K Ensing

Development and registration of chiral drugs.

In this review we describe the impact of chirality on drug development and registration in the United States, Japan and the European Community. Enantiomers may have differences in their pharmacological profiles, and, therefore, chiral drugs ask for special analytical and pharmacological attention during their development. However, the registration authorities have no clear policy towards the registration of chiral drugs. The absence of a clear policy regarding chirality causes a great deal of confusion and frustration at various levels and is not in the interest of industries developing newer and more beneficial drugs.

RADIORECEPTOR ASSAY - A TOOL FOR THE BIOANALYSIS OF DRUGS

Abstract Radioreceptor assays can be developed for every class of drugs whose pharmacological action is mediated by a drug-receptor interaction. They provide a measure for biological activity which can be related to pharmacological effect.

DEVELOPMENT OF A SENSITIVE RADIORECEPTOR ASSAY FOR OXYPHENONIUM IN PLASMA AND URINE

A radioreceptor assay (RRA) for oxyphenonium has been developed. It is based on competition between [3H]dexetimide and oxyphenonium for binding to muscarinic receptors from calf striata. The RRA is optimized towards incubation medium and to extraction by ion pair formation with sodium picrate. At least 4 times 10−10 m of oxyphenonium is necessary to permit a reliable assay. This corresponds to a detection limit of drug of 2 ng ml−1 urine. After extraction, drug at 100 pg ml−1 of plasma can be estimated using 4 ml samples. The method is applicable to monitoring the drug and to the determination of its pharmacokinetics after therapeutic dosing. Urine levels can also be monitored.

Application of Empore C-8 extraction disks for screening urine in systematic toxicological analysis

Solid-phase extraction (SPE) by means of disposable columns has become a widely accepted technique for sample pretreatment in toxicology, both for directed analyses and for screening analyses. However, the sample capacity in SPE is usually limited to a few millilitres. Therefore, we have investigated to what extent these problems can be overcome by using Empore extraction disks, consisting of chemically modified C-8 reversed-phase silica, embedded in an inert polytetrafluoroethylene (PTFE) matrix. Human urine was selected as the matrix and dexetimide and mepyramine were initially used as test drugs because these drugs were available in tritiated form. Additional drugs investigated included codeine, hexobarbital, imipramine, methamphetamine, and nitrazepam. In these investigations, the sample capacity for untreated urine was at least 25 mL, and analyte quantities up to 250 micrograms could be retained by these filters. Washing with water/methanol mixtures was successful in removing substantial amounts of endogenous interferences, and methanol proved to be an acceptable eluent. Thus, these disks seem to have interesting potential for toxicological analysis in that sample concentration and cleanup can be achieved at the same time.

SOLID-PHASE EXTRACTION OF MORPHINE FROM WHOLE-BLOOD BY MEANS OF BOND ELUT CERTIFY COLUMNS

The use of Bond Elut Certify columns for the isolation of morphine from whole blood was evaluated. In order to monitor possible losses and the elution profile of morphine, a small amount of the tritiated analogue was added to the samples. Four sample pretreatment methods, three protein precipitation methods and one sonication/dilution method, were tested. The latter one gave the best results. The blood sample was applied onto the column at pH 3.3 after sonication and dilution with 0.1 M phosphate buffer (pH 3.3). The retention of morphine was affected by the pH of the samples, and the loss of morphine during sample application was minimized at low pH (3.3). The interferences were removed by washing the column with the phosphate buffer, 0.01 M acetic acid (pH 3.3), and methanol, sequentially. Ammoniated methanol, 2 mL at 2%, was selected to elute morphine. As a result, more than 80% of 3H-morphine was recovered for concentrations of morphine ranging from 5 to 4000 ng/mL.

Centrifugation or filtration in quantitative radioreceptor assays.

In quantitative radioreceptor assays the amount of a drug present in the medium to be assayed is inversely related to the amount of receptor-bound radiolabelled ligand. Usually, separation of the bound and free fractions of radiolabelled ligand is done by filtration, in which the bound fraction can easily be collected. However, the filtration disturbs the equilibrium between bound and free fractions, which may lead to erroneous results. Because the decrease in bound radiolabelled ligand is accompanied by an increase in free labelled ligand, we decided also to measure this free fraction after separation by centrifugation and to compare these data with the filtration data. In these experiments a radioreceptor assay for anticholinergics was employed. The results indicate that both methods are compatible in precision when appropriate conditions are used whereas each method has its specific features.

DEVELOPMENT OF A RADIORECEPTOR ASSAY FOR THE D2-SELECTIVE DOPAMINE AGONIST N-0437

N-0437 is a recently developed dopamine (D2) agonist, theoretically attractive in the therapy of Parkinsons disease and glaucoma. Since its high potency allows small doses of the compound in clinical use and as extensive metabolism occurs in animals, a highly sensitive assay method was required for drug-monitoring purposes. To this end we developed a radioreceptor assay (RRA), a sensitive tool for the assessment of the samples (dopaminergic) bioactivity. The RRA is based on competition between N-0437 and its tritium-labeled analogue for binding to dopamine receptors. The assay has been optimized for the preparation of the receptor suspension and the incubation conditions. Direct application of the assay for biological samples was impossible because of matrix interferences. Therefore, a solid-phase extraction method was developed in which the combination of a polar Si column and dichloromethane as eluent resulted in an effective elimination of the interferences. Recoveries were better than 90 and 95% for plasma and urine, respectively, even at concentrations at the determination limit of the method (300 pg/ml). Relative standard deviations were less than 15%. Because RRAs are stereoselective, the method discriminates between active and inactive species.

INFLUENCE OF CHEMICAL-STRUCTURE OF TRICYCLIC TERTIARY DIMETHYLAMINES ON CHIRAL SEPARATION BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY AFTER DERIVATIZATION WITH (-)-MENTHYLCHLOROFORMATE

Abstract In the present study (−)-menthylchloroformate was used as a chiral derivatizing agent for promethazine, trimeprazine, trimipramine and N-(2-dimethylaminopropyl)iminodibenzyl. (−)-Menthylchloroformate reacted with the tertiary dimethylamine moiety in these tricyclic antihistamines and antidepressants, resulting in the formation of diastereoisomers. Owing to the reaction conditions, during the derivatization with (−)-menthylchloroformate, the possibility of racemization had to be established. For this purpose different ratios of (+) and (−)-promethazine were prepared. Enantiomeric separation of these mixtures took place on a Chiral α AGP column or, after derivatization with (−)-menthylchloroformate, on a C18 column. The results from these two independent separation systems were compared with trace racemization. No racemization was found during the experiments. To study the effects of changes in the molecular structures of the tertiary dimethylamines on the chromatographic behavior of the derivatization products, four tertiary dimethylamines [promethazine, trimipramine, trimeprazine and N-(2-dimethylaminopropyl) iminodibenzyl] were derivatized and analyzed. With these amines the effects on resolution and capacity factor of replacing a phenothiazine ring by an iminodibenzyl ring or insertion of a carbon molecule between the chiral centre in the chain and the place where (−)-menthylchloroformate reacts were studied. Not only the distance between the chiral centres in the diastereoisomers (a longer distance caused less resolution and higher capacity factors) but also the kind of ring influenced resolution and capacity factor. Finally, the influence of eluent composition on resolution and capacity factor was studied. Three different mixtures of methanol and acetic acid were tested. More acetic acid in the eluent caused a better resolution and higher capacity factor. The higher capacity factor, however, resulted in unacceptable retention times.

DETERMINATION OF ATROPINE IN PLASMA BY A DIRECT RADIORECEPTOR ASSAY

A highly sensitive radioreceptor assay for the anticholinergic atropine was developed and could be applied directly to plasma samples obtained from mini-pigs without any clean-up. Plasma samples were collected during 6 h after atropine was administered intravenously or endobronchially. The endobronchial plasma concentration-time curves were characterized by a very rapid rise of the concentration but with a subsequent much slower decrease than after intravenous administration. This indicates an initially high as well as prolonged uptake from the lungs.

Different Behavior Toward Muscarinic Receptor Binding Between Quaternary Anticholinergics and Their Tertiary Analogues

A number of corresponding tertiary and quaternary anticholinergic analogues were examined for their ability to inhibit specific 3H-dexetimide binding to calf brain muscarinic receptors. In all cases the tertiary antagonists (except pirenzepine) showed steep and monophasic inhibition curves, whereas those of the quaternary derivatives were shallow (thiazinamium, methylbenactyzine) or even biphasic (oxyphenonium, methylatropine, methylscopolamine). These observations show that the addition of a methyl group to the nitrogen atom changes the mode of interaction of the anticholinergics to muscarinic receptor binding sites. Whether there are separate binding sites present or differences in interaction mode for only the quaternary moiety is discussed.