Evaluation of Antibacterial Functionalized Dihydropyrimidine Photoaffinity Probes Toward Mechanism of Action Studies.

Antibiotic-resistant bacteria are a global health concern, necessitating the development of antibiotics working through new or underutilized mechanisms. Functionalized amino dihydropyrimidines have previously demonstrated potential as antibacterial agents, but they had limited potency, and their biological mechanism was not understood. To further evaluate their potential, focused libraries were prepared and screened for bacterial growth inhibition, and these compounds provided additional insights into the structure-activity relationships, allowing for the preparation of compounds that inhibited all strains of Staphylococcus aureus with an MIC of 2 µg/mL. After eliminating the proposed mechanism of dihydrofolate reductase inhibition, trifluoromethyl diazirine photoaffinity probes were synthesized to investigate their mechanism, and these were tested to ensure the photolabile group did not impact the antibacterial activity. Finally, the compounds were screened for hemolysis and mammalian cytotoxicity. While they lacked nonspecific membrane rupturing activity, many of the compounds showed significant mammalian cytotoxicity, indicating further development will be required to render them selective for bacteria.

Novel extended-release transdermal formulations of the psychedelic N,N-dimethyltryptamine (DMT).

There is considerable evidence from the literature that psychedelics, such as N,N-dimethyltryptamine (DMT), are safe and effective treatments for depression. However, clinical administration to induce psychedelic effects and expensive psychotherapy-assisted treatments likely limit accessibility to the average patient. There is emerging evidence that DMT promotes positive behavioral changes in vivo at sub-hallucinogenic dosages, and depending on the target indication, subjecting patients to high, bolus dosages may not be necessary. Due to rapid metabolic degradation, achieving target levels of DMT in subjects is difficult, requiring IV administration, which poses risks to patients during the intense hallucinogenic and subjective drug effects. The chemical and physical properties of DMT make it an excellent candidate for non-invasive, transdermal delivery platforms. This paper outlines the formulation development, in vitro, and in vivo testing of transdermal drug-in-adhesive DMT patches using various adhesives and permeation enhancers. In vivo behavioral and pharmacokinetic studies were performed with lead patch formulation (F5) in male and female Swiss Webster mice, and resulting DMT levels in plasma and brain samples were quantified using LC/MS/MS. Notable differences were seen in female versus male mice during IV administration; however, transdermal administration provided consistent, extended drug release at a non-hallucinogenic dose. The IV half-life of DMT was extended by 20-fold with administration of the transdermal delivery system at sub-hallucinogenic plasma concentrations not exceeding 60 ng/mL. Results of a translational head twitch assay (a surrogate for hallucinogenic effects in non-human organisms) were consistent with absence of hallucinations at low plasma levels achieved with our TDDS. Despite the reported low bioavailability of DMT, the non-invasive transdermal DMT patch F5 afforded an impressive 77 % bioavailability compared to IV at two dosages. This unique transdermal delivery option has the potential to provide an out-patient treatment option for ailments not requiring higher, bolus doses and is especially intriguing for therapeutic indications requiring non-hallucinogenic alternatives.

Transient Elevation of Plasma Glucocorticoids Supports Psilocybin-Induced Anxiolysis in Mice.

While correlations between drug-induced cortisol elevation, self-reported anxiety, and treatment outcomes have been reported for human studies during psilocybin-assisted psychotherapy, the mechanistic relationship between psychedelic-associated alterations in plasma glucocorticoid responses and the time course of anxious responsiveness remains unclear. Using rodents, both time-bound manipulation of glucocorticoid concentrations and assessment of anxiety-like behaviors can be achieved. Here, 3 mg/kg IP psilocybin was found to have anxiolytic-like effects in C57BL/6 male mice at 4 h after treatment. These effects were not altered by pretreatment with a 5-HT2A antagonist but were blunted by pretreatment with a glucocorticoid receptor antagonist or suppression of psilocybin-induced corticosterone elevations. Anxiolytic-like effects were also observed at 4 h following treatment with the nonpsychedelic 5-HT2A agonist lisuride at a dose causing a similar increase in plasma glucocorticoids as that seen with psilocybin, as well as following stress-induced (via repeated injection) glucocorticoid release alone. Psilocybin's anxiolytic-like effects persisted at 7 days following administration. The long-term anxiolytic effects of psilocybin were lost when psilocybin was administered to animals with ongoing chronic elevations in plasma corticosterone concentrations. Overall, these experiments indicate that acute, resolvable psilocybin-induced glucocorticoid release drives the postacute anxiolytic-like effects of psilocybin in mice and that its long-term anxiolytic-like effects can be abolished in the presence of chronically elevated plasma glucocorticoid elevations.

Expedited Evaluation of Conformational Stability-Heterogeneity Associations for Crude Polyclonal Antibodies in Response to Conjugate Vaccines.

Conjugate vaccines have been demonstrated to be a promising strategy for immunotherapeutic intervention in substance use disorder, wherein a hapten structurally similar to the target drug is conjugated to an immunogenic carrier protein. The antibodies generated following immunization with these species can provide long-lasting protection against overdose through sequestration of the abused drug in the periphery, which mitigates its ability to cross the blood-brain barrier. However, these antibodies exhibit a high degree of heterogeneity in structure. The resultant variations in chemical and structural compositions have not yet been clearly linked to the stability that directly affects their in vivo functional performance. In this work, we describe a rapid mass-spectrometry-based analytical workflow capable of simultaneous and comprehensive interrogation of the carrier protein-dependent heterogeneity and stability of crude polyclonal antibodies in response to conjugate vaccines. Quantitative collision-induced unfolding-ion mobility-mass spectrometry with an all-ion mode is adapted to rapidly assess the conformational heterogeneity and stability of crude serum antibodies collected from four different vaccine conditions, in an unprecedented manner. A series of bottom-up glycoproteomic experiments was performed to reveal the driving force underlying these observed heterogeneities. Overall, this study not only presents a generally applicable workflow for fast assessment of crude antibody conformational stability and heterogeneity at the intact protein level but also leverages carrier protein optimization as a simple solution to antibody quality control.

In vivo validation of psilacetin as a prodrug yielding modestly lower peripheral psilocin exposure than psilocybin.

Introduction: The use of the psychedelic compound psilocybin in conjunction with psychotherapy has shown promising results in the treatment of psychiatric disorders, though the underlying mechanisms supporting these effects remain unclear. Psilocybin is a Schedule I substance that is dephosphorylated in vivo to form an active metabolite, psilocin. Psilacetin, also known as O-acetylpsilocin or 4-acetoxy-N,N-dimethyltryptamine (4-AcO-DMT), is an unscheduled compound that has long been suggested as an alternative psilocin prodrug, though direct in vivo support for this hypothesis has thus far been lacking. Methods: This study employed liquid chromatography-tandem mass spectrometry (LC-MS/MS) to assess the time-course and plasma concentrations of psilocin following the intraperitoneal (IP) administration of psilacetin fumarate or psilocybin to male and female C57Bl6/J mice. Results: Direct comparisons of the time courses for psilocin exposure arising from psilocybin and psilacetin found that psilocybin led to 10-25% higher psilocin concentrations than psilacetin at 15-min post-injection. The half-life of psilocin remained approximately 30 min, irrespective of whether it came from psilocybin or psilacetin. Overall, the relative amount of psilocin exposure from psilacetin fumarate was found to be approximately 70% of that from psilocybin. Discussion: These findings provide the first direct support for the long-standing assumption in the field that psilacetin functions as a prodrug for psilocin in vivo. In addition, these results indicate that psilacetin fumarate results in lower peripheral psilocin exposure than psilocybin when dosed on an equimolar basis. Thoughtful substitution of psilocybin with psilacetin fumarate appears to be a viable approach for conducting mechanistic psychedelic research in C57Bl6/J mice.

The 46.1 Antibody Mediates Neurotensin Uptake into the CNS and the Effects Depend on the Route of Intravenous Administration.

Central nervous system (CNS) exposure to blood-borne biotherapeutics is limited by the restrictive nature of the brain vasculature. In particular, tightly sealed endothelial cells of the blood-brain barrier (BBB) prevent the uptake of protein and gene medicines. An approach to increase the bioavailability of such therapeutics is harnessing the BBB endothelial cells' own receptor-mediated transcytosis (RMT) mechanisms. Key to this process is a targeting ligand that can engage a BBB-resident RMT receptor. We recently identified an antibody, named 46.1, that accumulates in the mouse brain after intravenous injection. To further characterize the brain targeting and penetrating properties of clone 46.1, we conjugated neurotensin (NT) to an scFv-Fc form of the antibody (46.1-scFv-Fc-LongLinker-NT). While centrally administered NT decreases the core body temperature and locomotor activity, effects attributed to two spatially segregated brain areas, systemically administered NT has limited effects. Hence, NT can be used as a model therapeutic payload to evaluate the brain penetration of BBB-targeting antibodies and their capability to accumulate in discrete brain areas. We demonstrate that intravenously administered 46.1-scFv-Fc-LL-NT can elicit transient hypothermia and reduce drug-induced hyperlocomotion, confirming that 46.1 can deliver drug cargo to the CNS at pharmacologically relevant doses. Interestingly, when two intravenous administration routes in mice, retro-orbital and tail vein, were compared, only retro-orbital administration led to transient hypothermia. We further explored the retro-orbital route and demonstrated that the 46.1-scFv-Fc-LL-NT could enter the brain arterial blood supply directly from the retro-orbital/cavernous sinus. Taken together, the 46.1 antibody is capable of transporting drug cargo into the CNS, and at least of a portion of its CNS accumulation occurs via the cavernous sinus-arterial route.

Development of Cross-Reactive Antibodies for the Identification and Treatment of Synthetic Cannabinoid Receptor Agonist Toxicity.

Synthetic cannabinoid receptor agonists (SCRAs) are compounds that mimic the pharmacology of the psychoactive components in cannabis. These compounds are structurally diverse, inexpensive, commercially available, and difficult to identify with modern analytical methods, making them highly accessible for recreational use. Suspected SCRA toxicity, which can present with a breadth of cardiovascular, gastrointestinal, and neurological disturbances, is currently addressed through symptom management followed by a toxicological screening that often occurs long after patient discharge. Here, we report the development of four cross-reactive anti-SCRA bioconjugate vaccines as a platform for developing improved diagnostic and therapeutic interventions against SCRA intoxication, using SCRA-resembling small molecule haptens that combine common subregional motifs occurring within and across different generations of SCRA molecules. Using a combination of multiplexed competitive ELISA screening and chemoinformatic analyses, it was found that the antibodies resulting from vaccination with these bioconjugates demonstrated their ability to detect multiple SCRAs with a Tanimoto minimum common structure score of 0.6 or greater, at concentrations below 8 ng/mL. The scope of SCRAs detectable using these haptens was found to include both bioisosteric and non-bioisosteric variants within the core and tail subregions, as well as SCRAs bearing valine-like head subregions, which are not addressed by commercially available ELISA screening approaches. Vaccination with these bioconjugates was also found to prevent the changes in locomotion and body temperature that were induced by a panel of SCRAs at doses of 1 and 3 mg/kg. Further refinement of this genericized hapten design and cross-reactivity-prioritizing approach may enable the rapid detection of otherwise cryptic SCRAs that arise during overdose outbreaks, and could ultimately lead to identification of monoclonal antibody species applicable for overdose reversal.

Evaluation of Prescribing Patterns Following Surgical Procedures in Opioid Naïve Patients at a Veterans Affairs Teaching Hospital.

OBJECTIVES: To evaluate facility postoperative opioid prescribing patterns in comparison to published guidelines and adherence to opioid safety mandates. METHODS: This quality analysis was performed between November 2019 and March 2020. Patients were identified to have been opioid naïve prior to receiving a new opioid prescription postoperatively during the study period. Patient charts were reviewed, and patients were contacted to collect desired data. Statistical analysis was performed to evaluate distributions of morphine equivalent daily dose and opioid day supply prescribed across study subpopulations. RESULTS: Ninety-four of 100 prescriptions evaluated were determined to be within quantity or duration recommendations of the selected guideline. Statistical analysis found no significantly different distributions between the duration and quantity of opioid prescribed at discharge and patient-specific risk factors. Forty-eight patients did not use the entire quantity of the initial opioid prescription dispensed. Of those patients, 26 still had opioids within the home. Opioid risk review documentation was completed in 19 of 65 patients indicated for documentation. CONCLUSION: Most opioid prescriptions provided within the study period aligned with recommendations from author-selected guidelines. However, a review of risk prior to opioid prescribing frequently was not performed. The number of patients utilizing less than 50% of prescribed opioids, and few refills indicate that reductions in opioids prescribed would improve safety for both patients and the surrounding community without increasing the risk for the under-treatment of postoperative pain. Improved prescribing habits and patient safety will be targeted through provider education regarding risk review documentation in opioid naïve patients.

Complexes of Ghrelin GHS-R1a, GHS-R1b, and Dopamine D1 Receptors Localized in the Ventral Tegmental Area as Main Mediators of the Dopaminergic Effects of Ghrelin.

Ghrelin receptor, also known as growth hormone secretagogue receptor (GHS-R1a), is coexpressed with its truncated isoform GHS-R1b, which does not bind ghrelin or signal, but oligomerizes with GHS-R1a, exerting a complex modulatory role that depends on its relative expression. D1 dopamine receptor (D1R) and D5R constitute the two D1-like receptor subtypes. Previous studies showed that GHS-R1b also facilitates oligomerization of GHS-R1a with D1R, conferring GHS-R1a distinctive pharmacological properties. Those include a switch in the preferred coupling of GHS-R1a from Gq to Gs and the ability of D1R/D5R agonists and antagonists to counteract GHS-R1a signaling. Activation of ghrelin receptors localized in the ventral tegmental area (VTA) seems to play a significant role in the contribution of ghrelin to motivated behavior. In view of the evidence indicating that dopaminergic cells of the VTA express ghrelin receptors and D5R, but not D1R, we investigated the possible existence of functional GHS-R1a:GHS-R1b:D5R oligomeric complexes in the VTA. GHS-R1a:GHS-R1b:D5R oligomers were first demonstrated in mammalian transfected cells, and their pharmacological properties were found to be different from those of GHS-R1a:GHS-R1b:D1R oligomers, including weak Gs coupling and the ability of D1R/D5R antagonists, but not agonists, to counteract the effects of ghrelin. However, analyzing the effect of ghrelin in the rodent VTA on MAPK activation with ex vivo experiments, on somatodendritic dopamine release with in vivo microdialysis and on the activation of dopaminergic cells with patch-clamp electrophysiology, provided evidence for a predominant role of GHS-R1a:GHS-R1b:D1R oligomers in the rodent VTA as main mediators of the dopaminergic effects of ghrelin.SIGNIFICANCE STATEMENT The activation of ghrelin receptors localized in the ventral tegmental area (VTA) plays a significant role in the contribution of ghrelin to motivated behavior. We present evidence that indicates these receptors form part of oligomeric complexes that include the functional ghrelin receptor GHS-R1a, its truncated nonsignaling isoform GHS-R1b, and the dopamine D1 receptor (D1R). The binding of ghrelin to these complexes promotes activation of the dopaminergic neurons of the VTA by activation of adenylyl cyclase-protein kinase A signaling, which can be counteracted by both GHS-R1a and D1R antagonists. Our study provides evidence for a predominant role of GHS-R1a:GHS-R1b:D1R oligomers in rodent VTA as main mediators of the dopaminergic effects of ghrelin.

Family of Structurally Related Bioconjugates Yields Antibodies with Differential Selectivity against Ketamine and 6-Hydroxynorketamine.

The dissociative-hypnotic compound ketamine is being used in an increasingly wide range of therapeutic contexts, including anesthesia, adjunctive analgesia, treatment-resistant depression, but it also continues to be a notable substance of abuse. No specific antidotes exist for ketamine intoxication or overdose. Immunopharmacotherapy has demonstrated the ability to offer overdose protection through production of highly specific antibodies that prevent psychoactive drug penetration across the blood-brain barrier, although antiketamine antibodies have not yet been assessed or optimized for use in this approach. Moreover, generation of specific antibodies also provides an opportunity to address the role of 6-hydroxynorketamine metabolites in ketamine's rapid-acting antidepressant effect through selective restriction of metabolite access to the central nervous system. Hapten design is a critical element for tuning immune recognition of small molecules, as it affects the presentation of the target antigen and thus the quality and selectivity of the response. Here, we report the synthesis and optimization of carrier protein and conjugation conditions for an initial hapten, norketamine-N-COOH (NK-N-COOH), to optimize vaccination conditions and assess the functional consequences of such vaccination on ketamine-induced behavioral alterations occurring at dissociative-like (50 mg/kg) doses. Iterating from this initial approach, two additional haptens, ketamine-N-COOH (KET-N-COOH) and 6-hydroxynorketamine-N-COOH (HNK-N-COOH), were synthesized to target either ketamine or 6-hydroxynorketamine with greater selectivity. The ability of these haptens to generate antiketamine, antinorketamine, and anti-6-hydroxynorketamine immune responses in mice was then assessed using enzyme-linked immunosorbent assay (ELISA) and competitive surface plasmon resonance (SPR) methods. All three haptens provoked immune responses in vivo, although the KET-N-COOH and 6-HNK-N-COOH haptens yielded antibodies with 5- to 10-fold improvements in affinity for ketamine and/or 6-hydroxynorketamine, as compared to NK-N-COOH. Regarding selectivity, vaccines bearing a KET-N-COOH hapten yielded an antibody response with approximately equivalent Kd values against ketamine (86.4 ± 3.2 nM) and 6-hydroxynorketamine (74.1 ± 7.8 nM) and a 90-fold weaker Kd against norketamine. Contrastingly, 6-HNK-N-COOH generated the highest affinity and most selective antibody profile, with a 38.3 ± 4.7 nM IC50 against 6-hydroxynorketamine; Kd values for ketamine and norketamine were 33- to 105-fold weaker, at 1290 ± 281.5 and 3971 ± 2175 nM, respectively. Overall, these findings support the use of rational hapten design to generate antibodies capable of distinguishing between structurally related, yet mechanistically distinct, compounds arising from the same precursor molecule. As applied to the production of the first-reported anti-6-hydroxynorketamine antibodies to date, this approach demonstrates a promising path forward for identifying the individual and combinatorial roles of ketamine and its metabolites in supporting rewarding effects and/or rapid-acting antidepressant activity.

Catalysts for change: the cellular neurobiology of psychedelics.

The resurgence of interest in the therapeutic potential of psychedelics for treating psychiatric disorders has rekindled efforts to elucidate their mechanism of action. In this Perspective, we focus on the ability of psychedelics to promote neural plasticity, postulated to be central to their therapeutic activity. We begin with a brief overview of the history and behavioral effects of the classical psychedelics. We then summarize our current understanding of the cellular and subcellular mechanisms underlying these drugs' behavioral effects, their effects on neural plasticity, and the roles of stress and inflammation in the acute and long-term effects of psychedelics. The signaling pathways activated by psychedelics couple to numerous potential mechanisms for producing long-term structural changes in the brain, a complexity that has barely begun to be disentangled. This complexity is mirrored by that of the neural mechanisms underlying psychiatric disorders and the transformations of consciousness, mood, and behavior that psychedelics promote in health and disease. Thus, beyond changes in the brain, psychedelics catalyze changes in our understanding of the neural basis of psychiatric disorders, as well as consciousness and human behavior.

Pharmacy stakeholder reports on ethical and logistical considerations in anti-opioid vaccine development.

BACKGROUND: As opioid use disorder (OUD) incidence and its associated deaths continue to persist at elevated rates, the development of novel treatment modalities is warranted. Recent strides in this therapeutic area include novel anti-opioid vaccine approaches. This work compares logistical and ethical considerations surrounding currently available interventions for opioid use disorder with an anti-opioid vaccine approach. METHODS: The opinions of student pharmacists and practicing pharmacists assessing knowledge, perceptions, and attitudes toward current and future OUD management strategies were characterized using a staged, multi-modal research approach incorporating a focus group, pilot survey development and refinement, and final survey deployment. Survey responses were assessed using one- and two-way parametric and non-parametric analyses where appropriate, and multi-dimensional matrix profiles were compared using z-tests following an exhaustive combinatorial sum of differences calculation between items within each compared matrix. RESULTS: Focus group content analysis revealed a high level of agreeableness among participants regarding anti-opioid vaccine technology and a sense of shared ownership regarding solutions to the opioid epidemic at large. Pilot survey results demonstrated subject ability to consider both pragmatic and ethical considerations related to current therapeutics and novel interventions in a single instrument, with high endurance amongst engaged subjects. Access inequality was the most concerning ethical consideration identified for anti-opioid vaccines. Support for anti-opioid vaccine implementation across various clinical scenarios was strongest for voluntary use amongst individuals in recovery, and lowest for mandatory use in at-risk individuals. CONCLUSIONS: Ethical and logistical concerns surrounding anti-opioid vaccines were largely similar to those for current OUD therapeutics overall. Anti-opioid vaccines were endorsed as helpful potential additions to current OUD therapeutic approaches, particularly for voluntary use in the later stages of clinical progression.

Naloxone acceptance by outpatient veterans: A risk-prioritized telephone outreach event.

BACKGROUND: Opioid overdose is a major public health concern in the United States. Naloxone education and distribution can decrease the risk of overdose deaths. A previous study showed that a longitudinal, multi-attempt telephone intervention by a single pharmacy resident was effective for distributing naloxone to a high-risk veteran population. OBJECTIVES: The purpose of this project was to investigate whether a team-based, single-attempt telephone outreach event is effective for distributing naloxone to at-risk outpatient veterans. METHODS: The Risk Index for Overdose or Serious Opioid-Induced Respiratory Depression (RIOSORD) tool was used to identify patients with risk class ≥4. Pharmacy trainees contacted 164 patients and offered naloxone. The primary outcome was the proportion of patients with RIOSORD risk class ≥4 who had naloxone before versus after the intervention. RESULTS: The proportion of patients with RIOSORD class ≥4 who had a naloxone kit before and after the event was 0.28 and 0.63, respectively (difference = 0.35, p < 1 × 10-6). Per-protocol analysis showed that of 164 patients contacted, 67% were reached (n = 109) and 80 patients accepted naloxone, corresponding to a 73% acceptance rate for those reached. CONCLUSIONS: A team-based telephone outreach event is an effective method for distributing naloxone to at-risk outpatient veterans.

Anti-Opioid Antibodies in Individuals Using Chronic Opioid Therapy for Lower Back Pain.

In addition to the risk of developing opioid use disorder (OUD), known side-effects of long-term opioid use include chronic inflammation and hyperalgesia, which may arise from immune responses induced following chronic opioid use. To investigate this hypothesis, blood samples were obtained from individuals with chronic back pain who were either chronically taking prescription opioids or had minimal recent opioid exposure. Patient samples were analyzed using an enzyme-linked immunosorbent assay (ELISA) against hydrocodone- or oxycodone-hapten conjugates to assess the levels of antibodies present in the samples. While no specific response was seen in opioid-naïve subjects, we observed varying levels of anti-opioid IgM antibodies in the exposed subjects. In these subjects, antibody formation was found to be weakly correlated with current reported daily opioid dose. Other drugs of abuse found to elicit an immune response have been shown to generate advanced glycation end-products (AGEs) through reaction with glucose and subsequent modification of self-proteins. Investigations into this potential mechanism of anti-opioid antibody production identified reduced the formation of reactive intermediate species upon norhydrocodone reaction with glucose in comparison with nornicotine, thus identifying potentially important differences in hapten processing to yield the observed adaptive immune response.

Classics in Chemical Neuroscience: Buprenorphine.

Buprenorphine has not only had an interdisciplinary impact on our understanding of key neuroscience topics like opioid pharmacology, pain signaling, and reward processing but has also been a key influence in changing the way that substance use disorders are approached in modern medical systems. From its leading role in expanding outpatient treatment of opioid use disorders to its continued influence on research into next-generation analgesics, buprenorphine has been a continuous player in the ever-evolving societal perception of opioids and substance use disorder. To provide a multifaceted account on the enormous diversity of areas where this molecule has made an impact, this article discusses buprenorphine's chemical properties, synthesis and development, pharmacology, adverse effects, manufacturing information, and historical place in the field of chemical neuroscience.

DARK Classics in Chemical Neuroscience: Synthetic Cannabinoids (Spice/K2).

This Review covers the background, pharmacology, adverse effects, synthesis, pharmacokinetics, metabolism, and history of synthetic cannabinoid compounds. Synthetic cannabinoids are a class of novel psychoactive substances that act as agonists at cannabinoid receptors. This class of compounds is structurally diverse and rapidly changing, with multiple generations of molecules having been developed in the past decade. The structural diversity of synthetic cannabinoids is supported by the breadth of chemical space available for exploitation by clandestine chemists and incentivized by attempts to remain ahead of legal pressures. As a class, synthetic cannabinoid products have a more serious adverse effect profile than that of traditional phytocannabinoids, including notable risks of lethality, as well as a history of dangerous adulteration. Most synthetic cannabinoids are rapidly metabolized to active species with prolonged residence times and peripheral tissue distribution, and analytical confirmation of use of these compounds remains challenging. Overall, the emergence of synthetic cannabinoids serves as a noteworthy example of the pressing public health challenges associated with the increasing development of easily synthesized, structurally flexible, highly potent, psychoactive drugs.

Heroin vaccine: Using titer, affinity, and antinociception as metrics when examining sex and strain differences.

Anti-drug vaccines have potential as new interventions against substance use disorder (SUD). However, given the challenges seen with inter-individual variability in SUD vaccine trials to date, new interventions should ensure a robust immune response and safety profile among a diverse population. This requires accounting for sex and heritable genetic differences in response to both abused substances as well as the vaccination itself. To test response variability to our heroin-tetanus toxoid (Her-TT) immunoconjugate vaccine, we vaccinated male and female mice from several mouse strains including Swiss Webster (SW), BALB/c, and Jackson diversity mice (J:DO). Previous studies with vaccinated male SW mice demonstrated a rare hypersensitivity resulting in mice rapidly expiring with exposure to a low dose of heroin. Our results indicate that this response is limited to only male SW mice, and not to any other strain or female SW mice. Our data suggest that this hypersensitivity is not the result of an overactive cytokine or IgE response. Vaccination was similarly effective among the sexes for each strain and against repeated heroin challenge. Inbred BALB/c and J:DO mice were found to have the best vaccine response against heroin in antinociception behavioral assay. These results highlight the importance of incorporating both male and female subjects, along with different strains to mimic diverse human populations, as new SUD vaccines are being tested.

Ghrelin Receptor Influence on Cocaine Reward is Not Directly Dependent on Peripheral Acyl-Ghrelin.

The peptide hormone acyl-ghrelin and its receptor, GHSR1a, represent intriguing therapeutic targets due to their actions in metabolic homeostasis and reward activity. However, this pleotropic activity makes it difficult to intervene in this system without inducing unwanted effects. Thus, it is desirable to identify passive and active regulatory mechanisms that allow differentiation between functional domains. Anatomical restriction by the blood brain barrier represents one major passive regulatory mechanism. However, it is likely that the ghrelin system is subject to additional passive mechanisms that promote independent regulation of orexigenic behavior and reward processing. By applying acyl-ghrelin sequestering antibodies, it was determined that peripheral sequestration of acyl-ghrelin is sufficient to blunt weight gain, but not cocaine rewarding effects. However, both weight gain and reward-associated behaviors were shown to be blocked by direct antagonism of GHSR1a. Overall, these data indicate that GHSR1a effects on reward are independent from peripheral acyl-ghrelin binding, whereas centrally-mediated alteration of energy storage requires peripheral acyl-ghrelin binding. This demonstration of variable ligand-dependence amongst functionally-distinct GHSR1a populations is used to generate a regulatory model for functional manipulation of specific effects when attempting to therapeutically target the ghrelin system.

Enhancing Efficacy and Stability of an Antiheroin Vaccine: Examination of Antinociception, Opioid Binding Profile, and Lethality.

In recent years, drug conjugate vaccines have shown promise as therapeutics for substance use disorder. As a means to improve the efficacy of a heroin conjugate vaccine, we systematically explored 20 vaccine formulations with varying combinations of carrier proteins and adjuvants. In regard to adjuvants, we explored a Toll-like receptor 9 (TLR9) agonist and a TLR3 agonist in the presence of alum. The TLR9 agonist was cytosine-guanine oligodeoxynucleotide 1826 (CpG ODN 1826), while the TLR3 agonist was virus-derived genomic doubled-stranded RNA (dsRNA). The vaccine formulations containing TLR3 or TLR9 agonist alone elicited strong antiheroin antibody titers and blockade of heroin-induced antinociception when formulated with alum; however, a combination of TLR3 and TLR9 adjuvants did not result in improved efficacy. Investigation of month-long stability of the two lead formulations revealed that the TLR9 but not the TLR3 formulation was stable when stored as a lyophilized solid or as a liquid over 30 days. Furthermore, mice immunized with the TLR9 + alum heroin vaccine gained significant protection from lethal heroin doses, suggesting that this vaccine formulation is suitable for mitigating the harmful effects of heroin, even following month-long storage at room temperature.

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.