A Vaccine against Benzimidazole-Derived New Psychoactive Substances That Are More Potent Than Fentanyl.

New psychoactive substance (NPS) opioids have proliferated within the international drug market. While synthetic opioids are traditionally composed of fentanyl analogues, benzimidazole-derived isotonitazene and its derivatives are the current NPS opioids of concern. Hence, in this study, we implement immunopharmacotherapy wherein antibodies are produced with high titers and nanomolar affinity to multiple benzimidazole-derived NPS opioids (BNO). Notably, these antibodies blunt psychoactive and physiological repercussions from BNO exposure, which was observed through antinociception, whole-body plethysmography, and blood-brain biodistribution studies. Moreover, we detail previously unreported pharmacokinetics of these drugs, which explains the struggle of traditional pharmaceutical opioid antagonists against BNO substances. These findings provide further insight into the in vivo effects of BNO drugs and the development of effective broad-spectrum therapeutics against NPS opioids.

Paper-based lateral flow assay using rhodamine B-loaded polymersomes for the colorimetric determination of synthetic cannabinoids in saliva.

Synthetic cannabinoids are one of the many substances of abuse widely spreading in modern society. Medical practitioners and law enforcement alike highly seek portable, efficient, and reliable tools for on-site detection and diagnostics. Here, we propose a colorimetric lateral flow assay (LFA) combined with dye-loaded polymersome to detect the synthetic cannabinoid JWH-073 efficiently. Rhodamine B-loaded polymersome was conjugated to antibodies and fully characterized. Two LFA were proposed (sandwich and competitive), showing a high level of sensitivity with a limit of detection (LOD) reaching 0.53 and 0.31 ng/mL, respectively. The competitive assay was further analyzed by fluorescence, where the LOD reached 0.16 ng/mL. The application of the LFA over spiked synthetic saliva or real human saliva demonstrated an overall response of 94% for the sandwich assay and 97% for the competitive LFA. The selectivity of the system was assessed in the presence of various interferents. The analytical performance of the LFA system showed a coefficient of variation below 6%. The current LFA system appears as a plausible system for non-invasive detection of substance abuse and shows promise for synthetic cannabinoid on-site sensing.

Application of Biofunctionalized Magnetic Nanoparticles Based-Sensing in Abused Drugs Diagnostics.

Real-time detection of substance use is an approach of high interest leading to the optimization of behavioral interventions and drug abuse intervention. The current methods in use suffer many limitations and need high logistical and laboratory requirements. Biosensors have shown a great potential in overcoming these limitations. In the present study, the electrochemical biosensor composed of a screen-printed electrode (SPE) was designed for the detection of synthetic cannabinoid (SC). Antibody-immobilized magnetic nanoparticles were also used to create a surface on the transducer with magnetic interactions in order to detect JWH-073 as a SC model. The use of immobilized magnetic nanoparticles to create working surfaces makes the electrode a reusable SPE which can be reutilized after the cleansing. To examine and observe any possible changes on the surface due to its interaction with the analyte, different electrochemical techniques such as differential pulse voltammetry, cyclic voltammetry, and electrochemical impedance spectrometry were applied. Based on the obtained results, the linearity of the biosensor was found between 5 and 400 ng/mL, and the detection limit was calculated as 22 ng/mL (n = 6) using the 3 Sb/m formula. The biosensor functionality was studied in the presence of some related interferents that showed lower responses than JWH-073, thus demonstrating the good selectivity of the prepared biosensor. Finally, the sensory platform was used to test synthetic urine sample, and the results were compared with obtained results from liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF/MS), which showed that the proposed method could be utilized to identify abuse drugs.

Two immunoassays for the detection of 2C-B and related hallucinogenic phenethylamines.

INTRODUCTION: The use of new psychoactive substances as drugs of abuse has dramatically increased over the last years. Hallucinogenic phenethylamines gained particular popularity as they have both stimulating and psychedelic effects. Although generally perceived as safe, these illicit drugs pose a serious health risk; they have been linked to cases of severe poisoning or even deaths. Therefore, simple, cost-effective and reliable methods are needed for rapid determination of abused hallucinogens. METHODS: For this purpose, two haptens derived from 2C-H were designed, synthesized and subsequently attached to a carrier protein. Polyclonal antibodies obtained from a rabbit immunized with one of the prepared immunogens were used for the development of two immunoassays. RESULTS: In this study, a lateral flow immunoassay (LFIA) and an enzyme linked immunosorbent assay (ELISA) for the detection of 2C-B and related hallucinogenic phenethylamines in urine were developed. The presented LFIA is primarily suitable for on-site monitoring as it is simple and can provide a visual evidence of 2C-B presence within a few minutes. Its reasonable sensitivity (LODLFIA = 15 ±â€¯7 ng mL-1) allows detection of the drug presence in urine after acute exposure. For greater accuracy, highly sensitive ELISA (LODELISA = 6 ±â€¯3 pg mL-1) is proposed for toxicological quantitative analyses of positive samples captured by the LFIA. DISCUSSION: The comparison of the ELISA with the well-established UHPLC-MS-MS method shows excellent agreement of results, which confirms good potential of the ELISA to be used for routine analyses of 2C-B and related hallucinogenic phenethylamines of both main sub-families.

Drugs of Abuse Induced-Subversion of the Peripheral Immune Response and Central Glial Activity: Focus on Novel Therapeutic Approaches.

BACKGROUND: Drugs of abuse affect both central nervous system (CNS) and peripheral immune function. Besides the involvement of dopamine and glutamate systems, chronic exposure to drugs of abuse alters immune homeostasis, promoting a pro-inflammatory status. At the same time, impaired peripheral immunity leads to an increased susceptibility to infections in drug abusers. DISCUSSION: There is evidence that certain drugs, such as opioids, activate microglial cells and astrocytes which, in turn, provoke central neuroinflammation. Particularly, opioids bind the Toll-like receptor (TLR)-4 with increased expression of nuclear factor kappa-light-chain-enhancer of activated B cells and release of pro-inflammatory cytokines. Peripheral mediators released by immune cells also contribute to aggravate central neuroinflammation. CONCLUSION: These are based either on the inhibition of TLR-4 activation by drugs of abuse or on the correction of dopamine and glutamate pathways. Finally, a hypothetic nutraceutical intervention with polyphenols in view of their anti-inflammatory and anti-oxidant properties will be outlined as an adjuvant treatment for drugs of abuse-related disorders.

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.

Conjugate Vaccine Immunotherapy for Substance Use Disorder.

Substance use disorder, especially in relation to opioids such as heroin and fentanyl, is a significant public health issue and has intensified in recent years. As a result, substantial interest exists in developing therapeutics to counteract the effects of abused drugs. A promising universal strategy for antagonizing the pharmacology of virtually any drug involves the development of a conjugate vaccine, wherein a hapten structurally similar to the target drug is conjugated to an immunogenic carrier protein. When formulated with adjuvants and immunized, the immunoconjugate should elicit serum IgG antibodies with the ability to sequester the target drug to prevent its entry to the brain, thereby acting as an immunoantagonist. Despite the failures of first-generation conjugate vaccines against cocaine and nicotine in clinical trials, second-generation vaccines have shown dramatically improved performance in preclinical models, thus renewing the potential clinical utility of conjugate vaccines in curbing substance use disorder. This review explores the critical design elements of drug conjugate vaccines such as hapten structure, adjuvant formulation, bioconjugate chemistry, and carrier protein selection. Methods for evaluating these vaccines are discussed, and recent progress in vaccine development for each drug is summarized.

Development and Characterization of Monoclonal Antibodies Specific for 3-(1-naphthoyl) Indole Derivatives.

3-(1-naphthoyl) indole is one of the raw materials that synthesizes a synthetic cannabinoid such as 1-pentyl-3-(1-naphthoyl) indole (JWH-018) and 1-butyl-3-(1-naphthoyl) indole (JWH-073). It is important to detect the 3-(1-naphthoyl) indole derivatives rapidly, sensitively, and comprehensively. We developed two monoclonal antibodies (MAb) against 3-(1-naphthoyl) indole derivatives, named NT1 (IgG1) and NT2 (IgG1), which were possibly effective for detecting 3-(1-naphthoyl) indole derivatives. The cross-reactive ability of these MAbs was evaluated using a competitive enzyme-linked immunosorbent assay (ELISA). In the results, we found both of these antibodies recognize 3-(1-naphthoyl) indole and its derivatives. However neither of these antibodies recognize naphtoic acid, 4-methyl-naphtoic acid, and indole. Sixty to 100 nanomole per liter of 3-(1-naphthoyl) indole derivatives, such as 1-methyl-3-(1-naphthoyl) indole, 1-ethyl-3-(1-naphthoyl) indole, and 1-octyl-3-(1-naphthoyl) indole, can be detected using both of the obtained MAbs. Thus, the MAbs produced in this study could be a useful tool for the detection of 3-(1-naphthoyl) indole derivatives.

Quality control of antibodies for assay development.

Antibodies are used as powerful tools in basic research, for example, in biomarker identification, and in various forms for diagnostics, for example, identification of allergies or autoimmune diseases. Due to their robustness and ease of handling, immunoassays are favourite methods for investigation of various biological or medical questions. Nevertheless in many cases, additional analyses such as mass spectrometry are used to validate or confirm the results of immunoassays. To minimize the workload and to increase confidence in immunoassays, there are urgent needs for antibodies which are both highly specific and well validated. Unfortunately many commercially available antibodies are neither well characterized nor fully tested for cross-reactivities. Adequate quality control and validation of an antibody is time-consuming and can be frustrating. Such validation needs to be performed for every assay/application. However, where an antibody validation is successful, a highly specific and stable reagent will be on hand. This article describes the validation processes of antibodies, including some often neglected factors, as well as unspecific binding to other sample compounds in a multiparameter diagnostic assay. The validation consists of different immunological methods, with important assay controls, and is performed in relation to the development of a diagnostic test.

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.

Investigating hapten clustering as a strategy to enhance vaccines against drugs of abuse.

Vaccines for drugs of abuse have yet to achieve full clinical relevance, largely due to poor/inconsistent immune responses in patients. The use of multivalent scaffolding as a means to tailor drug-hapten density and clustering was examined in the context of drug-immune response modulation. A modular trivalent hapten containing a diglycine spacer, triAM1(Gly)2, was synthesized and shown to elicit anti-nicotine antibodies at equivalent affinity and concentration to the monovalent AM1 analog, despite in this instance having a lower effective hapten density. Augmenting this data, the corresponding monovalent hapten AM1(Gly)2 resulted in enhanced antibody affinity and concentration. Drug-hapten clustering represents a new vaccine paradigm, and, while examined only in the context of nicotine, it should be readily translatable to other drugs of abuse.

Hapten-specific naïve B cells are biomarkers of vaccine efficacy against drugs of abuse.

Vaccination against drugs of abuse shows efficacy in animal models, yet few subjects achieve effective serum antibody titers in clinical studies. A barrier to translation is the lack of pre-vaccination screening assays that predict the most effective conjugate vaccines or subjects amenable to vaccination. To address this obstacle, we developed a fluorescent antigen-based enrichment method paired with flow cytometry to characterize hapten-specific B cells. Using this approach, we studied naïve and activated B cells specific for structurally-related model haptens based on derivatization of the morphinan structure at the C6 position on oxycodone or at the C8 position on hydrocodone, and showing different pre-clinical efficacy against the prescription opioid oxycodone. Prior to vaccination, naïve B cells exhibited relatively higher affinity for the more effective C6-derivatized oxycodone-based hapten (6OXY) and the 6OXY-specific naïve B cell population contained a higher number of B cells with greater affinity for free oxycodone. Higher affinity of naïve B cells for hapten or oxycodone reflected greater efficacy of vaccination in blocking oxycodone distribution to brain in mice. Shortly after immunization, activated hapten-specific B cells were detected prior to oxycodone-specific serum antibodies and provided earlier evidence of vaccine failure or success. Analysis of hapten-specific naïve and activated B cells may aid rational vaccine design and provide screening tools to predict vaccine clinical efficacy against drugs of abuse or other small molecules.

Validation of a novel immunoassay for the detection of synthetic cannabinoids and metabolites in urine specimens.

Synthetic cannabinoid drugs do not cross react on traditional marijuana immunoassay tests, preventing their use in large scale drug screening programs. This paper describes the validation and performance characteristics of two enzyme linked immunosorbent assays designed to detect the use of two common synthetic cannabinoids in urine, JWH-018 and JWH-250. The JWH-018 assay has significant cross-reactivity with several synthetic cannabinoids and their metabolites, whereas the JWH-250 assay has limited cross-reactivity. The assays are calibrated at 5 ng/mL with the 5-OH metabolite of JWH-018 and the 4-OH metabolite of JWH-250. The method was validated with 114 urine samples for JHW-018 and 84 urine samples for JWH-250 and confirmed by using liquid chromatography tandem mass spectrometry, which tests for metabolites of JWH-018, JWH-019, JWH-073, JWH-250 and AM-2201. The accuracy was determined to be 98% with greater than 95% sensitivity and specificity for both assays.

Drugs of abuse effects on immunity and microbial pathogenesis.

Substance abuse remains a serious medical, public health, and social problem. The impact on destructive public health and health costs compounded with the negative consequences of drugs abuse poses a significant toll on the economy. Despite significant advancement of research in the field treatment of and care of patients with substance abuse has lagged behind because of limited education and training of clinicians on substance abuse problems. The goal of the special issue is to provide the current status on the mechanisms underlying the increased prevalence of opportunistic infections in the drug abuse population, to identify important areas where further research would be beneficial and to open new avenues of investigation for therapeutic development. We aimed these articles for the benefit of both basic and clinical researchers.

Allergic hypersensitivity to cannabis in patients with allergy and illicit drug users

Background: Cannabis is the illicit drug most widely used by young people in high-income countries. Allergy symptoms have only occasionally been reported as one of the adverse health effects of cannabis use. Objectives: To study IgE-mediated response to cannabis in drug users, atopic patients, and healthy controls. Methods: Asthmatic patients sensitised to pollen, and all patients sensitised to tobacco, tomato and latex, considered as cross-reacting allergens, were selected from a data base of 21,582 patients. Drug users attending a drug-rehabilitation clinic were also included. Controls were 200 non-atopic blood donors. Specific IgE determination, prick tests and specific challenge with cannabis extracts were performed in patients and controls. Results: Overall, 340 patients, mean age 26.9±10.7 years, were included. Males (61.4%) were the most sensitised to cannabis (p<0.001). All cannabis-sensitised patients were alcohol users. Eighteen (72%) of the patients allergic to tomato were sensitised to cannabis, but a positive specific challenge to cannabis was highest in patients sensitised to tobacco (13/21, 61.9%), (p<0.001). Pollen allergy was not a risk factor for cannabis sensitisation. Prick tests and IgE for cannabis had a good sensitivity (92 and 88.1%, respectively) and specificity (87.1 and 96%) for cannabis sensitisation. Conclusions: Cannabis may be an important allergen in young people. Patients previously sensitised to tobacco or tomato are at risk. Cannabis prick tests and IgE were useful in detecting sensitisation (AU)

Opioids and HIV/HCV infection.

Since human immunodeficiency virus (HIV) and hepatitis C virus (HCV) share the same modes of transmission and common risk factors for infection, co-infections with HIV and HCV are frequently found in injection drug users (IDUs). IDUs represent one of the largest reservoirs of HIV as well as HCV in the United States. These two pathogens are also likely to be responsible for the highest infectious disease morbidity and mortality rates among IDUs. IDUs frequently involve the abuse of heroin, the most common abused opiate. Opiates have been suggested to have a cofactor role in the immunopathogenesis of HIV disease, as they have the potential to compromise host immune responses and enhances microbial infections. Although in vitro studies have yielded relatively agreeable data that morphine, the active metabolite of heroin, exacerbate HIV infection/replication, epidemiologic studies as well as in vivo non-human primate investigations on the impact of opiate abuse on HIV disease progression have yielded the conflicting data. Given immunomodulation and immunocompromising effect as well as demonstrated impact to enhance HIV replication in vitro, it is reasonable to believe that opiate abuse is a facilitator in HIV and/or HCV disease progression. However, much remain to be learned about the mechanisms of opiate-mediated broad influence on host immunity and viral expression. Thus, more extensive studies are needed in order to determine the effects of different conditions of opiate abuse and to define the understanding of the role of opiate in modulating HIV and/or HCV disease progression.

Allergic hypersensitivity to cannabis in patients with allergy and illicit drug users.

BACKGROUND: Cannabis is the illicit drug most widely used by young people in high-income countries. Allergy symptoms have only occasionally been reported as one of the adverse health effects of cannabis use. OBJECTIVES: To study IgE-mediated response to cannabis in drug users, atopic patients, and healthy controls. METHODS: Asthmatic patients sensitised to pollen, and all patients sensitised to tobacco, tomato and latex, considered as cross-reacting allergens, were selected from a data base of 21,582 patients. Drug users attending a drug-rehabilitation clinic were also included. Controls were 200 non-atopic blood donors. Specific IgE determination, prick tests and specific challenge with cannabis extracts were performed in patients and controls. RESULTS: Overall, 340 patients, mean age 26.9±10.7 years, were included. Males (61.4%) were the most sensitised to cannabis (p<0.001). All cannabis-sensitised patients were alcohol users. Eighteen (72%) of the patients allergic to tomato were sensitised to cannabis, but a positive specific challenge to cannabis was highest in patients sensitised to tobacco (13/21, 61.9%), (p<0.001). Pollen allergy was not a risk factor for cannabis sensitisation. Prick tests and IgE for cannabis had a good sensitivity (92 and 88.1%, respectively) and specificity (87.1 and 96%) for cannabis sensitisation. CONCLUSIONS: Cannabis may be an important allergen in young people. Patients previously sensitised to tobacco or tomato are at risk. Cannabis prick tests and IgE were useful in detecting sensitisation.

Immunology as it pertains to drugs of abuse, AIDS and the neuroimmune axis: mediators and traffic.

The interactions between the immune and nervous systems are very complex, and yet our understanding of these interactions is still relatively limited. The neuroinflammatory reaction that can accompany HIV infection occurs because of a cascade of events that appears to require the migration of HIV-infected cells across the blood-brain barrier. In susceptible individuals, this leads to inflammatory processes which can include substantial changes in neuronal function. It is possible to consider the inflammatory events to be composed of two essential processes. The first process is cellular traffic, and the second, is the expression and recognition of the various pro-inflammatory and/or toxic mediators. The added complication of drug abuse adds complexity to the traffic and mediator release events, and depending on the specific drug being abused, the disease can be exacerbated in these individuals. An understanding of the fine details of these mediator and traffic processes should provide useful targets for therapeutic intervention to attenuate disease associated with HIV infection.