Carfentanil, the most potent of the fentanyl analogues, is at the forefront of synthetic opioid-related deaths, second to fentanyl. Moreover, the administration of the opioid receptor antagonist naloxone has proven inadequate for an increasing number of opioid-related conditions, often requiring higher/additional doses to be effective, as such interest in alternative strategies to combat more potent synthetic opioids has intensified. Increasing drug metabolism would be one strategy to detoxify carfentanil; however, carfentanil's major metabolic pathways involve N-dealkylation or monohydroxylation, which do not lend themselves readily to exogenous enzyme addition. Herein, we report, to our knowledge, the first demonstration that carfentanil's methyl ester when hydrolyzed to its acid was found to be 40,000 times less potent than carfentanil in activating the μ-opioid receptor. Physiological consequences of carfentanil and its acid were also examined through plethysmography, and carfentanil's acid was found to be incapable of inducing respiratory depression. Based upon this information, a hapten was chemically synthesized and immunized, allowing the generation of antibodies that were screened for carfentanil ester hydrolysis. From the screening campaign, three antibodies were found to accelerate the hydrolysis of carfentanil's methyl ester. From this series of catalytic antibodies, the most active underwent extensive kinetic analysis, allowing us to postulate its mechanism of hydrolysis against this synthetic opioid. In the context of potential clinical applications, the antibody, when passively administered, was able to reduce respiratory depression induced by carfentanil. The data presented supports further development of antibody catalysis as a biologic strategy to complement carfentanil overdose reversal.

The opioid epidemic remains a dire public health crisis with millions of people currently suffering from opioid use disorder (OUD) and tens of thousands dying each year. Synthetic opioids are most responsible for the crisis because of their extreme potency and ease of manufacture. Carfentanil for example has an estimated potency 10,000 times greater than morphine and thus is highly dangerous for human use. Herein, we report two synthetic opioid vaccines that elicited high-affinity antibodies against carfentanil and fentanyl with cross-reactivity to other synthetic opioids in mice and offered protection against opioid-induced respiratory depression, the primary cause of overdose deaths. These vaccines also successfully diminished drug biodistribution to the brain and shielded against opioid analgesic effects. Collectively, these findings provide new insights into the development of immunotherapeutic strategies aimed at opioid abuse and overdose.

SARS-CoV-2 virus has recently given rise to the current COVID-19 pandemic where infected individuals can range from being asymptomatic, yet highly contagious, to dying from acute respiratory distress syndrome. Although the world has mobilized to create antiviral vaccines and therapeutics to combat the scourge, their long-term efficacy remains in question especially with the emergence of new variants. In this work, we exploit a class of compounds that has previously shown success against various viruses. A salicylanilide library was first screened in a SARS-CoV-2 activity assay in Vero cells. The most efficacious derivative was further evaluated in a prophylactic mouse model of SARS-CoV-2 infection unveiling a salicylanilide that can reduce viral loads, modulate key cytokines, and mitigate severe weight loss involved in COVID-19 infections. The combination of anti-SARS-CoV-2 activity, cytokine inhibitory activity, and a previously established favorable pharmacokinetic profile for the lead salicylanilide renders salicylanilides in general as promising therapeutics for COVID-19.

A double conjugation strategy was implemented to produce an anti-fentanyl vaccine, which was predicated upon preformed-antibody-assisted antigen presentation. The new vaccine was found to reduce the psychoactive effects of fentanyl without the addition of the immunostimulant CpG oligodeoxynucleotide.

Clostridioides difficile infection (CDI) causes serious and sometimes fatal symptoms like diarrhea and pseudomembranous colitis. Although antibiotics for CDI exist, they are either expensive or cause recurrence of the infection due to their altering the colonic microbiota, which is necessary to suppress the infection. Here, we leverage a class of known membrane-targeting compounds that we previously showed to have broad inhibitory activity across multiple Clostridioides difficile strains while preserving the microbiome to develop an efficacious agent. A new series of salicylanilides was synthesized, and the most potent analog was selected through an in vitro inhibitory assay to evaluate its pharmacokinetic parameters and potency in a CDI mouse model. The results revealed reduced recurrence of CDI and diminished disturbance of the microbiota in mice compared to standard-of-care vancomycin, thus paving the way for novel therapy that can potentially target the cell membrane of C. difficile to minimize relapse in the recovering patient.

Heroin overdose and addiction remain significant health and economic burdens in the world today costing billions of dollars annually. Moreover, only limited pharmacotherapeutic options are available for treatment of heroin addiction. In our efforts to combat the public health threat posed by heroin addiction, we have developed vaccines against heroin. To expand upon our existing heroin-vaccine arsenal, we synthesized new aryl and alkyl sulfonate ester haptens; namely aryl-mono-sulfonate (HMsAc) and Aryl/alkyl-di-sulfonate (H(Ds)2) as carboxyl-isosteres of heroin then compared them to our model heroin-hapten (HAc) through vaccination studies. Heroin haptens were conjugated to the carrier protein CRM197 and the resulting CRM-immunoconjugates were used to vaccinate Swiss Webster mice following an established immunization protocol. Binding studies revealed that the highest affinity anti-heroin antibodies were generated by the HMsAc vaccine followed by the HAc and H(Ds)2 vaccines, respectively (HMsAc > HAc≫HDs2). However, neither the HMsAc nor H(Ds)2 vaccines were able to generate high affinity antibodies to the psychoactive metabolite 6-acetyl morphine (6-AM), in comparison to the HAc vaccine. Blood brain bio-distribution studies supported these binding results with vaccine efficiency following the trend HAc > HMsAc ≫ H(Ds)2 The work described herein provides insight into the use of hapten-isosteric replacement in vaccine drug design.

The nearly insurmountable adversity that accompanies opioid use disorder (OUD) creates life-altering complications for opioid users. To worsen matters, existing small-molecule drugs continue to inadequately address OUD due to their engagement of the opioid receptor, which can leave the user to deal with side effects and financial hardships from their repeated use. An alternative therapeutic approach utilizes endogenously generated antibodies through active vaccination to reduce the effect of opioids without modulating the opioid receptor. Here, we explore different adjuvants and storage conditions to improve opioid vaccine efficacy and shelf life. Our results revealed that inulin-based formulations (Advax) containing a CpG oligodeoxynucleotide (ODN) acted as effective adjuvants when combined with a heroin conjugate: immunized mice showed excellent recovery from heroin-induced antinociception accompanied by high titer, high opioid affinity serum antibodies similar to the immunopotentiating properties of traditional alum-based adjuvants. Moreover, nonhuman primates vaccinated with a heroin/fentanyl combination vaccine demonstrated potent antibody responses against opioids when formulated with both inulin and alum adjuvants. Finally, storing a freeze-dried opioid vaccine formulation maintained efficacy for up 1 year at room temperature. The results from our studies represent an advance toward a clinically feasible opioid vaccine.

Synthetic cannabinoids (SCs) constitute a significant portion of psychoactive substances forming a major public health risk. Due to the wide variety of SCs, broadly neutralizing antibodies generated by active immunization present an intriguing pathway to combat cannabinoid use disorder. Here, we probed hapten design for antibody affinity and cross reactivity against two classes of SCs. Of the 10 haptens screened, 3 vaccine groups revealed submicromolar IC50, each targeting 5–6 compounds in our panel of 22 drugs. Moreover, SCs were successfully sequestered when administered by vaping or intraperitoneal injection, which was confirmed within animal models by observing locomotion, body temperature, and pharmacokinetics. We also discovered synergistic effects to simultaneously blunt two drug classes through an admixture vaccine approach. Collectively, our study provides a comprehensive foundation for the development of vaccines against SCs.

One proposed factor contributing to the increased opioid overdose deaths is the increased frequency of synthetic opioids, including fentanyl and fentanyl analogs. A treatment strategy currently under development to address the ongoing opioid crisis is immunopharmacotherapies or opioid-targeted vaccines. The present study determined the effectiveness and selectivity of a fentanyl-tetanus toxoid conjugate vaccine to alter the behavioral effects of fentanyl and a structurally dissimilar mu-opioid agonist oxycodone in male rhesus monkeys (n=3-4). Fentanyl and oxycodone produced dose-dependent suppression of behavior in an assay of schedule-controlled responding and antinociception in an assay of thermal nociception (50°C). Acute naltrexone (0.032 mg/kg) produced an approximate 10-fold potency shift for fentanyl to decrease operant responding. The fentanyl vaccine was administered at weeks 0, 2, 4, 9, 19, and 44 and fentanyl or oxycodone potencies in both behavioral assays were redetermined over the course of 49 weeks. The vaccine significantly and selectively shifted fentanyl potency at least 10-fold in both assays at several time points over the entire experimental period. Mid-point titer levels were significantly correlated with fentanyl antinociceptive potency shifts. Antibody affinity for fentanyl as measured by a competitive binding assay increased over time to around 3-4 nM. The fentanyl vaccine also significantly increased fentanyl plasma levels approximately 6-fold consistent with the hypothesis that the vaccine sequesters fentanyl in the blood. Overall, these results support the continued development and evaluation of this fentanyl vaccine to address the ongoing opioid crisis. Highlights Vaccine blunted fentanyl rate-suppression potency ∼ 10-fold Vaccine blunted fentanyl antinociceptive potency ∼25-fold Fentanyl vaccine was as effective as acute 0.032 mg/kg naltrexone Vaccine was selective for fentanyl and not oxycodone Antibody immune response ∼ 3 nM affinity for fentanyl

The current opioid crisis remains a significant public health issue and there is a critical need for biomedical research to develop effective and easily deployable candidate treatments. One emerging treatment strategy for opioid use disorder includes immunopharmacotherapies or opioid-targeted vaccines. The present study determined the effectiveness of a fentanyl-tetanus toxoid conjugate vaccine to alter fentanyl self-administration using a fentanyl-vs.-food choice procedure in male and female rats under three experimental conditions. For comparison, continuous 7-day naltrexone (0.01–0.1 mg/kg/h) and 7-day clonidine (3.2–10 μg/kg/h) treatment effects were also determined on fentanyl-vs.-food choice. Male and female rats responded for concurrently available 18% diluted Ensure® (liquid food) and fentanyl (0–10 μg/kg/infusion) infusions during daily sessions. Under baseline and saline treatment conditions, fentanyl maintained a dose-dependent increase in fentanyl-vs.-food choice. First, fentanyl vaccine administration significantly blunted fentanyl reinforcement and increased food reinforcement for 15 weeks in non-opioid dependent rats. Second, surmountability experiments by increasing the unit fentanyl dose available during the self-administration session 10-fold empirically determined that the fentanyl vaccine produced an approximate 22-fold potency shift in fentanyl-vs.-food choice that was as effective as the clinically approved treatment naltrexone. Clonidine treatment significantly increased fentanyl-vs.-food choice. Lastly, fentanyl vaccine administration prevented the expression of withdrawal-associated increases in fentanyl-vs.-food choice following introduction of extended 12 h fentanyl access sessions. Overall, these results support the potential and further consideration of immunopharmacotherapies as candidate treatments to address the current opioid crisis.

Background One emerging strategy to address the opioid crisis includes opioid-targeted immunopharmacotherapies. This study compared effectiveness of a heroin-tetanus toxoid (TT) conjugate vaccine to antagonize heroin, 6-acetylmorphine (6-AM), morphine, and fentanyl antinociception in rats. Methods Adult male and female Sprague Dawley rats received three doses of active or control vaccine at weeks 0, 2, and 4. Vaccine pharmacological selectivity was assessed by comparing opioid dose-effect curves in 50°C warm-water tail-withdrawal procedure before and after active or control heroin-TT vaccine. Route of administration [subcutaneous (SC) vs. intravenous [IV)] was also examined as a determinant of vaccine effectiveness. Continuous naltrexone treatment (0.0032-0.032 mg/kg/h) effects on heroin, 6-AM, and morphine antinociceptive potency was also determined as a benchmark for minimal vaccine effectiveness. Results The heroin-TT vaccine decreased potency of SC heroin (5-fold), IV heroin (3-fold), and IV 6-AM (3-fold) for several weeks without affecting IV morphine or SC and IV fentanyl potency. The control vaccine did not alter potency of any opioid. Naltrexone dose-dependently decreased antinociceptive potency of SC heroin, and treatment with 0.01 mg/kg/h naltrexone produced similar, approximate 8-fold decreases in potencies of SC and IV heroin, IV 6-AM, and IV morphine. The combination of naltrexone and active vaccine was more effective than naltrexone alone to antagonize SC heroin but not IV heroin. Conclusions The heroin-TT vaccine formulation examined is less effective, but more selective, than chronic naltrexone to attenuate heroin antinociception in rats. Furthermore, these results provide an empirical framework for future preclinical opioid vaccine research to benchmark effectiveness against naltrexone.

Preferential interactions of proteins with water and osmolytes play a major role in controlling the thermodynamics of protein solutions. While changes in protein stability and shifts in phase behavior are often reported with the addition of osmolytes, the underlying protein interactions with water and/or osmolytes are typically inferred rather than measured directly. In this work, Kirkwood-Buff integrals for protein-water interactions (G12) and protein-osmolyte interactions (G23) were determined as a function of osmolyte concentration from density measurements of antistreptavidin immunoglobulin gamma-1 (AS-IgG1) in ternary aqueous solutions for a set of common neutral osmolytes: sucrose, trehalose, sorbitol, and poly(ethylene glycol) (PEG). For sucrose and PEG solutions, both protein-water and protein-osmolyte interactions depend strongly on osmolyte concentrations (c3). Strikingly, both osmolytes change from being preferentially excluded to preferentially accumulated with increasing c3. In contrast, sorbitol and trehalose solutions do not show large enough preferential interactions to be detected by densimetry. G12 and G23 values are used to estimate the transfer free energy for native AS-IgG1 (Δμ2N) and compared with existing models. AS-IgG1 unfolding via calorimetry shows a linear increase in midpoint temperatures as a function of trehalose, sucrose, and sorbitol concentrations, but the opposite behavior for PEG. Together, the results highlight limitations of existing models and common assumptions regarding the mechanisms of protein stabilization by osmolytes. Finally, PEG preferential interactions destabilize the Fab regions of AS-IgG1 more so than the CH2 or CH3 domains, illustrating preferential interactions can be specific to different protein domains.

A detailed understanding of the mechanism of unfolding, aggregation, and associated rheological changes is developed in this study for β-Lactoglobulin at different pH values through concomitant measurements utilizing dynamic light scattering (DLS), optical microrheology, Raman spectroscopy, and differential scanning calorimetry (DSC). The diffusion interaction parameter kD emerges as an accurate predictor of colloidal stability for this protein consistent with observed aggregation trends and rheology. Drastic aggregation and gelation were observed at pH 5.5. Under this condition, the protein’s secondary and tertiary structures changed simultaneously. At higher pH (7.0 and 8.5), oligomerizaton with no gel formation occurred. For these solutions, tertiary structure and secondary structure transitions were sequential. The low frequency Raman data, which is a good indicator of hydrogen bonding and structuring in water, has been shown to exhibit a strong correlation with the rheological evolution with temperature. This study has, for the first time, demonstrated that this low frequency Raman data, in conjunction with the DSC endotherm, can be been utilized to deconvolve protein unfolding and aggregation/gelation. These findings can have important implications for the development of protein-based biotherapeutics, where the formulation viscosity, aggregation, and stability strongly affects efficacy or in foods where protein structuring is critical for functional and sensory performance.

The correlation between molecular changes and microstructural evolution of rheological properties has been demonstrated for the first time in a mixed anionic/zwitterionic surfactant-based wormlike micellar system. Utilizing a novel combination of DLS-microrheology and Raman Spectroscopy, the effect of electrostatic screening on these properties of anionic (SLES) and zwitterionic (CapB) surfactant mixtures was studied by modulating the NaCl concentration. As Raman Spectroscopy delivers information about the molecular structure and DLS-microrheology characterizes viscoelastic properties, the combination of data delivered allows for a deeper understanding of the molecular changes underlying the viscoelastic ones. The high frequency viscoelastic response obtained through DLS-microrheology has shown the persistence of the Maxwell fluid response for low viscosity solutions at high NaCl concentrations. The intensity of the Raman band at 170 cm−1 exhibits very strong correlation with the viscosity variation. As this Raman band is assigned to hydrogen bonding, its variation with NaCl concentration additionally indicates differences in water structuring due to potential microstructural differences at low and high NaCl concentrations. The microstructural differences at low and high NaCl concentrations are further corroborated by persistence of a slow mode at the higher NaCl concentrations as seen through DLS measurements. The study illustrates the utility of the combined DLS, DLS-optical microrheology and Raman Spectroscopy in providing new molecular structural insights into the self-assembly process in complex fluids.

Soft matter / complex fluid systems are ubiquitous across a range of industrial and consumer sectors with common examples being inks, paints, drilling fluids, cosmetics, personal care products and foodstuffs. In many instances, the final product format of these complex fluids are gels or soft solids and the processing and product functionality attributes of such materials are often dependent on their rheological response and viscosity. The rheology evolution in such complex fluid systems as a function of formulation parameters (e.g. pH, ionic strength) is intimately connected to corresponding changes in the microand mesostructure and intermolecular and intramolecular associations and interactions. Most insights developed into understanding the self-assembly and rheology evolution process in such systems has primarily focused on elucidating the associated microand mesostructural changes through various scattering (light, x-ray, neutron) and imaging techniques (cryo-TEM, SEM, AFM). Furthermore detailed insights into the associated chemical conformational/structural changes and various non-covalent interactions (e.g. H-bonds, hydrophobic interactions) leading to the selfassembly process have been very limited. An understanding of the changes in the molecular level structural changes as self-assembly and gelation progresses will provide new mechanistic insights that will allow better optimization and control over performance controlling formulation design rules in industrial complex fluids. In this talk, we provide new structural/interaction insights into the self-assembly and gelation mechanism of complex fluids through combination of a number of well-established analytical techniques namely dynamic light scattering (DLS), Raman spectroscopy and optical microrheology. The talk will illustrate the utility of the combination of mesoscale structureproperty elucidation techniques such as DLS/microrheology with the high resolution chemical structure/conformation elucidation techniques such as Raman spectroscopy in generating novel mechanistic insights that will allow the performance engineering of complex fluids and soft matter systems. This will be exemplified through studies into the selfassembly/gelation mechanisms in three very different complex fluids-mixed anionic/zwitterionic surfactant wormlike micelles, polyvinyl alcohol and a thermoreversible, gel-forming agarose biopolymer.

Determination of the physicochemical properties of protein therapeutics and their aggregates is critical for developing formulations that enhance product efficacy, stability, safety and manufacturability. Analytical challenges are compounded for materials: (1) that are formulated at high concentration, (2) that are formulated with a variety of excipients, and (3) that are available only in small volumes. In this article, a new instrument is described that measures protein secondary and tertiary structure, as well as molecular size, over a range of concentrations and formulation conditions of low volume samples. Specifically, characterization of colloidal and conformational stability is obtained through a combination of two well-established analytical techniques: dynamic light scattering (DLS) and Raman spectroscopy, respectively. As the data for these two analytical modalities are collected on the same sample at the same time, the technique enables direct correlation between them, in addition to the more straightforward benefit of minimizing sample usage by providing multiple analytical measurements on the same aliquot non-destructively. The ability to differentiate between unfolding and aggregation that the combination of these techniques provides enables insights into underlying protein aggregation mechanisms. The article will report on mechanistic insights for aggregation that have been obtained from the application of this technique to the characterization of lysozyme, which was evaluated as a function of concentration and pH.

Molecular recognition is central to the design of therapeutics, chemical catalysis and sensors. Motifs for doing so most commonly involve biological structures such as antibodies and aptamers. The key to such biological recognition consists of a folded and constrained heteropolymer that, via intra-molecular forces, forms a unique three dimensional structure that creates a binding pocket or an interface able to recognize a specific molecule. In this work, we demonstrate that synthetic heteropolymers can be alternatively constrained by adsorption around a nanoparticle, and specifically a single walled carbon nanotube (SWNT), forming a corona phase and resulting in a new form of molecular recognition of specific molecules. The phenomenon is shown to be generic, with new heteropolymer recognition complexes demonstrated for three distinct examples: Riboflavin, l-thyroxine, and estradiol, each predicted using a 2D …

Understanding molecular recognition is of fundamental importance in applications such as therapeutics, chemical catalysis and sensor design. The most common recognition motifs involve biological macromolecules such as antibodies and aptamers. The key to biorecognition consists of a unique three-dimensional structure formed by a folded and constrained bioheteropolymer that creates a binding pocket, or an interface, able to recognize a specific molecule. Here, we show that synthetic heteropolymers, once constrained onto a single-walled carbon nanotube by chemical adsorption, also form a new corona phase that exhibits highly selective recognition for specific molecules. To prove the generality of this phenomenon, we report three examples of heteropolymer-nanotube recognition complexes for riboflavin, L-thyroxine and oestradiol. In each case, the recognition was predicted using a two-dimensional thermodynamic model of surface interactions in which the dissociation constants can be tuned by perturbing the chemical structure of the heteropolymer. Moreover, these complexes can be used as new types of spatiotemporal sensors based on modulation of the carbon nanotube photoemission in the near-infrared, as we show by tracking riboflavin diffusion in murine macrophages.