relationship between added and bound 14C-phenobarbital as depicted in Figure 32.4.
Flunisolide is a fast-acting corticoid designed for the treatment of allergic rhinitis, asthma, and other allied respiratory disorders in humans*. As the quantum of drug delivered by inhalation (i.e., the usual route of administration of the drug), is invariably small, the plasma-levels attained can also be fairly small. Hence, there is a dire need for a sensitive method of plasma concentration evaluation which is satisfied by radioimmunoassay.
Synthesis of Hapten Immunogen and Antiserum Production : The hapten, flunisolide-bovine-se-rum-albumin conjugate is prepared by coupling the 21-hemisuccinate of flunisolide to bovine-serum-albumin with a water-soluble carbodiimide coupling reagent*. The reaction mixture is dialysed exhaustively against normal saline to cause purification and the extent of conjugation is estimated by measuring the protein concen-tration**. However, the flunisolide residues are determined by UV-absorption method.
An emulsion of the hapten (i.e., conjugate) in normal saline is prepared by mixing with an equal volume of Freund’s complete adjuvant. The prepared emulsion is injected subcutaneously into four different sites in New Zealand albino rabbits. Six weeks after the initial injection, all the animals are placed on a regimen of weekly booster shots. After a period of six months, antiserum from these animals are harvested and dilutions of 1 : 10,000 to 1 : 30,000 produced 50% binding or more and is employed in the RIA.
RIA-Procedure : The following steps are to be adopted in a sequential manner, namely :
· Flunisolide standards required for the preparation of the standard curve are obtained by dilution of a stock solution of 10 mg of it in 10 ml of ethanol,
· A series of standard solution viz., 20, 50, 100, 200, 300, 500 and 600 pg per 0.1 ml in tris-(hydroxymethyl)-aminomethane/hydrochloric acid buffer and stored duly at 0 °C temperature,
· An ethanolic solution of 3H-Flunisolide is diluted with tris-(hydroxymethyl)-aminomethane/hydro-chloric acid buffer and 0.1% gelatin such that 0.1 ml portion contains 8,000 to 10,000 cmp activity,
· The antiserums are diluted in the said buffer with 0.1% gelatin to give rise to a total binding of between 35-50%,
· The charcoal stock solution is diluted as and when required with the aforementioned buffer immedi-ately before, use,
· RIA is conducted by mixing together various dilutions of antiserum, buffer solution, 3H-Flunisolide and various dilutions of flunisolide standard solutions in a set of test tubes,
· A second set of test tubes containing various dilutions of antiserum, buffer solution, 3H-Flunisolide and various dilutions of the plasma being analysed of flunisolide content are prepared separately,
· The two sets of test tubes are incubated at temperature of 0 °C after adding constant volume of charcoal suspension to each of the tubes and mixing them thoroughly on a Vortex Mixer,
· The incubation is done overnight,
· The tubes are then centrifuged at 2500 rpm for 4 minutes and immediately 0.5 ml of the supernate is transferred into scintillation vials, and
· The scintillation fluid is added and the solutions are counted for 10 minutes in Scintillation Counter***.
The percentage inhibition is calculated and the values obtained from the first set of tubes is used to plot a standard curve. The concentrations of flunisolide from the standard curve values from their calulated percent-age inhibition value as depicted in figure 32.5 below :
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