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.

A Retrospective Analysis of Urine Drugs of Abuse Immunoassay True Positive Rates at a National Reference Laboratory.

Urine drug screens are commonly performed to identify drug use or monitor adherence to drug therapy. The purpose of this retrospective study was to evaluate the true positive and false positive rates of one of our in-house urine drug screen panels. The urine drugs of abuse panel studied consists of screening by immunoassay then positive immunoassay results were confirmed by mass spectrometry. Reagents from Syva and Microgenics were used for the immunoassay screen. The screen was performed on a Beckman AU5810 random access automated clinical analyzer. The percent of true positives for each immunoassay was determined. Agreement with previously validated GC-MS or LC-MS-MS confirmatory methods was also evaluated. There were 8,825 de-identified screening results for each of the drugs in the panel, except for alcohol (N = 2,296). The percent of samples that screened positive were: 10.0% for amphetamine/methamphetamine/3,4-methylenedioxy-methamphetamine (MDMA), 12.8% for benzodiazepines, 43.7% for opiates (including oxycodone) and 20.3% for tetrahydrocannabinol (THC). The false positive rate for amphetamine/methamphetamine was ∼14%, ∼34% for opiates (excluding oxycodone), 25% for propoxyphene and 100% for phencyclidine and MDMA immunoassays. Based on the results from this retrospective study, the true positive rate for THC drug use among adults were similar to the rate of illicit drug use in young adults from the 2013 National Survey; however, our positivity rate for cocaine was higher than the National Survey.

Decrease of lymphoproliferative response by amphetamine is mediated by dopamine from the nucleus accumbens: influence on splenic met-enkephalin levels.

Despite the mesocorticolimbic dopaminergic pathway being one of the main substrates underlying stimulating and reinforcing effects induced by psychostimulant drugs, there is little information regarding its role in their effects at the immune level. We have previously demonstrated that acute exposure to amphetamine (5 mg/kg, i.p.) induced an inhibitory effect on the splenic T-cell proliferative response, along with an increase in the methionine(met)-enkephalin content at limbic and immune levels, 4 days after drug administration. In this study, we investigated if a possible dopamine mechanism underlies these amphetamine-induced effects by administering D1 and D2 dopaminergic antagonists or a dopaminergic terminal neurotoxin before the drug. Pre-treatment with either SCH-23390 (0.1 mg/kg, i.p.) or raclopride (0.1 mg/kg, i.p.), a D1 or D2 dopaminergic receptor antagonist, respectively, abrogated the effects of amphetamine on the lymphoproliferative response and on met-enkephalin levels of the spleen. The amphetamine-induced increase in limbic met-enkephalin content was suppressed by SCH-23390 but not by raclopride pre-treatment. Finally, an intra-accumbens 6-hydroxy-dopamine injection administered 2 weeks previously prevented amphetamine-induced effects on the lymphoproliferative response and on met-enkephalin levels in the prefrontal cortex and spleen. These findings strongly suggest that D1 and D2 dopaminergic receptors are involved in amphetamine-induced effects at immune level as regards the lymphoproliferative response and the changes in spleen met-enkephalin content, whereas limbic met-enkephalin levels were modulated only by the D1 dopaminergic receptors. In addition, this study showed that a mesolimbic component modulated amphetamine-induced effects on the immune response, as previously shown at a behavioral level.

Insights into scFv:drug binding using the molecular dynamics simulation and free energy calculation.

Molecular dynamics simulations and free energy calculation have been performed to study how the single-chain variable fragment (scFv) binds methamphetamine (METH) and amphetamine (AMP). The structures of the scFv:METH and the scFv:AMP complexes are analyzed by examining the time-dependence of their RMSDs, by analyzing the distance between some key atoms of the selected residues, and by comparing the averaged structures with their corresponding crystallographic structures. It is observed that binding an AMP to the scFv does not cause significant changes to the binding pocket of the scFv:ligand complex. The binding free energy of scFv:AMP without introducing an extra water into the binding pocket is much stronger than scFv:METH. This is against the first of the two scenarios postulated in the experimental work of Celikel et al. (Protein Science 18, 2336 (2009)). However, adding a water to the AMP (at the position of the methyl group of METH), the binding free energy of the scFv:AMP-H2O complex, is found to be significantly weaker than scFv:METH. This is consistent with the second of the two scenarios given by Celikel et al. Decomposition of the binding energy into ligand-residue pair interactions shows that two residues (Tyr175 and Tyr177) have nearly-zero interactions with AMP in the scFv:AMP-H2O complex, whereas their interactions with METH in the scFv:METH complex are as large as -0.8 and -0.74 kcal mol(-1). The insights gained from this study may be helpful in designing more potent antibodies in treating METH abuse.

In silico experiments of single-chain antibody fragment against drugs of abuse.

Three sets of in silico experiments have been conducted to elucidate the binding mechanics of two drugs, (+)-methamphetamine (METH) and amphetamine (AMP) to the single-chain variable fragment (scFv) recently engineered from anti-METH monoclonal antibody mAb6H4 (IgG, κlight chain, K(d)=11nM). The first set of in silico experiments are long time equilibration runs of scFv:drug complexes and of drug-free scFv both in the solution. They demonstrate how the solution structures of scFv deviate from its crystallographic form with or without drug molecules bound to it. They lead to the prediction that the Arrhenius activation barrier is nearly zero for transitions from the dissociated state to the bound state. The second set of in silico experiments are nonequilibrium dynamics of pulling the drug molecules out of the binding pocket of scFv and the equilibration runs for drugs to fall back into the binding pocket. They demonstrate that extra water molecules (in addition to the two crystallographic waters) exist inside the binding pocket, underneath the drug molecules. These extra waters must have been evaporated from the binding pockets during the crystallization process of the in vitro experiments of structural determination. The third set of in silico experiments are nonequilibrium steered molecular dynamics simulations to determine the absolute binding free energies of METH and AMP to scFv. The center of mass of a drug molecule (METH or AMP) is steered (pulled) towards (forward) and away from (reverse) the binding site, sampling forward and reverse pulling paths. Mechanic work is measured along the pulling paths. The work measurements are averaged through the Brownian dynamics fluctuation dissipation theorem to produce the free-energy profiles of the scFv:drug complexes as a function of the drug-scFv separation. These experiments lead to the theoretical prediction of absolute binding energies of METH and AMP that are in agreement with the in vitro experimental results.

Efectos hematológicos, inmunológicos y neuroquímicos del tratamiento crónico con anfetamina en la rata macho / Hematological, immunological and neurochemical effects of chronic amphetamine treatment in male rats

In the present study, we have analyzed the effect of chronic amphetamine sulfate(AMPH) treatment on haematological, immunological and neurochemical parametersin the male rat. AMPH increased the total peripheral leukocyte count, andaltered its differential counts, decreasing lymphocytes and increasing neutrophils.Flow cytometry study showed that the decline in circulating lymphocytes wascaused by the loss of a particular lymphocyte subset, B-cell, which reduced both inpercentage and in absolute number by 50%. T-cell population increased by 15% butnot in absolute number, however there was no difference in either CD4+ or CD8+ Tlymphocyte subsets between experimental groups. Neurochemically, AMPHreduced norepinephrine (NE) and serotonin (5-HT) contents in the hypothalamusand increased dopamine (DA) content in the striatum. Chronic AMPH increased ina dose-dependent manner serum corticosterone levels, had no effect on circulatingcatecholamines, reduced adrenal weights, and did not affect spleen weights althoughreduced their cellularities. These results show that chronic AMPH have importanteffects on immune function, particularly on humoral immune response because itreduced the circulating B cell population by half. In addition, AMPH plays animportant role in the redistribution and trafficking of leukocytes, and both effectsseem to be mediated by sympathetic innervation of the lymphoid organs(AU)

En este estudio analizamos el efecto del tratamientocrónico con sulfato de anfetamina(AMPH) administrada a través de sonda gástrica,sobre parámetros hematológicos, inmunológicosy neuroquímicos de la rata macho. LaAMPH incrementó el numero total de leucocitosperiféricos y alteró su formula diferencial,disminuyendo los linfocitos e incrementandolos neutrófilos. El estudio de Citometría deFlujo mostró que el descenso linfocítico afectófundamentalmente a la población de células B,que se redujo en un 50% tanto en porcentajecomo en valores absolutos. La población decélulas T se incrementó en un 15%, pero nohubo diferencias entre las subpoblacionesTCD4+ o TCD8+. La AMPH redujo los contenidosde norepinefrina y serotonina en elhipotálamo e incrementó el contenido dedopamina en el estriado. Además elevó enforma proporcional a la dosis, los niveles plasmáticosde corticosterona, pero no tuvo efectosobre las catecolaminas circulantes. También,el tratamiento crónico con AMPH redujolos pesos de las adrenales, no modificó el delos bazos aunque sí redujo su celularidad. Losresultados muestran que la anfetamina tieneimportantes efectos sobre el sistema inmuneparticularmente sobre la respuesta inmunehumoral , ya que reduce a la mitad la poblaciónde células B, y sobre la redistribución y tráficolinfocitarios, y que estos efectos parecen estarmediados por la inervación simpática de losórganos linfoides(AU)

Effects of amphetamine on NK-related cytotoxicity in rats differing in locomotor reactivity and social position.

The effect of i.p. administration of 1mg/kg of amphetamine (AMPH) on natural killer cells cytotoxicity (NKCC) and number of large granular lymphocytes (LGL-NK) together with plasma corticosterone (CORT) level and WBC was evaluated in male Wistar rats differing in two behavioral features: locomotor reactivity to novelty (high, HR and low, LR responders) and social position (dominants, D and subordinates, S). In the majority of animals AMPH evoked (30 min after administration) an increase in NKCC and LGL (NK) number accompanied by lymphopenia, neutrocytosis, monocytosis, and an increase in CORT level. Changes in NKCC (LU20) showed substantial individual variability: in HR group approximately 513Delta%, p <0.01 (relative to the control); LR group approximately 56Delta%, p >.05; D group approximately 441Delta%, p >0.001; S group approximately 216Delta%, p >0.05; HR/D group approximately 643Delta%, p <.001; HR/S group approximately 414Delta%, p <.001; LR/D group approximately 191Delta%, p >.05; and LR/S group approximately -19Delta%, p .05. The increase in CORT level, lymphopenia, and neutrocytosis indicated a stress-like reaction to AMPH. No significant correlation between NKCC and CORT level was found. The results obtained indicate that AMPH can evoke an increase in NK-related cytotoxic activity quantitatively related to high behavioral reactivity to novelty and social dominance, however NKCC is not related to the AMPH-induced CORT changes.

Choice of an ELISA assay for screening postmortem blood for amphetamine and/or methamphetamine.

The object of this study was to evaluate the suitability of the Neogen Corp. microtiter plate enzyme-linked immunoassays (ELISA) for the screening of postmortem blood for amphetamine and methamphetamine and to choose the more appropriate assay for screening. Forty-seven postmortem whole blood specimens were obtained from drug-involved deaths, which had been screened and confirmed positive for methamphetamine and/or amphetamine. Eighty-five negative specimens were obtained from non-amphetamines-involved deaths, 17 of which involved decomposition. Specimens were tested using the Neogen Amphetamine Ultra and Neogen Methamphetamine/MDMA microtiter plate ELISA assays. No matrix effects were found for whole blood in these assays, and a dilution of 1:5 was chosen to facilitate pipetting and to bring the IC50 of the microtiter plate ELISA assay within the range of amphetamines concentrations encountered in medical examiner specimens. True positives, true negatives, false positives, and false negatives were determined relative to gas chromatography-mass spectrometry (GC-MS) and graphed for the ELISA. From these graphs and the receiver operating curves (ROC), the optimal cut-off for the Neogen Methamphetamine/MDMA ELISA was 50 ng/mL methamphetamine equivalents and the optimum cut-off for the Neogen Amphetamine Ultra ELISA was 100 ng/mL amphetamine equivalents. The Neogen Methamphetamine ELISA had a sensitivity of 93.6% +/- 3.5% and a specificity of 77.6% +/- 4.5% versus GC-MS at the cut-off of 50-ng/mL methamphetamine equivalents. The Neogen Amphetamine Ultra ELISA had a sensitivity of 95.7% +/- 3.0% and a specificity of 72.9% +/- 5.2% versus GC-MS at the 100-ng/mL amphetamine equivalents cut-off. The areas under the ROCs were equivalent for the two ELISA assays.

The effect of amphetamine sensitization on mouse immunoreactivity.

Recent studies indicate a role of the immune system in the behavioral effects of amphetamine in rodents. In the present study we attempted to find a connection between the behavioral changes induced by repeated, intermittent administration of amphetamine and some immunological consequences of sensitization to amphetamine in mice. Male Albino Swiss mice were treated repeatedly (for 5 days) with amphetamine (1 mg/kg, i.p.). On day 9, they received a challenge dose of amphetamine (1 mg/kg). Acute administration of amphetamine increased their locomotor activity by ca. 40%. In animals treated repeatedly with amphetamine, the challenge dose of the psychostimulant induced behavioral sensitization, i.e. the higher locomotor activation as compared with that after its first administration to mice. Immune functions were evaluated by the ability of splenocytes to proliferate and to produce cytokines such as interferon gamma (IFN-gamma), interleukin (IL)-4 and IL-10. Acute amphetamine administration significantly decreased, by ca. 30% and 25%, the proliferation of splenocytes in response to an optimal and a suboptimal dose of concanavalin A (Con A), respectively, and increased their ability to produce IL-4. Chronic intermittent treatment with amphetamine significantly decreased, by ca. 65% and 50%, the proliferative response of T cells to an optimal and a suboptimal dose of Con A, respectively, and diminished by 20% the metabolic activity of splenocytes. The above data showed that both acute and chronic amphetamine administration diminished some aspects of the cell-mediated immunity; nevertheless, immunosuppression was particularly evident in amphetamine-sensitized mice. Our findings seem to indicate possible importance of monitoring and correcting immune changes in the therapy of amphetamine addiction.

Evaluation of six commercial amphetamine and methamphetamine immunoassays for cross-reactivity to phenylpropanolamine and ephedrine in urine.

We evaluated six commercially available amphetamine (A) and methamphetamine (MA) immunoassays for their relative cross-reactivities to isomers of phenylpropanolamine (PPA) and ephedrine (E) in urine: Syva EMIT, Abbott fluorescence polarization (FPIA), Roche, and Diagnostic Products Corporation (DPC) radioimmunoassays for A and MA. Two stereoisomers of PPA and four stereoisomers of E were tested using (1) drug-free urine spiked at 1,000 mg/L or 100 mg/L of each compound and (2) 60 clinical urine specimens not containing A or MA but having varying amounts of PPA and/or E. Specimens responding greater than the 1-mg/L A or MA cutoff were defined as positive. All specimens spiked at 100 mg/L were negative by all immunoassays. All specimens spiked at 1,000 mg/L were positive by EMIT and negative by FPIA, Roche A, and DPC A; 1,000 mg/L/-E and d-pseudoephedrine were also positive by Roche MA and DPC MA. Three of the 60 clinical specimens tested positive by EMIT and one specimen tested positive by DPC A and DPC MA.