Studies on the toxicological detection of the designer drug 4-bromo-2,5-dimethoxy-beta-phenethylamine (2C-B) in rat urine using gas chromatography-mass spectrometry

The phenethylamine-derived designer drug 4-bromo-2,5-dimethoxy-beta-phenethylamine (2C-B) is known to be extensively metabolized in various species including humans. In rat urine, 2C-B was found to be excreted mainly via its metabolites. In the current study, the toxicological detection of these metabolites in the authors' systematic toxicological analysis (STA) procedure was examined. The STA procedure using full-scan GC-MS allowed proving an intake of a common drug abusers' dose of 2C-B by detection of the O-demethyl deaminohydroxy and two isomers of the O-demethyl metabolites in rat urine. Assuming similar metabolism, the described STA procedure should be suitable for proof of an intake of 2C-B in human urine.     

Proof of a 1-(3-chlorophenyl)piperazine (mCPP) intake: use as adulterant of cocaine resulting in drug-drug interactions?

Since 2005, increasing numbers of seizures of the designer drug of abuse 1-(3-chlorophenyl)piperazine (mCPP) have been reported. This paper describes the unequivocal proof of a mCPP intake. Differentiation from the intake of its precursor drugs trazodone and nefazodone was performed by a systematic toxicological analysis (STA) procedure using full-scan GC-MS after acid hydrolysis, liquid-liquid extraction and microwave-assisted acetylation. The found mCPP/hydroxy-mCPP ratio indicated altered metabolism of this cytochrome (CYP) 2D6 catalyzed reaction compared to published studies using the same procedure. Although this might be ascribed to a poor metabolizer (PM) phenotype, genotyping revealed no PM genotype but indications for an intermediate metabolizer genotype. However, a PM phenotype could also be caused by drug-drug interactions with CYP2D6 inhibitors or substrates such as the co-consumed cocaine and diltiazem and/or diltiazem metabolites, respectively. In conclusion, the presented data indicate a possible relevance of CYP2D6 polymorphism and/or drug interactions to mCPP toxicokinetics, which is important for risk assessment of this new designer drug of abuse, in particular if it is used as adulterant of CYP2D6 substrates such as cocaine.     

Hair testing for drugs of abuse

Hair testing for drugs of abuse is a developing technology, which offers the possibility of longer detection times than is commonly obtained with urine analysis. It is the main method for evaluation of an individual's drugs of abuse history. In many countries hair analysis is routinely used to detect drug abuse in forensic cases, occupational and traffic medicine and clinical toxicology. Hair analysis in pregnant women, neonates and infants is a useful tool for the detection of drug exposure in utero. In Croatia hair testing for drugs of abuse is performed at the Institute for Medical Research and Occupational Health. Three-year experience in drugs of abuse analysis in hair is described. In 331 hair samples (270 from adolescents and 61 from adults) opiates and metabolites, cocaine, methadone, and amphetamines were analyzed by gas chromatography/mass spectrometry. Most prevalent drugs of abuse in adolescents were amphetamines, and in adults heroin. From the examples cited and samples analyzed it is evident that hair testing is emerging as a reliable biological marker for cumulative account of individual exposure to drugs of abuse.     

Sex differences and ovarian hormones in animal models of drug dependence

Increasing evidence indicates the presence of sex differences in many aspects of drug abuse. Most studies reveal that females exceed males during the initiation, escalation, extinction, and reinstatement (relapse) of drug-seeking behavior, but males are more sensitive than females to the aversive effects of drugs such as drug withdrawal. Findings from human and animal research indicate that circulating levels of ovarian steroid hormones account for these sex differences. Estrogen (E) facilitates drug-seeking behavior, while progesterone (P) and its metabolite, allopregnanalone (ALLO), counteract the effects of E and reduce drug seeking. Estrogen and P influence other behaviors that are affiliated with drug abuse such as drug-induced locomotor sensitization and conditioned place preference. The enhanced vulnerability to drug seeking in females vs. males is also additive with the other risk factors for drug abuse (e.g., adolescence, sweet preference, novelty reactivity, and impulsivity). Finally, treatment studies using behavioral or pharmacological interventions, including P and ALLO, also indicate that females show greater treatment effectiveness during several phases of the addiction process. The neurobiological basis of sex differences in drug abuse appears to be genetic and involves the influence of ovarian hormones and their metabolites, the hypothalamic pituitary adrenal (HPA) axis, dopamine (DA), and gamma-hydroxy-butyric acid (GABA). Overall, sex and hormonal status along with other biological risk factors account for a continuum of addiction-prone and -resistant animal models that are valuable for studying drug abuse prevention and treatment strategies.     

Structural Characterization of a Therapeutic Anti-Methamphetamine Antibody Fragment: Oligomerization and Binding of Active Metabolites

Vaccines and monoclonal antibodies (mAb) for treatment of (+)-methamphetamine (METH) abuse are in late stage preclinical and early clinical trial phases, respectively. These immunotherapies work as pharmacokinetic antagonists, sequestering METH and its metabolites away from sites of action in the brain and reduce the rewarding and toxic effects of the drug. A key aspect of these immunotherapy strategies is the understanding of the subtle molecular interactions important for generating antibodies with high affinity and specificity for METH. We previously determined crystal structures of a high affinity anti-METH therapeutic single chain antibody fragment (scFv6H4, K_D = 10 nM) in complex with METH and the (+) stereoisomer of 3,4-methylenedioxymethamphetamine (MDMA, or “ecstasy”). Here we report the crystal structure of scFv6H4 in homo-trimeric unbound (apo) form (2.60Å), as well as monomeric forms in complex with two active metabolites; (+)-amphetamine (AMP, 2.38Å) and (+)-4-hydroxy methamphetamine (p-OH-METH, 2.33Å). The apo structure forms a trimer in the crystal lattice and it results in the formation of an intermolecular composite beta-sheet with a three-fold symmetry. We were also able to structurally characterize the coordination of the His-tags with Ni²⁺. Two of the histidine residues of each C-terminal His-tag interact with Ni²⁺ in an octahedral geometry. In the apo state the CDR loops of scFv6H4 form an open conformation of the binding pocket. Upon ligand binding, the CDR loops adopt a closed formation, encasing the drug almost completely. The structural information reported here elucidates key molecular interactions important in anti-methamphetamine abuse immunotherapy.     

Leg-to-body geometry determines eyestalk reactions to substrate til

Summary The effect of oscillatory movement of single legs (about C-B axis) on compensatory eyestalk reactions and on leg counterforce as normally produced by substrate tilt has been investigated with the legs at different states of flexion (M-C joint) and leg-to-body orientation (T-C joint). Eye response and force reaction released by standard C-B movement increase with increasing angle of M-C (Figs. 2, 4) and decrease with decrease of T-C angle (Fig. 3). The effects demonstrate a multiplying influence of M-C signal and T-C signal on C-B signals. The stump of an autotomized leg releases eye responses of similar magnitude to unimpaired single legs (Fig. 5).The data are discussed in relation to neurophysiological findings and with respect to the geometrical implications for the mechanism of substrate orientation (Fig. 6).We wish to give thanks to the Max-Planck-Institut (FS, DMN) and to the Carnegie Trust (DMN) for financial support. We thank Renate Alton for technical assistance and Dorothee Maier for her help with the data analysis. Particular thanks should be expressed to the Istituto di Zoologia, Universita di Firenze (Director: Prof. Dr. L. Pardi) for promoting this research.     

The Drosophila Eip78c Gene Is Not Vital but Has a Role in Regulating Chromosome Puffs

We have generated a number of chromosomal aberrations that disrupt the early-late ecdysone-induced 78C puff gene (Eip78C, ecdysone-induced protein, FlyBase name for the E78 gene of STONE and THUMMEL 1993), which encodes the two members of the nuclear hormone receptor superfamily Eip78C-A and Eip78C-B. The aberrations include deletions of the ligand-binding/dimerization domain of both, inversions that split Eip78C-A but retain residual Eip78C-B expression, and a small deletion specific for Eip78C-B. We find that wild-type Eip78C functions are completely dispensable for normal development under laboratory conditions. However, we show that Eip78C-B is required for the maximal puffing activity of a subset of late puffs (63E and 82F) since these puffs are reduced in size in Eip78C-B mutant backgrounds. Paradoxically the same late puffs are reduced, as well as at least one other, when the Eip78C-B cDNA is overexpressed from a heat shock promoter. These data indicate either that Eip78C function is redundant or that it plays a subtle modulating role in the regulation of chromosome puffing.     

真菌代谢产物的药物发现——资源、问题和策略

本期菌物学报“真菌生物活性代谢产物的药物发现专刊”刊登了7篇深度论述的综述性文章,19篇研究论文及2篇简报。内容涉及植物内生真菌产紫杉醇/紫杉烷的研究现状及最新进展、虫草菌素产生菌的培养策略及其药理学研究进展、灵芝三萜和胞外多糖生物合成的调控策略、淡水和海水真菌活性代谢产物研究进展等。本期内容还涉及内生真菌的分离和鉴定、海洋真菌的代谢产物分离、大型真菌的培养、代谢产物的分离及其药理学研究、酚类化合物测定方法、蛋白质组学以及生物转化等研究,基本体现了我国真菌代谢产物新药发现研究的最新进展,对真菌代谢产物合成调控、活性代谢产物分离和结构鉴定以及药理学研究等都具有重要的参考价值。     

Gas chromatography–ion trap mass spectrometry method for the simultaneous measurement of MDMA (ecstasy) and its metabolites,MDA, HMA,and HMMA in plasma and urine

The investigation of 3,4-methylenedioxymethamphetamine (MDMA; ecstasy) abuse requires very robust methods with high sensitivity and wide linearity ranges for the quantification of this drug of abuse and its main metabolites in body fluids. An optimized gas chromatography–ion trap mass spectrometry (GC–IT/MS) methodology with electron impact ionization addressing these issues is presented. The sample preparation involves an enzymatic hydrolysis of urine and plasma for conjugate cleavage, a SPE extraction, and a derivatization process. The method was fully validated in rat plasma and urine. Linearity for a wide concentration range was achieved for MDMA, and the metabolites 3,4-methylenedioxyamphetamine (MDA), 4-hydroxy-3-methoxyamphetamine (HMA) and 4-hydroxy-3-methoxymethamphetamine (HMMA). Limits of quantification were 2 ng/mL in plasma and 3.5 ng/mL in urine using a Selected Ion Monitoring detection mode. Selectivity, accuracy, precision, and recovery met the required criteria for the method validation. This GC–IT/MS method provides high sensitivity and adequate performance characteristics for the simultaneous quantification of MDMA, MDA, HMA and HMMA in the studied matrices.     

Methamphetamine-Induced Depression of Monoamine Synthesis in the Rat: Development of Tolerance

Animals treated with high doses of amphetamines have been used as a model of schizophrenia due to the similarities between the psychosis associated with this mental disorder and that induced by chronic amphetamine abuse. When administered to naive rats in high doses, the amphetamine-like CNS stimulant methamphetamine produces drastic alterations in the neurochemical parameters of the neostriatal monoaminergic systems. These alterations are characterized by a decrease in the activities of the rate-limiting enzymes for dopamine and serotonin synthesis, as well as a decrease in the concentrations of both neurotransmitters and their metabolites. However, tolerance develops to these neurochemical effects when drug administration occurs in a pattern similar to that encountered during chronic amphetamine abuse. The results indicate that the neurochemical alterations produced by amphetamines in naive and tolerant animals differ widely. This suggests that the administration of high doses of amphetamine-like central stimulants to naive rats may not be an appropriate model for studying the neurochemical changes associated with psychosis and amphetamine abuse.     

A high-throughput cell-based toxicity analysis of drug metabolites using flow cytometry

The effects of liver enzymes on drug activities are important considerations in the drug discovery process. Frequently, liver microsomes are used to simulate first-pass metabolism in the liver; however, there are significant disadvantages to the microsome system. As an alternative, a simple cell-based, high-throughput system that allows for examination of metabolite activity is described. Using multiparameter flow cytometry and the low-volume, high-sample format of 96-well plates, it is possible to rapidly evaluate a dose-response curve for metabolites based on variables including initial compound concentrations, hepatocyte cell line metabolic activities, and time. Using HepG2 cells as a surrogate for hepatic metabolism of a potential therapeutic, the impact of metabolites on Jurkat cell death was measured by both propidium iodide dye exclusion and cell cycle analysis. While this system is not proposed to supplant liver microsome studies, this alternative assay provides a highly adaptable, low-cost, and high-throughput measure of drug metabolism.     

Advances in new psychoactive substances identification: the U.R.I.To.N. Consortium

Identification of new psychoactive substances (NPS) in biological and non-biological samples represents a hard challenge for forensic toxicologists. Their great chemical variety and the speed with which new NPS are synthesised and spread make stringent the need of advanced tools for their detection based on multidisciplinary approaches. For this reason, in August 2016, the “Unit of Research and Innovation in Forensic Toxicology and Neuroscience of Addiction” (U.R.I.To.N.) was founded by the Forensic Toxicology Division of the University of Florence. In this Research Unit, various professionals (i.e. forensic toxicologists, chemists, physicians) collaborate to study all the aspects of drugs of abuse, especially NPS. Herein, we describe the multidisciplinary approach comprising liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS), gas chromatography hyphenated to mass spectrometry (GC–MS) and solution nuclear magnetic resonance analysis that allowed the identification of three NPS such as 1-(benzofuran-5-yl)-N-methylpropan-2-amine, 2-amino-1-(4-bromo-2,5-dimethoxyphenyl)ethan-1-one (bk-2C-B), and 3-(2-aminopropyl)indole (α-methyltryptamine) in seized materials.     

The effects of N-dodecane pretreatment on the metabolism and distribution of benzo(A) pyrene in the isolated perfused rabbit lung

B(a)P is an ubiquitous potent carcinogen that has been associated with the increased incidence of human bronchiogenic carcinoma in occupational and urban populations. An isolated perfused rabbit lung preparation (IPL) was used to study the influence of pretreatment with a cocarcinogen, n-dodecane, on the metabolism of benzo (a)-pyrene (B(a)P). ¹⁴C-B(a)P was administered intratracheally to the IPL following biweekly inhalation exposure of the rabbits to n-dodecane. Results from n-dodecane pretreatment group were compared to those from the no-pretreatment group and an intratracheally administered B(a)P pretreatment group (B(a)P IT). Metabolites were isolated from serial blood samples up to 3 hours after administration of the ¹⁴C-B(a)P to the IPL. Patterns of B(a)P metabolites were determined using thin layer and high performance liquid chromatography, and liquid scintillation counting. The rates of appearance of B(a)P metabolites in the blood and the B(a)P metabolic pattern for n-dodecane groups were compared to B(a)P IT and no-pretreatment groups. The rates of appearance of metabolites in the blood (ng/hr/g of lung) were similar for B(a)P IT and n-dodecane pretreatment groups, but were five and four times greater, respectively, when compared to rates of appearance of metabolites for the no-pretreatment group. The B(a)P metabolic pattern of n-dodecane pretreatment showed an increase in the dihydrodiol and nonextractable formation and a decrease in the monohydroxy and diones compared to the no-pretreatment group. The metabolite pattern for B(a)P IT pretreatment group was similar to the metabolite pattern for the n-dodecane pretreatment group. The significance of the findings is that a known cocarcinogen, n-dodecane, appears to be as good an enzyme inducer as B(a)P in stimulating metabolism of B(a)P in lung. Therefore, man's risk in developing cancer may not be solely a result of exposure to a carcinogen, but may be dependent upon other constituents in his environment.     

Drug Abuse—Recommendations for California Treatment and Research Facilities

The California Legislature has directed the Regents of the University of California to collect and act as an information exchange on research and services relating to drug abuse, and to provide advice with respect to fields in which research is needed.The current report, prepared under that directive, outlines the method by which data on drug abuse research and treatment facilities will be collected, and how this data will be prepared so that appropriate recommendations can be made to the state legislature.This initial report also outlines areas of immediate concern in the area of drug abuse for the benefit of the state legislature. These areas include current state policies which interfere with investigators competing for federal research funds; pharmacological misclassification of various agents of drug abuse (including marijuana, cocaine and mescaline); lack of awareness of the major adolescent drug abuse problem in California, namely that associated with methamphetamine abuse; the inconsistent and destructive effects of current Nalline clinic programs, and legal restraints which interfere with the proper treatment of drug abusers by physicians trained in treating such patients.     

Detection of new exemestane metabolites by liquid chromatography interfaced to electrospray-tandem mass spectrometry

Exemestane is an irreversible aromatase inhibitor used for anticancer therapy. Unfortunately, this drug is also misused in sports to avoid some adverse effects caused by steroids administration. For this reason exemestane has been included in World Anti-Doping Agency prohibited list. Usually, doping control laboratories monitor prohibited substances through their metabolites, because parent compounds are readily metabolized. Thus metabolism studies of these substances are very important. Metabolism of exemestane in humans is not clearly reported and this drug is detected indirectly through analysis of its only known metabolite: 17β-hydroxyexemestane using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and gas chromatography coupled to mass spectrometry (GC-MS). This drug is extensively metabolized to several unknown oxidized metabolites. For this purpose LC-MS/MS has been used to propose new urinary exemestane metabolites, mainly oxidized in C6-exomethylene and simultaneously reduced in 17-keto group. Urine samples from four volunteers obtained after administration of a 25mg dose of exemestane were analyzed separately by LC-MS/MS. Urine samples of each volunteer were hydrolyzed followed by liquid-liquid extraction and injected into a LC-MS/MS system. Three unreported metabolites were detected in all urine samples by LC-MS/MS. The postulated structures of the detected metabolites were based on molecular formulae composition obtained through high accuracy mass determination by liquid chromatography coupled to hybrid quadrupole-time of flight mass spectrometry (LC-QTOF MS) (all mass errors below 2ppm), electrospray (ESI) product ion spectra and chromatographic behavior.     

Neurochemical Consequences of Dysphoric State During Amphetamine Withdrawal in Animal Models: A Review

Chronic abuse of amphetamines, such as d-amphetamine (AMPH) and d-methamphetamine, results in psychological dependence, a condition in which the drug produces a feeling of satisfaction and a drive that requires periodic or continuous administration of the drug to produce overwhelming pleasure or to avoid discomfort such as dysphoria. The dysphoric state of AMPH withdrawal has been recognized as depressive syndromes, such as anhedonia, depression, anxiety, and social inhibition, in early drug abstinence. Medication for treatment of the dysphoric state is important for AMPH abusers to avoid impulsive self-injurious behavior or acts that are committed with unconscious or uncontrolled suicidal ideation. However, successful treatments for AMPH withdrawal remain elusive, since the exact molecular basis of the expression of dysphoria has not been fully elucidated. This review focuses on the molecular aspects of AMPH withdrawal as indexed by neurochemical parameters under a variety of injection regimens (for example, levels of brain monoamines and their metabolites, and γ-aminobutyric acid, expression of genes and proteins involved in neuronal activity, and monoamine metabolism and availability) in rodent models which exhibit significant phenotypic features relevant to the syndromes of AMPH withdrawal in humans.     

Metabolism of the new designer drug alpha-pyrrolidinopropiophenone (PPP) and the toxicological detection of PPP and 4'-methyl-alpha-pyrrolidinopropiophenone (MPPP) studied in rat urine using gas chromatography-mass spectrometry

R,S-alpha-pyrrolidinopropiophenone (PPP) is a new designer drug with assumed amphetamine-like effects which has appeared on the illicit drug market. The aim of this study was to identify the PPP metabolites using solid-phase extraction, ethylation or acetylation as well as to develop a toxicological detection procedure in urine using solid-phase extraction, trimethylsilylation and gas chromatography-mass spectrometry (GC-MS). Analysis of urine samples of rats treated with PPP revealed that PPP was extensively metabolized by hydroxylation of the pyrrolidine ring with subsequent dehydrogenation to the corresponding lactam, hydroxylation of the aromatic ring in position 4' or double dealkylation of the pyrrolidine ring to the corresponding primary amine (cathinone) partly followed by reduction of the keto group to the corresponding secondary alcohol (norephedrines). As cathinone and the norephedrine diastereomers are also formed after intake of other drugs of abuse or medicaments, special attention must be paid to the detection of the unequivocal metabolite 2"-oxo-PPP as an unambiguous proof for the intake of PPP. The hydroxy groups were found to be partly conjugated. Based on these data, PPP could be detected in urine via its metabolites by full-scan GC-MS using mass chromatography for screening and library search for identification by comparison of the spectra with reference spectra. The same toxicological detection procedure can be applied to other designer drugs of the pyrrolidinophenone type, like MOPPP, MDPPP, MPHP, and MPPP. The detection of the latter will also be presented here.     

Mechanisms of Metabonomic for a Gateway Drug: Nicotine Priming Enhances Behavioral Response to Cocaine with Modification in Energy Metabolism and Neurotransmitter Level

Nicotine, one of the most commonly used drugs, has become a major concern because tobacco serves as a gateway drug and is linked to illicit drug abuse, such as cocaine and marijuana. However, previous studies mainly focused on certain genes or neurotransmitters which have already been known to participate in drug addiction, lacking endogenous metabolic profiling in a global view. To further explore the mechanism by which nicotine modifies the response to cocaine, we developed two conditioned place preference (CPP) models in mice. In threshold dose model, mice were pretreated with nicotine, followed by cocaine treatment at the dose of 2 mg/kg, a threshold dose of cocaine to induce CPP in mice. In high-dose model, mice were only treated with 20 mg/kg cocaine, which induced a significant CPP. ¹H nuclear magnetic resonance based on metabonomics was used to investigate metabolic profiles of the nucleus accumbens (NAc) and striatum. We found that nicotine pretreatment dramatically increased CPP induced by 2 mg/kg cocaine, which was similar to 20 mg/kg cocaine-induced CPP. Interestingly, metabolic profiles showed considerable overlap between these two models. These overlapped metabolites mainly included neurotransmitters as well as the molecules participating in energy homeostasis and cellular metabolism. Our results show that the reinforcing effect of nicotine on behavioral response to cocaine may attribute to the modification of some specific metabolites in NAc and striatum, thus creating a favorable metabolic environment for enhancing conditioned rewarding effect of cocaine. Our findings provide an insight into the effect of cigarette smoking on cocaine dependence and the underlying mechanism.     

Study on metabolites of 2,5-dimethoxy-4-bromamphetamine (DOB) in human urine using gas chromatography-mass spectrometry

The study of the biotransformation of a new synthetic drug 2,5-dimethoxy-4-bromamphetamine (DOB) and identification of its metabolites in urine of a poisoned person is described using gas chromatography mass spectrometry (GC-MS) with various ways of derivatization. It has been confirmed that one of its metabolic pathways leads to the corresponding 2-O-desmethyl and 5-O-desmethyl metabolites when the latter is prevailing. It is important to know the metabolism of this neurotoxic and hallucinogenic substance as it is a prerequisite for developing reliable toxicological diagnostic procedures and for assessment of toxicological risks.     

Patterns of childhood trauma and psychological distress among injecting heroin users in China

Background

Childhood trauma has been reported as a possible cause of future substance abuse in some countries. This study reports the prevalence of childhood trauma and examines its association with psychological distress among injecting drug users from mainland China.

Methodology

The study was conducted in three government-operated drug rehabilitation facilities in Shanghai, China in 2007. The Early Trauma Inventory Self Report-Short Form (ETISR-SF) was used to evaluate 4 types (general, emotional, physical and sexual) and severity of childhood trauma, and the Symptom Checklist-90-Revised (SCL-90-R) to evaluate psychological distress.

Principal Findings

Among 341 injecting drug users who completed the study, about 80% reported one or more types of childhood trauma, specifically 53% general trauma, 56% physical abuse, 36% emotional abuse and 26% sexual abuse. Compared to female injecting drug users, males reported significantly higher scores of general trauma and physical abuse, but lower sexual abuse scores. Hierarchical linear regression analyses showed that greater physical and emotional abuse in childhood predict greater current psychopathological distress among these injecting drug users in China.

Conclusions

The results reveal a high prevalence of childhood trauma among injecting drug users in China, and it is comparable to other similar studies in Western countries. It is important to consider the role of childhood trauma in the prevention and treatment of substance abuse.