Cross-reactivity of designer drugs, including cathinone derivatives, in commercial enzyme-linked immunosorbent assays.

Since the introduction of synthetic heroin, designer drugs have been increasing in prevalence in the United States drug market over the past few decades. Recently, 'legal highs' sold as 'bath salts' have become a household term for one such class of designer drugs. While a number of federal and state bans have been enacted, the abuse of these designer drugs still continues. Few assays have been developed for the comprehensive detection of such compounds, so it is important to investigate how they may or may not react in presumptive screens, i.e. pre-existing commercial immunoassays. In this experiment, 16 different ELISA reagents were evaluated to determine the cross-reactivity of 30 designer drugs, including 24 phenylethylamines (including 8 cathinone derivatives), 3 piperazines, and 3 tryptamines. Cross-reactivity towards most drugs was <4% in assays targeting amphetamine or methamphetamine. Compounds such as MDA, MDMA, ethylamphetamine, and α-methyltryptamine demonstrated cross-reactivities in the range of 30-250%, but data were consistent with both manufacturer's inserts and published literature. When tested against the Randox Mephedrone/Methcathinone kit, cathinone derivatives demonstrated cross-reactivity at concentrations as low as 150 ng/ml. Since this same reagent did not cross-react with other amphetamine-like compounds, it opens the possibility to screen post-mortem specimens without the interference of putrefactive amines. All other assays demonstrated essentially no cross-reactivity towards any of the analytes evaluated. Given these results, a great need exists for more broad-range screening techniques to be applied when analyzing biological specimens by immunoassays for drugs of abuse, specifically the more recent designer drugs.

Evaluating the relationship between postmortem and antemortem morphine and codeine concentrations in whole blood.

The purpose of this study was to examine the relationship between antemortem (AM) and postmortem (PM) morphine and codeine concentrations in whole blood. In addition, the effects of antemortem to death interval as well as the postmortem interval were considered during the interpretive process. The cases of seven human subjects are presented here with an average postmortem interval of 28 h (13.5-48 h) and an average antemortem to death interval of 97 min (ranging from 9 to 300 min). Drug concentrations were obtained from AM blood collected from local hospitals in Miami, FL, and postmortem blood was obtained from the Miami-Dade County Medical Examiner Department in Miami, FL. The results obtained for this study indicated that factors such as metabolism and postmortem interval can affect postmortem drug concentrations in an unpredictable manner. Four out of seven morphine cases appeared to be affected by postmortem redistribution, and five of seven codeine and two of two 6-monoacetylmorphine cases were affected as well.

Brain biomarkers for identifying excited delirium as a cause of sudden death.

Excited delirium (ED) syndrome is a serious medical condition associated with acute onset of agitated violent behavior that often culminates in a sudden unexplained death. While the contribution of restraint, struggle and the use of conductive energy devices (CED) to the cause and manner of death raise controversy, a CNS dysfunction of dopamine signaling may underlie the delirium and fatal autonomic dysfunction. We conducted a mortality review for a case series of ninety excited delirium deaths and present results on the association of a 2-protein biomarker signature. We conducted quantitative analyses of the dopamine transporter and heat shock protein 70 validated for specificity and degree of interindividual variation. Incident circumstances, force measures, autopsy and toxicology results were determined for all subjects. A majority of the victims in this case series tested positive for cocaine in blood and brain, although four had no licit or illicit drugs or alcohol measured at autopsy. Mean core body temperature where recorded was 40.7 degrees C. The expression of the heat shock protein HSPA1B transcript was elevated 1.8-4-fold in postmortem brain. The elevation of Hsp70 in autopsy brain specimens confirms that hyperthermia is an associated symptom and often a harbinger of death in these cases. Dopamine transporter levels were below the range of values measured in age-matched controls, providing pathologic evidence for increased risk of chaotic dopamine signaling in excited delirium. When combined with descriptions of the decedents' behavior prior to death, a 2-protein biomarker signature can serve as a reliable forensic tool for identifying the excited delirium syndrome at autopsy.

Fatality due to acute alpha-methyltryptamine intoxication.

In February 2003, the Miami-Dade County Medical Examiner Department reported the first known death in the country related to alpha-methyltryptamine (AMT). AMT is an indole analogue of amphetamine investigated in the 1960s as an antidepressant, stimulant, and monoamine oxidase inhibitor. Today, AMT is recognized as a powerful psychedelic drug among high school and college-aged men and women. Its popularity is partly due to the multitude of anecdotal websites discussing AMT as well as its legality and availability for purchase via the Internet prior to April 2003. Emergency designation of AMT as a Schedule 1 controlled substance by the Drug Enforcement Administration occurred shortly after the death in Miami-Dade County. The case in Miami involved a young college student who, prior to death, advised his roommate that he was "taking hallucinating drugs" and as a result had "discovered the secret of the universe". Approximately 12 h later, the roommate discovered the deceased lying in bed unresponsive. An empty 1-g vial of AMT was recovered from the scene and sent to the toxicology laboratory. Initial screening of urine by enzyme-multiplied immunoassay technique was positive for amphetamines, and the basic drug blood screen detected a small peak later identified by mass spectrometry as AMT. For quantitation, AMT was isolated using solid-phase extraction, derivatized with pentafluoropropionic anhydride, and analyzed using gas chromatography-mass spectrometry. Quantitative analysis was based upon m/z 276, 303, and 466 for AMT and m/z 306, 333, and 496 for the internal standard, 5-methoxy-alpha-methyltryptamine. A linear calibration curve from 50 to 500 ng/mL was used to calculate the concentration of AMT in the samples and controls. Blood, tissue, and gastric specimens were diluted to bring the observed concentration within the limits of the standard curve. Matrix matched controls were extracted and analyzed with each run. Postmortem iliac vein blood revealed 2.0 mg/L, gastric contents (48 g collected at autopsy) contained 9.6 mg total of AMT, liver contained 24.7 mg/kg, and the brain contained 7.8 mg/kg. An additional Medical Examiner case from another jurisdiction revealed 1.5 mg/L in antemortem serum.

Stability of ethanol in postmortem blood and vitreous humor in long-term refrigerated storage.

Ethanol concentrations in postmortem blood and vitreous humor samples collected at the Miami-Dade Medical Examiner Department over 5-6 years ago were reexamined to assess whether vitreous humor is a more reliable specimen for the analysis of ethanol in samples stored long term. The average change in 50-mL polypropylene tubes containing blood was 0.06 gm/dL (35% loss). On the other hand, vitreous humor samples collected in 10-mL gray-top Vacutainer tubes yielded an average change of 0.01 gm/dL (6.1% loss). This study demonstrates that vitreous humor may be a reliable matrix for ethanol analysis following prolonged refrigerated storage of the samples.

Fatal cold medication intoxication in an infant.

The case history and toxicological findings of an infant fatality involving pseudoephedrine, brompheniramine, and dextromethorphan are presented. Concentrations of brompheniramine and dextromethorphan were measured in both postmortem blood and liver specimens using a gas chromatograph equipped with a nitrogen-phosphorus detector. Brompheniramine and dextromethorphan were 0.40 mg/L and 0.50 mg/L, respectively, in the blood sample and 0.16 mg/kg and 0.57 mg/kg in the liver sample. The concentration of pseudoephedrine in blood and liver specimens was measured using gas chromatography-mass spectrometry and was determined to be 14.4 mg/L in the blood and 16 mg/kg in the liver. Additionally, a baby bottle allegedly administered to the infant was collected as evidence and sent to the Medical Examiner's Office for evaluation. The amounts of total brompheniramine, dextromethorphan, and pseudoephedrine remaining in the baby bottle were 1.4 mg, 9.4 mg, and 40 mg, respectively.

Dopamine transport function is elevated in cocaine users.

Dopaminergic transmission has been suggested to be a primary mechanism mediating reinforcement, withdrawal and craving associated with psychostimulant addiction. Pyscho-stimulants attenuate dopamine transporter (DAT) clearance efficiency, resulting in a net increase in synaptic dopamine levels. Re-uptake rate is determined by the number of functional DAT molecules at the membrane surface. Previous in vivo imaging studies in humans and in vitro studies in post-mortem human brain have demonstrated that chronic cocaine abuse results in a neuroadaptive increase in DAT-binding site density in the limbic striatum. Whether this increase in DAT availability represents an increase in the functional activity of the transporter is unknown. Here, we present evidence that DAT function is elevated by chronic cocaine abuse. The effect of increasing post-mortem interval on the functional viability of synaptosomes was modeled in the baboon brain. Baboon brains sampled under conditions similar to human brain autopsies yielded synaptosomal preparations that were viable up to 24 h post-mortem. Dopamine (DA) uptake was elevated twofold in the ventral striatum from cocaine users as compared to age-matched drug-free control subjects. The levels of [3H]DA uptake were not elevated in victims of excited cocaine delirium, who experienced paranoia and marked agitation prior to death. In keeping with the increase in DAT function, [3H]WIN 35,428 binding was increased in the cocaine users, but not in the victims of excited delirium. These results demonstrate that DA uptake function assayed in cryopreserved human brain synaptosomes is a suitable approach for testing hypotheses of the mechanisms underlying human brain disorders and for studying the actions of addictive drugs in man.

Medication Development of Ibogaine as a Pharmacotherapy for Drug Dependencea.

The potential for deriving new psychotherapeutic medications from natural sources has led to renewed interest in rain forest plants as a source of lead compounds for the development of antiaddiction medications. Ibogaine is an indole alkaloid found in the roots of Tabernanthe iboga (Apocynaceae family), a rain forest shrub that is native to equatorial Africa. Ibogaine is used by indigenous peoples in low doses to combat fatigue, hunger and in higher doses as a sacrament in religious rituals. Members of American and European addict self-help groups have claimed that ibogaine promotes long-term drug abstinence from addictive substances, including psychostimulants and cocaine. Anecdotal reports attest that a single dose of ibogaine eliminates withdrawal symptoms and reduces drug cravings for extended periods of time. The purported antiaddictive properties of ibogaine require rigorous validation in humans. We have initiated a rising tolerance study using single administration to assess the safety of ibogaine for the treatment of cocaine dependency. The primary objectives of the study are to determine safety, pharmacokinetics and dose effects, and to identify relevant parameters of efficacy in cocaine-dependent patients. Pharmacokinetic and pharmacodynamic characteristics of ibogaine in humans are assessed by analyzing the concentration-time data of ibogaine and its desmethyl metabolite (noribogaine) from the Phase I trial, and by conducting in vitro experiments to elucidate the specific disposition processes involved in the metabolism of both parent drug and metabolite. The development of clinical safety studies of ibogaine in humans will help to determine whether there is a rationale for conducting efficacy trials in the future.