Abstracts of the 24th international isotope society (UK group) symposium: synthesis and applications of labelled compounds 2015.

The 24th annual symposium of the International Isotope Society's United Kingdom Group took place at the Møller Centre, Churchill College, Cambridge, UK on Friday 6th November 2015. The meeting was attended by 77 delegates from academia and industry, the life sciences, chemical, radiochemical and scientific instrument suppliers. Delegates were welcomed by Dr Ken Lawrie (GlaxoSmithKline, UK, chair of the IIS UK group). The subsequent scientific programme consisted of oral presentations, short 'flash' presentations in association with particular posters and poster presentations. The scientific areas covered included isotopic synthesis, regulatory issues, applications of labelled compounds in imaging, isotopic separation and novel chemistry with potential implications for isotopic synthesis. Both short-lived and long-lived isotopes were represented, as were stable isotopes. The symposium was divided into a morning session chaired by Dr Rebekka Hueting (University of Oxford, UK) and afternoon sessions chaired by Dr Sofia Pascu (University of Bath, UK) and by Dr Alan Dowling (Syngenta, UK). The UK meeting concluded with remarks from Dr Ken Lawrie (GlaxoSmithKline, UK).

A time-resolved fluorescence immunoassay for the determination of a novel respiratory therapeutic agent, AR-C68397XX (Viozan) in human plasma.

A dissociation-enhanced lathanide fluorescence immunoassay (DELFIA) method has been developed for the determination of AR-C68397XX, a novel respiratory therapeutic agent, in human plasma. The method is a 'direct' immunoassay and provides an alternative to the solid phase extraction RIA described in a previous publication, which employs the same specific antiserum. The DELFIA method is suitable for the determination of the analyte at pg ml(-1) concentrations. The non-isotopic label was prepared by complexation of a DTPA derivative of AR-C68397XX with free europium cation (Eu3+). Plasma samples were diluted at least 5-fold prior to analysis to eliminate matrix interference. The calibration range is 10-2000 pg ml(-1), and the LOQ of the method is 50 pg ml(-1) using 50 microl of diluted human plasma sample.

A RIA combined with SPE for the determination of a dual D2-receptor and beta2-adrenoceptor agonist, AR-C68397XX, in human plasma.

A radioimmunoassay has been developed for the determination of AR-C68397XX, a dual D2-receptor and beta2-adrenoceptor agonist, in human plasma. The method incorporates solid phase sample extraction and is suitable for the determination of the analyte at pg ml(-1) concentrations. The antiserum was raised in Suffolk cross sheep following primary and booster immunisations with an immunogen prepared by conjugating a carboxyphenylmethyl derivative of AR-C68397XX, to bovine serum albumin. The radioligand was prepared by the 125I-labelled iodination of a derivative of AR-C68397XX. The solid phase extraction procedure, using octadecyl sorbent, was introduced to remove matrix interferences in the plasma and to enhance method sensitivity. The calibration range is 20-500 pg ml(-1), using 0.5 ml of undiluted human plasma sample.

A radioimmunoassay combined with solid-phase extraction for the determination (pg ml-1) of AR-C15849XX) in human plasma.

A radioimmunoassay has been developed for the determination of AR-C15849KF, a CCK-8 analogue, in human plasma. The method incorporates solid-phase sample extraction, is suitable for the determination of the analyte at pg ml-1 concentrations and is based on a method developed and validated for dog plasma. The solid-phase extraction, using ion-exchange aminopropyl and octadecyl sorbents sequentially, was retained for this procedure to remove matrix interferences in the plasma and to enhance method sensitivity. The calibration range is 10-500 pg ml-1, using a 1 ml sample of undiluted human plasma. The method has been successfully used to generate early human pharmacokinetic data during a programme of exploratory development.

A radioimmunoassay for the determination of tipredane in human plasma.

A sensitive method for the determination of tipredane in human plasma has been developed. Prior to radioimmunoassay, proteins are removed from plasma by precipitation with acetonitrile. A [125I]iodohistamide derivative of tipredane is used as a heterogeneous radioligand. The primary antiserum is raised in sheep against a tipredane (O-carboxymethyl) oxime-bovine serum albumin conjugate. Donkey anti-sheep IgG antiserum is used as the secondary antiserum in a double antibody separation procedure. The limit of quantification of the method is 1 ng ml-1 for 500 microliters of plasma. There is no significant interference from either established or putative metabolites of tipredane, endogenous steroids or a wide range of other drugs. The assay was used to determine the concentrations of tipredane in plasma samples collected in pilot clinical studies.

A radioimmunoassay combined with solid-phase extraction for the determination of a novel anti-obesity agent, ARL 15849XX, in dog plasma.

A radioimmunoassay has been developed for the determination of ARL 15849XX, a cholecystokinin-8 (CCK-8) analogue, in dog plasma. The method incorporates solid-phase sample extraction and is suitable for the determination of the analyte at picogram per millilitre concentrations. The antiserum was raised in Suffolk-cross sheep following primary and booster immunisations with an immunogen prepared by conjugating ARL 16935XX, an analogue of ARL 15849KF, to bovine serum albumin. The radioligand was prepared by the no-carrier-added 125I iodination of a non-sulphated derivative, ARL 15745XX. The solid-phase extraction procedure, carried out using ion-exchange aminopropyl and octadecyl sorbents sequentially, was introduced to remove matrix interferences in the plasma and to enhance the method sensitivity. The calibration range is 20-1000 pg ml-1, using a 1 ml sample of undiluted dog plasma.

Determination of dopexamine hydrochloride in human blood by high-performance liquid chromatography with electrochemical detection.

A method is described for the determination of dopexamine hydrochloride at concentrations of 5 to 100 ng/ml in human blood using electrochemical detection. The method uses a Hypersil ODS column and a mobile phase containing heptane sulphonate, orthophosphoric acid, diisopropylamine and disodium EDTA. Blood samples are stabilised immediately after collection by the use of dipotassium EDTA and a high concentration of sodium metabisulphite. The sample preparation procedure consists of a simple de-proteinisation with perchloric acid. The method is accurate, with inter-assay accuracies ranging from 100 to 104%, and is free of interference by blood from different individuals. Known and potential metabolites of dopexamine hydrochloride and a wide range of drugs do not interfere with the method. The method is precise with inter-assay coefficients of variation of 10.6% at 5 ng/ml and of less than 4.2% at higher concentrations. Stabilised blood samples may be stored for over six months at -25 degrees C prior to analysis.

Regiospecific deuteriation and tritiation of various drugs using a homogeneous rhodium trichloride catalyst.

The deuteriation and tritiation of a number of drugs containing carboxyl, amide, aralkylamine, and anilide functional groups have been investigated using homogeneous rhodium trichloride as a catalyst. Good incorporation of deuterium was observed and the regiospecificity for ortho exchange was very high for most of the drugs studied. Similarly, with tritium, good incorporation (specific activities) and regiospecificities were achieved in many cases. Satisfactory results were also obtained from the small number of heterocyclic-containing drugs that were included in the present study.

A radioimmunoassay method for the determination of nedocromil sodium in plasma and urine.

A radioimmunoassay method for the determination of nedocromil sodium (FPL 59002 disodium salt) in human plasma and urine is described. The method employs a primary antiserum raised in a sheep, and a mono-tyramide derivative of nedocromil sodium labelled with iodine-125 as a heterogeneous radioligand. Free and bound radioligand are separated using a secondary anti-sheep IgG antiserum. All three reagents are added simultaneously to samples containing nedocromil sodium prior to an overnight incubation. The method has a limit of detection of 0.25 ng ml(-1), when plasma sample volumes of 100 microl are analysed, and is accurate and precise. Inter-assay relative standard deviations (N = 18) of 15.1, 5.0 and 5.6% were found at concentrations in plasma of 0.5, 2.0 and 6.0 ng ml(-1) respectively. The method is specific as indicated by negligible cross-reaction of the anti-nedocromil sodium antiserum with a range of drugs. The method is applicable to the analysis of samples from subjects who have inhaled nedocromil sodium from a pressurised aerosol.

Radioimmunoassay of sodium cromoglycate in human plasma.

A sensitive radioimmunoassay method for sodium cromoglycate in human plasma is described. The lowest quantifiable concentration of sodium cromoglycate is 0.93 nmol/l when 0.1 ml plasma samples are analysed. Direct analysis of sodium cromoglycate concentrations in plasma samples collected up to several hours after the administration of single therapeutic doses of the compound is possible. An antiserum raised in a sheep, radioligand heterogeneously labelled with iodine-125, and a second antibody technique for the separation of bound and free radioligand are employed. The inter-assay coefficient of variation is less than 14% (n = 23). The range of the method is limited; both 0.01 and 0.1 ml volumes of plasma must be analysed to encompass the concentration range 0.93-139 nmol/l which may be encountered in plasma samples from patients and human volunteers. The method is specific for sodium cromoglycate as indicated by a low cross-reactivity of the anti-cromoglycate antiserum with a number of drugs.

Formation of the A/B cis ring junction of ecdysteroids in the locust, Schistocerca gregaria.

1. The mechanism of formation of the A/B cis ring junction of ecdysteroids in the locust Schistocerca gregaria, was investigated by incorporation of [4-14C,3 alpha-3H], [4-14C,4 alpha-3H] and [4-14C,4 beta-3H]cholesterol into 20-hydroxyecdysone in fifth-instar larvae and into ecdysteroid conjugates in ovaries of maturing adult females. 2. In both systems there was retention of the 4 alpha-3H atom in the ecdysteroid and elimination of the 3 alpha- and 4 beta-3H atoms. 3. The 3H retained in the ecdysone formed from [4 alpha-3H]cholesterol in the ovarian system was probably located at C-4. The results are interpreted by postulating the involvement of a 3-oxo-delta 4 intermediate in ecdysteroid biosynthesis in insects.

Mechanism of formation of the A/B cis ring junction of ecdysteroids in Polypodium vulgare.

1. The fates of the alpha-, 4 alpha- and 4 beta-hydrogen atoms of cholesterol during formation of the A/B cis ring junction of ecdysteroids was investigated by administration of [4-14C, 3 alpha-3H], [4-14C, 4 alpha-3H]- and [4-14C, 4 beta-3H]cholesterol species to the fern, Polypodium vulgare, and isolation of the 20-hydroxyecdysone formed in each case. 2. The 3H was retained in the ecdysteroid formed from each substrate. 3. Location of the 3H in the 20-hydroxyecdysone indicated that migration of 3H from the 3 alpha- and 4 beta-positions to C-4 and C-5, respectively, had occurred, whereas the 4 alpha-3H atom was retained at C-4. 4. A possible mechanism for the formation of the A/B cis ring junction of ecdysteroids in P. vulgare is presented.

Mechanism of dealkylation of clionasterol in the insect Tenebrio molitor.

1. 25-3H- and 26-14C-labelled (24S)-24-ethylcholest-5-en-3beta-ol (clionasterol) were synthesized from (24S)-24-ethylcholesta-5,25-dien-3beta-ol. 2. These labelled substrates were mixed and administered, together with the hypocholesterolemic agent, triparanol citrate, to Tenebrio molitor larvae. 3. The 3H label from the clionasterol substrate was retained in both the desmosterol and the cholesterol isolated from the larvae. 4. Location of this 3H label in the desmosterol showed that dealkylation of the clionasterol involved 3H migration from C-25 to C-24. A possible mechanism for dealkylation is presented.

Stereochemistry of olefinic bond formation in defensive steroids of Acilius sulcatus (Dytiscidae).

The defensive secretion of Acilius sulcatus contains a number of pregnane derivates: cortexone, 20alpha-hydroxy-4-pregnen-3-one, together with the unusual delta4,6 dienes, 6,7-dehydrocortexone, 20alpha-hydroxy-4,6-pregnadien-3-one and 4,6-pregnadien-3,20-dione. The synthesis of all these steroids except cortexone is described. Complete separation of the steroids of Acilius can be achieved by high-performance liquid chromatography on the reversed-phase column system. During biosynthesis of the Acilius steroids from cholesterol, introduction of the delta4 and delta6 bonds involves elimination of the 4beta and 7beta hydrogens, respectively. Possible mechanisms of formation of the delta4,6 steroids are discussed.