Vaccine-driven pharmacodynamic dissection and mitigation of fenethylline psychoactivity.

Fenethylline, also known by the trade name Captagon, is a synthetic psychoactive stimulant that has recently been linked to a substance-use disorder and 'pharmacoterrorism' in the Middle East. Although fenethylline shares a common phenethylamine core with other amphetamine-type stimulants, it also incorporates a covalently linked xanthine moiety into its parent structure. These independently active pharmacophores are liberated during metabolism, resulting in the release of a structurally diverse chemical mixture into the central nervous system. Although the psychoactive properties of fenethylline have been reported to differ from those of other synthetic stimulants, the in vivo chemical complexity it manifests upon ingestion has impeded efforts to unambiguously identify the specific species responsible for these effects. Here we develop a 'dissection through vaccination' approach, called DISSECTIV, to mitigate the psychoactive effects of fenethylline and show that its rapid-onset and distinct psychoactive properties are facilitated by functional synergy between theophylline and amphetamine. Our results demonstrate that incremental vaccination against a single chemical species within a multi-component mixture can be used to uncover emergent properties arising from polypharmacological activity. We anticipate that DISSECTIV will be used to expose unidentified active chemical species and resolve pharmacodynamic interactions within other chemically complex systems, such as those found in counterfeit or illegal drug preparations, post-metabolic tissue samples and natural product extracts.

Cross-reactivity studies and predictive modeling of "Bath Salts" and other amphetamine-type stimulants with amphetamine screening immunoassays.

INTRODUCTION: The increasing abuse of amphetamine-like compounds presents a challenge for clinicians and clinical laboratories. Although these compounds may be identified by mass spectrometry-based assays, most clinical laboratories use amphetamine immunoassays that have unknown cross-reactivity with novel amphetamine-like drugs. To date, there has been a little systematic study of amphetamine immunoassay cross-reactivity with structurally diverse amphetamine-like drugs or of computational tools to predict cross-reactivity. METHODS: Cross-reactivities of 42 amphetamines and amphetamine-like drugs with three amphetamines screening immunoassays (AxSYM(®) Amphetamine/Methamphetamine II, CEDIA(®) amphetamine/Ecstasy, and EMIT(®) II Plus Amphetamines) were determined. Two- and three-dimensional molecular similarity and modeling approaches were evaluated for the ability to predict cross-reactivity using receiver-operator characteristic curve analysis. RESULTS: Overall, 34%-46% of the drugs tested positive on the immunoassay screens using a concentration of 20,000 ng/mL. The three immunoassays showed differential detection of the various classes of amphetamine-like drugs. Only the CEDIA assay detected piperazines well, while only the EMIT assay cross-reacted with the 2C class. All three immunoassays detected 4-substituted amphetamines. For the AxSYM and EMIT assays, two-dimensional molecular similarity methods that combined similarity to amphetamine/methamphetamine and 3,4-methylenedioxymethampetamine most accurately predicted cross-reactivity. For the CEDIA assay, three-dimensional pharmacophore methods performed best in predicting cross-reactivity. Using the best performing models, cross-reactivities of an additional 261 amphetamine-like compounds were predicted. CONCLUSIONS: Existing amphetamines immunoassays unevenly detect amphetamine-like drugs, particularly in the 2C, piperazine, and ß-keto classes. Computational similarity methods perform well in predicting cross-reactivity and can help prioritize testing of additional compounds in the future.

Cross-reactivities of various phenethylamine-type designer drugs to immunoassays for amphetamines, with special attention to the evaluation of the one-step urine drug test Instant-View™, and the Emit® assays for use in drug enforcement.

Cross-reactivities of 76 kinds of phenethylamine-type designer drugs and related compounds to the urine drug tests Instant-View ™ (IV) (the Methamphetamine (MA) test, the Amphetamine 300 test, and the MDMA test) have been investigated. An on-site urine test kit consisting of these three IV tests has been evaluated for the on-site screening of MA users, and the kit has been found to have satisfactory specificity for drug enforcement purposes by separately detecting both MA and its metabolite amphetamine. The cross-reactivity profiles of Emit(®) II Plus Amphetamines Assay, Emit(®) II Plus Ecstasy assay, and Emit(®) d.a.u.(®) Amphetamine Class assay have also been investigated and discussed.

Roche DAT immunoassay: sensitivity and specificity testing for amphetamines, cocaine, and opiates in oral fluid.

Laboratory testing of oral fluid for drugs of abuse continues to expand in the workplace, legal, treatment, and health settings. In this study, we assessed recently developed homogeneous Roche DAT screening assays for amphetamines, cocaine metabolite [benzoylecgonine (BZE)], methamphetamines, and opiates in oral fluid. Precision and accuracy were assessed using control samples at +/-25% of cutoff. Sensitivity, specificity, and agreement compared to liquid chromatography-tandem mass spectrometry (LC-MS-MS) was assessed by analysis of oral fluid specimens collected from 994 subjects enrolled in a drug treatment or probation and parole drug-testing program. An additional 180 research specimens from Kroll Laboratories were analyzed for amphetamine and methamphetamine. Screening cutoff concentrations (ng/mL) were as follows: amphetamines, 40; cocaine metabolite, 3; methamphetamines, 40; and opiates, 10. LC-MS-MS analyses were performed with the following cutoff concentrations (ng/mL): amphetamine, 40; BZE, 2.0; methamphetamine, 40; and codeine or morphine, 10. The percent coefficient of variation ranged from 3.4% to 7.3%. Sensitivity and specificity of the Roche DAT assays compared to LC-MS-MS were > 94%, and agreement was > 96% for the four assays. The performance of the Roche DAT assays suggests these new homogeneous screening assays will be an attractive alternative to existing more labor-intensive enzyme immunoassays.

Matrix effect and cross-reactivity of select amphetamine-type substances, designer analogues, and putrefactive amines using the Bio-Quant direct ELISA presumptive assays for amphetamine and methamphetamine.

The aim of this study was to evaluate the Bio-Quant Direct ELISA assays for amphetamine and methamphetamine in the routine presumptive screening of biological fluids. Standard concentration curves of the target analytes were assayed to assess sensitivity, and known concentrations of common amphetamine-type substances (ephedrine, pseudoephedrine, phentermine), designer analogues (MDA, MDMA, MDEA, MBDB, PMA, 4-MTA, 2CB), and putrefactive amines (phenylethylamine, putrescine, tryptamine, tyramine) were analyzed to determine cross-reactivity. Results of the standard curve studies show the capacity of both Direct ELISA kits to confidently detect down to 3 ng/mL interday (PBS matrix; CVs 6.3-15.5%). Cross-reactivity relative to that of 50 ng/mL preparations of the target compounds demonstrated that the Direct ELISA kit for amphetamine also detected MDA (282%), PMA (265%), 4-MTA (280%), and phentermine (61%), and the Direct ELISA for methamphetamine also assayed positive for MDMA (73%), MDEA (18%), pseudoephedrine (19%), MBDB (8%), and ephedrine (9%). Matrix studies demonstrated that both ELISA kits could be applied to screening of blood, urine, and saliva to a concentration of 6 ng/mL or lower. In conclusion, the Bio-Quant Direct ELISA kits for amphetamine and methamphetamine are fast and accurate and have demonstrated themselves to be useful tools in routine toxicological testing.

Fluorescence polarization immunoassay detection of amphetamine, methamphetamine, and illicit amphetamine analogues.

The Abbott Diagnostics Amphetamine/Methamphetamine II and Amphetamine Class reagents were evaluated on the Abbott TDx for cross-reactivity to amphetamine and methamphetamine stereoisomers, several of their metabolites, and various illicit analogues, including 2-methoxyamphetamine, 4-hydroxymethamphetamine, 2,5-dimethoxy-amphetamine (DMA), 4-bromo-2,5-dimethoxyamphetamine (DOB), 4-bromo-2,5-dimethoxy-beta-phenethylamine (BDMPEA), 3,4,5-trimethoxyamphetamine (TMA), 3,4-methylenedioxy-amphetamine (MDA) N,N-dimethyl-3,4-methylenedioxy-amphetamine, N-hydroxy-3,4-methylenedioxyamphetamine (N-OH MDA), 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyethylamphetamine (MDEA), 2,5-dimethoxy-4-ethylamphetamine (DOE), 2,5-dimethoxy-4-methylamphetamine (DOM), and mescaline in concentrations ranging from 100 to 100,000 ng/mL. Results demonstrate the utility of this assay for detection of several of the above compounds; unfortunately many are still not detectable. Significant differences were observed between the Amphetamine/Methamphetamine II and Amphetamine Class reagents, particularly regarding their cross-reactivity to over-the-counter medications. Detection of the drugs amphetamine, methamphetamine, and the illicit analogues is not enhanced with the Amphetamine Class reagents, and unless detection of the over-the-counter compounds is of interest, these reagents are a poor choice compared to the Amphetamine/Methamphetamine II reagents. Cross-reactivity of some of the illicit analogues is such that the assay can reliably be used for the routine screening of these compounds.

Immunoassays of amphetamines: immunogen structure vs antibody specificity.

Various immunoassays have been developed for the detection of amphetamines. These have varying degrees of cross-reactivity to other drug and food components. Information on the immunogen structures used, and the specificities of the antibodies obtained, have allowed formulation of a "structure-specificity" pattern delineated on the basis of immunochemistry and stereochemistry. The 'structure-specificity' relationship should be useful to future developments of these immunoassays. Specifically, immunoassays intended to detect either amphetamine or methamphetamine with minimal cross-reaction, should employ immunogens with amphetamine (or methamphetamine) derivatized via the para position of the phenyl ring. Such assays should show minimal cross-reaction with other secondary (or tertiary) amines but should strongly cross-react with phenyl ring substituted analogs. On the other hand, assays intended for detection of both amphetamine and methamphetamine should employ amphetamine (rather than methamphetamine) derivatized via its amino group as an immunogen. Such assays should show minimal cross-reaction with other tertiary amines and phenyl-substituted amphetamine/methamphetamine.

The effect of fluorescein labels on the affinity of antisera to small haptens.

Small haptens such as methylamphetamine (MW 149) cannot, on their own, induce an immune response. It is also unlikely that they fill the binding site of any antibody that recognises them. Under such circumstances any attached label might be expected to enter the area of the binding site and exert an influence on overall binding. To investigate the possible influence of the label on binding, a range of fluorescein-labelled derivatives, differing in bridge length, were prepared. Antiserum binding of these labelled derivatives was then compared to that of the unlabelled drug. Evidence is presented which suggests that, with small haptens, the closeness of the fluorescein molecule can markedly influence antibody binding. Significant differences were found in titre, sensitivity, and assay kinetics. These overall effects appear to be brought about by the change in affinity of the antibody for the labelled hapten.

An improved label for amphetamine fluoroimmunoassay.

This paper describes the synthesis of amphetamine derivatives, functionalized at the para-position of the phenyl ring of the drug. These derivatives were used to prepare fluorescein-labelled amphetamine to replace the label in a previously described polarisation fluoroimmunoassay that is highly specific for amphetamine. The original label was obtained using material donated by industrial sources and therefore not generally available. When the new label was used in the immunoassay, specificity was similar but the sensitivity was improved by a factor of four. This increase in sensitivity is explained by elimination of bridge-binding effects.

Amphetamine radioimmunoassay: chemical tuning of specificity.

Four [125I]iodine labelled amphetamine derivatives were synthesised. Three of these proved useful in the development of radioimmunoassays for amphetamine. The assays, all using the same antiserum, had widely differing specificities for compounds closely related to amphetamine. Thus, chemical modification of the radiotracer was used to tune radioimmunoassay specificity without recourse to the production of different antisera.

Amphetamine EMIT--structure versus reactivity.

Over 60 different amphetamine-related amines were analyzed by EMIT (Enzyme Multiplied Immunoassay Technique) at several different concentrations. An attempt is made to relate structural differences to the affinity of the compounds for the EMIT amphetamine antibody. The effects of substituent placement on the amphetamine molecule are studied. Addition of a single alkyl group to the nitrogen of amphetamine increased reactivity, whereas all other structural changes had the reverse effect, decreasing reactivity.

Specificity of an antibody directed against d-methamphetamine. Studies with rigid and nonrigid analogs.

The specificity of an antibody directed against d-(S)-methamphetamine (MA) was determined by competitive binding assay with more than 50 compounds-metabolites, homologs, and analogs of amphetamine. The antibody appears to be specific both for the side chain and the aromatic ring of d-(S)-amphetamine (A). The basic requirements for a compound to be bound to the antibody are (a) an aromatic ring, (b) a basic nitrogen, and (c) a two-carbon chain between the aromatic ring and the nitrogen. A transoid conformation for the phenethylamine skeleton is preferred. The interaction of the antibody with compounds differing from MA or A in side-chain substitutions was directly proportional to the closeness of their structure to MA and/or A. The antibody exhibited greatly reduced affinity for ring-substituted analogs of A; the p-hydroxy metabolite of A did not bind to the antibody. A radioimmunoassay of A is described; it was utilized to study the disposition of A in dogs.