Cocaine Analog Research Articles

Probe electrospray ionization coupled to a quadrupole time-Of-flight: A feasibility study for the detection of cocaine and its derivatives in oral fluid

Probe Electrospray Ionization (PESI) is an atmospheric pressure ionization method that can be directly coupled with a mass spectrometer to allow ultrafast analyses without chromatographic separation and with minimal sample preparation. Using the particular case of cocaine and its metabolites in human oral fluid, the main objective of the present study was to test the feasibility of a new hybrid system combining a PESI source and a quadrupole time-of-flight (QTOF). The best results were obtained for a sample preparation with a simple dilution of 100 µL of oral fluids in an ethanol / 10 mM ammonium formate buffer (50/50) and 10 µL deposited on a dedicated sample plate and introduced into the PESI source. For HRMS acquisition, an approach consisting in a full-mass scan (“untargeted approach” from 100 to 500 m/z; MS1) followed by a targeted scheduled MSMS acquisition (precursor ions of the 3 molecules of interest and their 3 internal standards; MS2) gave the best signals. The total time of analysis was 0.45 min and the method was validated according to ISO15189 standard for a 5–100 ng/mL range, including accuracy and precision (inter-day and intra-day precision and bias values were lower than 15 %), matrix effect, carryover and specificity (no interference with a mixture of 119 psychotropic drugs spiked at 1 mg/L). The LLOD values were 1 ng/mL for the 3 cocaine derivatives and 83 Driving Under the Influence of Drug (DUID) cases sent to our Lab for the determination of illicit drugs in oral fluid were analyzed using the PESI-QTOF method and compared to a LC-MS/MS method. A perfect agreement was observed between the 2 methods, whether the cases were positive or negative. Cocaine was detected in 51 out of these 83 real cases (61.6 %). BZE and EME were also simultaneously detected in 50 of them (98.5 %). This feasibility study reports the first analytical method based on a coupling of a PESI source to a QTOF mass spectrometer. Adding the major advantage of high specificity through HRMS is a step forward for PESI technology.

Voltammetric Characterization and Quantification of the Cocaine Analog Nitracaine

AbstractSynthetic cocaine analogs are designer drugs that recently emerged as non‐controlled substitutes for their parent drug. Among them, nitracaine is of particular concern for its psychoactive effect. In this work, we present a thorough characterization of the electrochemical behavior of nitracaine, with a final quantification performed by differential pulse voltammetry (DPV) in ethanol/lithium perchlorate 0.1 M. The selectivity of the method and reproducibility of results were assessed. LOQ of 0.3 μg mL−1 and a linear dynamic range of up to 400 μg mL−1 were obtained. In addition, recoveries from 85 % to 101 % were achieved on both simulated and real samples. For nitracaine analysis in urine, a clean‐up and a preconcentration step by solid phase extraction (SPE) using the adsorbent Florisil have been developed and optimized through the Design of Experiments (DoE) strategy, thus achieving an enrichment factor of 20.

Structure of the human dopamine transporter and mechanisms of inhibition

The neurotransmitter dopamine has central roles in mood, appetite, arousal and movement1. Despite its importance in brain physiology and function, and as a target for illicit and therapeutic drugs, the human dopamine transporter (hDAT) and mechanisms by which it is inhibited by small molecules and Zn2+ are without a high-resolution structural context. Here we determine the structure of hDAT in a tripartite complex with the competitive inhibitor and cocaine analogue, (–)-2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane2 (β-CFT), the non-competitive inhibitor MRS72923 and Zn2+ (ref. 4). We show how β-CFT occupies the central site, approximately halfway across the membrane, stabilizing the transporter in an outward-open conformation. MRS7292 binds to a structurally uncharacterized allosteric site, adjacent to the extracellular vestibule, sequestered underneath the extracellular loop 4 (EL4) and adjacent to transmembrane helix 1b (TM1b), acting as a wedge, precluding movement of TM1b and closure of the extracellular gate. A Zn2+ ion further stabilizes the outward-facing conformation by coupling EL4 to EL2, TM7 and TM8, thus providing specific insights into how Zn2+ restrains the movement of EL4 relative to EL2 and inhibits transport activity.

Preclinical evaluation of [18F]FP-CIT, the radiotracer targeting dopamine transporter for diagnosing Parkinson’s disease: pharmacokinetic and efficacy analysis

BackgroundN-(3-fluoropropyl)-2β-carboxymethoxy-3β-(4-iodophenyl) nortropane (FP-CIT), the representative cocaine derivative used in dopamine transporter imaging, is a promising biomarker, as it reflects the severity of Parkinson’s disease (PD). 123I- and 18F-labeled FP-CIT has been used for PD diagnosis. However, preclinical studies evaluating [18F]FP-CIT as a potential diagnostic biomarker are scarce. Among translational research advancements from bench to bedside, translating preclinical findings into clinical practice is one-directional. The aim of this study is to employ a circular approach, beginning back from the preclinical stage, progressing to the supplementation of [18F]FP-CIT, and subsequently returning to clinical application. We investigated the pharmacokinetic properties of [18F]FP-CIT and its efficacy for PD diagnosis using murine models.ResultsBiodistribution, metabolite and excretion analyses were performed in mice and PD models were induced in rats using 6-hydroxydopamine (6-OHDA). The targeting efficiency of [18F]FP-CIT for the dopamine receptor was assessed through animal PET/CT imaging. Subsequently, correlation analysis was conducted between animal PET/CT imaging results and immunohistochemistry (IHC) targeting tyrosine hydroxylase. Rapid circulation was confirmed after [18F]FP-CIT injection. [18F]FP-CIT reached the highest uptake of 23.50 ± 12.46%ID/g in the striatum 1 min after injection, and it was rapidly excreted within 60 min. The major metabolic organs of [18F]FP-CIT were confirmed to be the intestines, liver, and kidneys. Its uptake in the intestine was approximately 5% ID/g. The uptake in the liver gradually increased, with excretion beginning after reaching a maximum after 60 min. The kidneys exhibited rapid elimination after 10 min. In the excretion study, rapid elimination was verified, with 21.46 ± 9.53% of the compound excreted within a 6 h period. Additionally, the efficacy of [18F]FP-CIT PET was demonstrated in the PD model, with a high correlation with IHC for both the absolute value (R = 0.803, p = 0.0017) and the ratio value (R = 0.973, p = 0.0011).ConclusionsThis study fills the gap regarding insufficient preclinical studies on [18F]FP-CIT, including its ADME, metabolites, and efficiency. The pharmacological results, including accurate diagnosis, rapid circulation, and [18F]FP-CIT excretion, provide complementary evidence that [18F]FP-CIT can be used safely and efficiently to diagnose PD in clinics, although it is already used in clinics.

Opioid, cannabinoid, cocaine, and methamphetamine epidemics: History, risk factors associated with them, and characteristics of drug action

In the last century, mankind has made significant progress in the search and study of new pain-relieving drugs through the targeted synthesis of chemical analogs of natural opioids, cannabinoids, cocaine, and amphetamines. Experience with the medical use of new synthetic opioids, cannabinoids, and amphetamines has shown not only their high analgesic efficacy but also their high danger because of the risks of drug dependence and addiction, which have caused drug epidemics. The paper described the history of relevant drug epidemics, and the pharmacodynamics and pharmacokinetics of the most dangerous drugs, as well as the risks associated with drug epidemics, were outlined. Specifically, the risk of drug dependence to opioids, cannabinoids, amphetamine, and cocaine was not recognized until too late. Therefore, the drug crisis was initially largely iatrogenic. In recent decades, controls on the prescription of narcotic drugs have tightened; thus, drug addiction as iatrogeny occurs less frequently. However, criminal elements of society have established clandestine production of drugs and their realization in the youth and LGBT community disguising as new, fashionable designer drugs and devices for their use. Moreover, new synthetic drugs differ from natural drugs in their stronger psychostimulant effects, ability to cause addiction after the first use, and high risk of fatal poisoning. Thus far, no drugs were the treatment of opioid, cannabinoid, amphetamine, and cocaine abuse. However, the development of specific vaccines for these drugs has begun in recent years. Active immunization of drug abusers with specially created vaccines is expected to assist doctors in treating drug abusers in the future.

Quantification of 108 illicit drugs and metabolites in bile matrix by LC–MS/MS for the toxicological testing of sudden death cases

Sudden death could occur after assumption of illicit drugs for recreational purposes in adults or after intoxication in children, and toxicological testing would help identify the cause of the death. Analytical methods sensitive and specific for the quantification of a great number of drugs and metabolites in at least 2 matrices should be used. Bile, collected postmortem, may be considered a specimen alternative to blood and urine to perform toxicological testing because of its extended detection window. The present study proposed a LC–MS/MS method to quantify 108 drugs and metabolites in bile. Compounds belonging to the drugs of abuse classes of amphetamines, benzodiazepines, cocaine derivatives, barbiturates, opioids, z-drugs, and psychedelics were analyzed. The sample preparation is simple and does not require solid-phase extraction. The proposed method showed an appropriate selectivity, specificity, accuracy, and precision of the calibrators and quality controls tested (precision < 15%; accuracy < 100 ± 15%). The sensitivity allowed to identify low amounts of drugs (e.g., morphine limit of detection = 0.2 µg/L; limit of quantification = 1.1 µg/L). There is no significant matrix effect, except for buprenorphine and 11-Nor-9-carboxy-Δ9-tetrahydrocannabinol. Carry-over was not present. Analytes were stable at least for 1 month at − 20 °C. Analyzing 13 postmortem specimens, methadone (50%), and cocaine (37.5%) resulted to be the most prevalent consumed substances; the concentrations quantified in bile resulted to be higher than the ones in blood suggesting bile as a potential new matrix for identifying illicit drugs and their metabolites.

Mariani wine: What's really in it? Analysis of the most popular tonic drink of the 19th century after 100 years of storage

During the Belle Epoque, the use of cocaine, well known to the South American populations, spread among the European elite in the form of a tonic drink created from coca leaves macerated in Bordeaux wine, the Mariani wine. Nowadays, bottles in circulation are extremely rare but the investigations of a Parisian historian led him to the discovery of a bottle of wine that belonged to Angelo Mariani's estate at the end of the 19th century. Our laboratory was asked to analyse the contents of the bottle in order to search for the main active compounds and to estimate the state of preservation. The analysis of the centenarian product collected was aimed at identifying the alcohols and solvents by HS-GC/FID and HS-GC/MS after dilution in water, and the main alkaloids contained in all the samples by LC-HRMS screening and LC-MS/MS quantification. The Mariani wine presented an alcohol content of 15.7°. The screening and the subsequent dosage revealed the following substances: cocaine (86 ng/mL), benzoylecgonine (383 ng/mL), cocaethylene (17 ng/mL), ecgonine methyl ester (130 ng/mL), caffeine, cuscohygrine, cinnamoylcocaine and synephrine. The alkaloids found in the Mariani wine confirm the real presence of coca leaves in this drink. Their low concentrations may indicate drug instability. The presence of cocaethylene demonstrates that it can be formed without passing through the liver, which was supposed to be the site of production when cocaine and ethanol are present in the body at the same time. These appear to be the first report in the literature presenting the analysis of Mariani wine with the dosage of cocaine derivatives.

Cocaine hydrochloride, cocaine methiodide and methylenedioxypyrovalerone (MDPV) cause distinct alterations in the structure and composition of the gut microbiota

Cocaine is a highly addictive psychostimulant drug of abuse that constitutes an ongoing public health threat. Emerging research is revealing that numerous peripheral effects of this drug may serve as conditioned stimuli for its central reinforcing properties. The gut microbiota is emerging as one of these peripheral sources of input to cocaine reward. The primary objective of the present study was to determine how cocaine HCl and methylenedioxypyrovalerone, both of which powerfully activate central reward pathways, alter the gut microbiota. Cocaine methiodide, a quaternary derivative of cocaine that does not enter the brain, was included to assess peripheral influences on the gut microbiota. Both cocaine congeners caused significant and similar alterations of the gut microbiota after a 10-day course of treatment. Contrary to expectations, the effects of cocaine HCl and MDPV on the gut microbiota were most dissimilar. Functional predictions of metabolic alterations caused by the treatment drugs reaffirmed that the cocaine congeners were similar whereas MDPV was most dissimilar from the other two drugs and controls. It appears that the monoamine transporters in the gut mediate the effects of the treatment drugs. The effects of the cocaine congeners and MDPV on the gut microbiome may form the basis of interoceptive cues that can influence their abuse properties.

Low doses of pharmaceutical formulations loaded with UFMG-V4N2 immunogen induce the production of IgG anti-cocaine antibodies and provide evidence of cerebral protection in the preclinical model

Anti-cocaine vaccines are a promising therapeutic strategy for treating cocaine use disorders. Here we hypothesize that nanoemulsions (NE) or suspensions (SS) loaded with the calix [4]arene-based immunogen UFMG-V4N2 can induce the production of anti-cocaine antibodies and decrease the passage of radiolabeled cocaine analog [99mTc]Trodat-1 through of the brain barrier. UFMG-V4N2 was characterized (solubility, morphology, DSC, XRD) and loaded into NEs and SSs using excipients approved for human use. Immunogenic efficacy was assessed by quantifying the titers and determining the specificity of anti-cocaine IgG antibodies, and by assessing the inhibition of [99mTc]Trodat-1 trafficking across the mice brain-barrier. UFMG-V4N2 is an amorphous, thermally stable molecule with very low hydrophilicity. The immunogenicity of NE or SS was similar, but aluminum phosphate and the lower dose of UFMG-V4N2 induced higher anti-cocaine IgG antibody titers, minimizing [99mTc]Trodat-1 uptake in the brain. Therefore, the UFMG-V4N2 may represent an alternative for the treatment of cocaine use disorder.

A Review of the Usage and Post-surgical Outcomes of Anesthesia for Laser In Situ Keratomileusis (LASIK) Procedure.

When laser in situ keratomileusis (LASIK) surgery is employed for myopia, hyperopia, and astigmatism, the process requires the usage of anesthetics to ensure that there is minimal patient harm and negative consequences once the procedure is complete. Statistical analysis was conducted as part of this review to evaluate the application of and distinctions between the different analgesics used for LASIK surgery by compiling and filtering information from multiple research studies. Topically administered oxybuprocaine and proparacaine were found to be the most commonly used anesthetics for LASIK, according to the data included in the review. It was also determined that there were no significant differences in terms of patient outcomes and drug concentrations when proparacaine was substituted for oxybuprocaine. This is particularly intriguing given their different chemical compositions. Temporary dry eyes were the most commonly reported adverse effect of LASIK when the anesthetic was employed. Perhaps cocaine derivatives produce similar anesthetic and post-surgical effects, but further investigations are needed to verify this hypothesis.

Cocaine derivatives–synthesis, analytical characterization and evaluation of their cross reactivity in immunochemical assays

Synthetic cocaine derivatives are currently marketed on various internet websites as legal cocaine alternatives. These psychoactive substances include phenyltropanes (troparil, dichloropane, RTI-55), 1,4-dialkyl piperazines, phenyl piperidines, benztropines, and dimethocaine. Although they have been sold as “research chemicals” for more than a decade, they have not been studied systematically. Their consumption evades forensic-toxicological perception through the absence of specific identification methods. For a holistic forensic investigation of synthetic cocaine derivatives, the availability of reference compounds is a limiting factor. Moreover, synthetic side products, stereoisomers, degradation products, or deuterated references have not been isolated and characterized. Therefore, the immunochemical cross-reactivity of cocaine derivatives will be investigated after their synthesis and analytical characterization. Diverse phenyltropanes, e.g., troparil, dichloropane, and phenyl piperidines, were synthesized using documented methods. In addition, stereoisomers and deuterated references as well as metabolites were also produced. The reactions were carefully monitored, and all products were purified and characterized with NMR spectroscopy, high-resolution mass spectrometry, and polarimetry. In addition, the synthesized substances and their metabolites, which are structurally closely related to cocaine and benzoylecgonine, were tested in a CEDIA cocaine assay to evaluate their cross-reactivity. In cooperation with the Institute of Pharmacy and Molecular Biotechnology in Heidelberg, the aimed phenyltropanes and phenyl piperidines were successfully synthesized and analytically characterized. All synthesized compounds were tested in the CEDIA cocaine assay in concentrations of 100 and 1000 ng/mL. Altogether none of the compounds showed significant cross-reactivity to create a false-positive result. The availability of reference compounds is often a bottleneck of research in forensic chemistry and toxicology. Therefore, the synthesis and characterization of reference compounds such as cocaine derivatives can support the development of methods for their identification and quantification. In addition, investigating authorities can gain better insides into the quality of the sold substances and the production methods in clandestine labs. Due to their structural similarities with cocaine and benzoylecgonine, a cross-reactivity in immunochemical assays was presumed but can be excluded for the CEDIA cocaine assay. All aimed synthetic cocaine derivatives were successfully synthesized and characterized. These substances will support the development of analytical methods and support their coverage in the daily forensic routine. The tested cocaine derivatives showed no significant cross-reactivity in the CEDIA cocaine immunoassay. While immunochemical assays are very helpful for the initial identification of drug users by police and forensic-toxicological laboratories, the cross-reactivity should be evaluated in other test systems.

Characterisation of the monoamine transport inhibition of 28 new psychoactive stimulants

The objective of this study was to characterize new psychoactive stimulants’ effects on the inhibition of the monoamine transporters for dopamine (DAT), serotonine (SER) and norepinephrine (NET). Still, new psychoactive substances (NPS) are continuously emerging in Europe and globally. A large group of NPS is stimulants, drugs including cathinones, phenylethylamines, MDMA-analogues and derivatives of cocaine. Many stimulants affect the reuptake and storage of neurotransmitters in neurons. A major site of stimulant action is inhibition of the reuptake transporters DAT, SERT and NET. These proteins transport monoamine neurotransmittors across the nerve synaptic membrane, thereby ceasing the synaptic activity. Inhibition of the reuptake of the neurotransmitter will prolong the synaptic activity. It has been shown that the relative potency in inhibiting the DAT, SERT and NET transporters correlate with the observed drug effects and also their abuse potential. Especially the DAT/SERT inhibition ratio gives an indication of the abuse potential. The DAT/NET/SERT inhibition potency for a set of NPS stimulant drugs were determined by a rapid and automated method to measure inhibition of neurotransmitter transport, using a fluorescent dye mix. Transporter inhibition studies were conducted using cell lines expressing either the human DAT (in CHO-K1 cells), SERT (in HEK293 cells) or NET (in MDCK cells) transporters. The signal from the dye transported into the cells was measured using a TECAN Spark plate reader. Inhibition was measured at 15 concentrations in triplicate and repeated in three independent experiments. In total eighteen cathinones, six phenylethylamines and four arylcyclohexylamines were characterized. DAT, NET and SERT transport inhibiting curves were generated and IC50 values were determined for all substances. For DAT the potency varied between an IC50 of 1.03 nM for the cathinone MDPiHP to > 50,000 nM for the phenytlethylamine TMA-6. For SERT inhibition the potency was in general lower. The phenylethylamines were the extremes with a range from 79.6 nM for methamnetamine to > 36,000 nM for TMA-6. The DAT/SERT inhibition ratios for the tested substances varied from 0.4 to 3080. Four substances exhibited an MDMA-like ratio below 1. Two substances had an inhibition ratio varying between > 1 and < 10, close to cocaine at 3.37. Nineteen of the tested substances had a ratio between > 10 and 330. The highest inhibition ratios were identified for MDPiHP (705) and 3F-α-PiHP (3080). For the cathinone N-butyl Hexedrone, it was not possible to determine the ratio. With the exception of TH-PVP all other cathinones had a higher DAT/SERT inhibition ratio than cocaine and ten even more than amphetamine, indicating a high abuse potential. The substance with the highest potency for NET transport inhibition was MDPiHP at IC50 27 nM, which was the only one with higher potency than amphetamine. Three cathinones, MDPT (tBuoNE), N-butyl Hexedrone and TH-PVP had IC50 value for NET > 11,000 nM. For most of the characterized substances, the potency preference was DAT > NET > SERT, but there are exceptions [deschloroketamine, 3F-PCP, TMA-6, methamnetamine, 5-MMPA, TH-PVP, eutylone, N-ethyl heptylone, MDPT (tBuONE) and 3,4-methylenedioxy PV8]. With the large variation among the stimulants in potency, it is therefore, essential to characterize DAT, SERT and NET inhibition. These results show that many of the emerging NPS stimulants are potent inhibitors of the monoamine transporters, although some lack activity or have a partial inhibitory effect.

The compulsion zone explains the self-administration of cocaine, RTI-55 and bupropion in rats

Rats that reliably self-administered cocaine also reliably self-administered the cocaine analog RTI-55 and bupropion. The inter-injection intervals of these dopamine transporter (DAT) inhibitors were regular at a given unit dose and increased as a function of unit dose. However, the mean rate of intake differed widely, ranging from 731 to 459 to 2.1 nmol/kg∙min−1 for bupropion, cocaine and RTI-55 respectively, a dramatic 348-fold range. An analysis of inter-injection intervals as a function of unit dose generated values for the mean satiety threshold of 50.6, 5.1 and 0.7 nmol/kg and t1/2 of 56.7, 9.3 and 255.6 min for bupropion, cocaine and RTI-55, respectively. The difference in rate of intake of bupropion and RTI-55 relative to cocaine is a product of their 0.1 and 7.3 fold difference in PD potency and their 6.1 and 27.5 fold difference in t1/2. Additionally, the relative durations of lever-pressing following termination of drug access correlated with the t1/2 estimates. It is hypothesized this duration represents the time required for the drug concentration to fall from the satiety threshold below the priming threshold (the minimum DAT inhibitor level that will induce lever-pressing). This indicates that the time needed for an animal to cease lever pressing following termination of access to the DAT inhibitor is predominately a function of the PK properties of the agonist. The self-administration behavior paradigm in the context of the compulsion zone theory can be used as a bioassay to determine the PK/PD properties of indirect dopamine receptor agonists.

Calix[n]arene-based immunogens: A new non-proteic strategy for anti-cocaine vaccine

IntroductionCocaine use disorder is a significant public health issue without a current specific approved treatment. Among different approaches to this disorder, it is possible to highlight a promising immunologic strategy in which an immunogenic agent may reduce the reinforcing effects of the drug if they are able to yield sufficient specific antibodies capable to bind cocaine and/or its psychoactive metabolites before entering into the brain. Several carriers have been investigated in the anti-cocaine vaccine development; however, they generally present a very complex chemical structure, which potentially hampers the proper assessment of the coupling efficiency between the hapten units and the protein structure. ObjectivesThe present study reports the design, synthesis and preclinical evaluation of two novel calix[n]arene-based anti-cocaine immunogens (herein named as V4N2 and V8N2) by the tethering of the hydrolysis-tolerant hapten GNE (15) on calix[4]arene and calix[8]arene moieties. MethodsThe preclinical assessment corresponded to the immunogenicity and dose–response evaluation of V4N2 and V8N2. The potential of the produced antibodies to reduce the passage of cocaine analogue through the blood–brain-barrier (BBB), modifying its biodistribution was also investigated. ResultsBoth calix[n]arene-based immunogens elicited high titers of cocaine antibodies that modified the biodistribution of a cocaine radiolabeled analogue (99mTc-TRODAT-1) and decreased cocaine-induced behavior, according to an animal model. ConclusionThe present results demonstrate the potential of V4N2 and V8N2 as immunogens for the treatment of cocaine use disorder.

Cocaine vaccine dAd5GNE protects against moderate daily and high-dose "binge" cocaine use.

The cocaine vaccine dAd5GNE is comprised of a disrupted serotype 5 adenovirus gene therapy vector covalently conjugated to the cocaine analog GNE. The vaccine evokes a high titer of circulating anti-cocaine antibodies that prevent cocaine from reaching its cognate receptors in the central nervous system. Prior studies have demonstrated the efficacy of dAd5GNE in models of occasional, moderate cocaine use. However, previous studies have not sufficiently evaluated the efficacy of dAd5GNE in models of the repetitive and high-dose “binge” use patterns common in human addicts. In the present study, we evaluated the capacity of dAd5GNE vaccination to protect against “binge” cocaine use and circumstances where vaccinated addicts attempt to override the vaccine. We modeled repetitive daily cocaine use in vaccinated Balb/c mice and African green monkeys, and evaluated high-dose “binge” scenarios in Balb/c mice. In each model of daily use the dAd5GNE vaccine prevented cocaine from reaching the central nervous system. In the high-dose “binge” model, vaccination decreased cocaine-induced hyperactivity and reduced the number of cocaine-induced seizures. Based on this data and our prior data in rodents and nonhuman primates, we have initiated a clinical trial evaluating the dAd5GNE anti-cocaine vaccine as a potential therapy for cocaine addicts who wish to stop cocaine use. If dAd5GNE vaccination is safe and produces high anti-cocaine antibody titers in the clinic, we hypothesize that the vaccine will restrict the access of cocaine to the central nervous system and inhibit cocaine-induced “highs” even in the context of moderate daily and high-dose “binge” use that might otherwise cause a drug-induced overdose.

Vaccination against cocaine using a modifiable dendrimer nanoparticle platform

Pharmacological therapies for the treatment of cocaine addiction have had disappointing efficacy, and the lack of recent developments in the clinical care of cocaine-addicted patients indicates a need for novel treatment strategies. Recent studies have shown that vaccination against cocaine to elicit production of antibodies that reduce concentrations of free drug in the blood is a promising method to protect against the effects of cocaine and reduce rates of relapse. However, the poorly immunogenic nature of cocaine remains a major hurdle to active immunization. Therefore, we hypothesized that strategies to increase targeted exposure of cocaine to the immune system may produce a more effective vaccine. To specifically direct an immune response against cocaine, in the present study we have conjugated a cocaine analog to a dendrimer-based nanoparticle carrier with MHC II-binding moieties that previously has been shown to activate antigen-presenting cells necessary for antibody production. This strategy produced a rapid, prolonged, and high affinity anti-cocaine antibody response without the need for an adjuvant. Surprisingly, additional evaluation using multiple adjuvant formulations in two strains of inbred mice found adjuvants were either functionally redundant or deleterious in the vaccination against cocaine using this platform. The use of conditioned place preference in rats after administration of this vaccine provided proof of concept for the ability of this vaccine to diminish cocaine reward. Together these data demonstrate the intrinsic efficacy of an immune-targeting dendrimer-based cocaine vaccine, with a vast potential for design of future vaccines against other poorly immunogenic antigens by substitution of the conjugated cargo.

Novel Fluorescent Ligands Enable Single-Molecule Localization Microscopy of the Dopamine Transporter.

The dopamine transporter (DAT) is critical for spatiotemporal control of dopaminergic neurotransmission and is the target for therapeutic agents, including ADHD medications, and abused substances, such as cocaine. Here, we develop new fluorescently labeled ligands that bind DAT with high affinity and enable single-molecule detection of the transporter. The cocaine analogue MFZ2-12 (1) was conjugated to novel rhodamine-based Janelia Fluorophores (JF549 and JF646). High affinity binding of the resulting ligands to DAT was demonstrated by potent inhibition of [3H]dopamine uptake in DAT transfected CAD cells and by competition radioligand binding experiments on rat striatal membranes. Visualization of binding was substantiated by confocal or TIRF microscopy revealing selective binding of the analogues to DAT transfected CAD cells. Single particle tracking experiments were performed with JF549-conjugated DG3-80 (3) and JF646-conjugated DG4-91 (4) on DAT transfected CAD cells enabling quantification and categorization of the dynamic behavior of DAT into four distinct motion classes (immobile, confined, Brownian, and directed). Finally, we show that the ligands can be used in direct stochastic optical reconstruction microscopy (dSTORM) experiments permitting further analyses of DAT distribution on the nanoscale. In summary, these novel fluorescent ligands are promising new tools for studying DAT localization and regulation with single-molecule resolution.

Stereoselective Synthesis of Tropanes via a 6π-Electrocyclic Ring-Opening/ Huisgen [3+2]-Cycloaddition Cascade of Monocyclopropanated Heterocycles.

The synthesis of tropanes via a microwave‐assisted, stereoselective 6π‐electrocyclic ring‐opening/ Huisgen [3+2]‐cycloaddition cascade of cyclopropanated pyrrole and furan derivatives with electron‐deficient dipolarophiles is demonstrated. Starting from furans or pyrroles, 8‐aza‐ and 8‐oxabicyclo[3.2.1]octanes are accessible in two steps in dia‐ and enantioselective pure form, being versatile building blocks for the synthesis of pharmaceutically relevant targets, especially for new cocaine analogues bearing various substituents at the C‐6/C‐7 positions of the tropane ring system. Moreover, the 2‐azabicyclo[2.2.2]octane core (isoquinuclidines), being prominently represented in many natural and pharmaceutical products, is accessible via this approach.

Compulsion zone theory of the self‐administration paradigm explains different drug durations of extinction responding with different cocaine analogues.

The compulsion zone theory of cocaine self‐administration states that lever‐pressing behavior is a drug‐induced behavior that is induced only when drug concentrations are between the priming and satiety thresholds. This explains responding after the termination of access to cocaine (the extinction phase of each session). Therefore, self‐administered drugs with longer durations of action would be expected to induce protracted extinction responding.To test this hypothesis, extinction responding was measured in rats after self‐administering 3 indirect dopamine agonists: cocaine (n = 11 rats) or the cocaine‐mimetic drugs, RTI‐55 (n = 7 rats), and bupropion (n = 7 rats). These drugs are known to have different half‐lives, with a rank order of RTI‐55, bupropion, and cocaine from longest to shortest. Across these drugs, we observed the number of lever‐presses correlated with the rank order, there were, on average, 20 presses for cocaine, 35 bupropion presses 53 RTI‐55 lever presses after the termination of access to each drug, respectively. However, lever‐pressing behavior would extinguish on an average of 18 minutes with cocaine, and 27 minutes with bupropion, while responding induced by RTI‐55 persisted for several hours, extinguishing within an average of 288 minutes. These durations were significantly different from one another (p &lt; 0.01, t‐test) and correlated with the established rank order of the half‐life of each drug.Additionally, we tested apomorphine (n=12 rats) a direct dopamine receptor agonist that is reliably self‐administered and has a short half‐life. Apomorphine‐induced responding extinguished in an average of 7 minutes. Apomorphine showed comparable lever presses to cocaine with an average of 23 per extinction. This indicates the extinction lever‐pressing is a method of measurement not limited to dopamine reuptake inhibitors but can be observed in direct‐acting agonists.These data provide further evidence that the lever‐pressing behavior after the termination of drug access is a drug‐induced response drug‐induced. The different durations of extinction responding, and numbers of extinction presses are not consistent with the idea that extinction occurs because animals learn that the drug is no longer available. Importantly, the compulsion zone theory applies to both self‐administered dopamine reuptake inhibitors other than cocaine, as well as direct‐acting dopamine receptor agonists. Thus, drugs with a longer half‐life take longer to transit the compulsion zone and induce lever‐pressing behavior for a correspondingly longer time. Further study into extinction responding may yield more information on the pharmacokinetic characteristics of self‐administered drugs.Support or Funding InformationSupported by NIDA grant U01DA039550Percent of active extinction sessions over time, with the cut‐off criteria data. Active sessions were grouped into 10‐minute bins. We see a rightward shift in extinction sessions of drugs with longer t1/2.Figure 1Durations of normalized sessions with the criteria applied. These durations were significantly different relative to the half‐life of each drug after the criteria are applied.*indicates significance (p &lt; 0.01, t‐test)Figure 2

Cocaine analogue conjugated magnetic nanoparticles for labeling and imaging dopaminergic neurons.

Molecular imaging of the dopamine transporter (DAT) with Positron Emission Tomography (PET) or Single Photon Emission Computed Tomography (SPECT) has been widely used in studies of neurological and psychiatric disorders. Nevertheless, there is a great interest in expanding molecular imaging to include magnetic resonance technology, because of the superior spatial resolution this technology may provide. Here we present a magnetic nanoparticle (NP) that specifically targets dopaminergic neurons and allows DAT imaging with magnetic resonance imaging (MRI). The nanoparticle (namely, NP-DN) is composed of an iron oxide core and a polyethylene glycol (PEG) coating to which a DAT specific dopaminergic neurolabeler (DN) is conjugated. NP-DN displayed long-term stability with favorable hydrodynamic size and surface charge suitable for in vivo application. In vitro studies showed NP-DN was non-toxic, displayed specificity towards DAT-expressing neurons, and demonstrated a 3-fold increase in DAT labeling over non-targeted NP. Our study shows NP-DN provides excellent contrast enhancement for MRI and demonstrates great potential for neuroimaging.