Fentanyl-Type Antagonist of the µ-Opioid Receptor: Important Role of Axial Chirality in the Active Conformation.

In recent years, synthetic opioids have emerged as a predominant cause of drug-overdose-related fatalities, causing the "opioid crisis." To design safer therapeutic agents, we accidentally discovered µ-opioid receptor (MOR) antagonists based on fentanyl with a relatively uncomplicated chemical composition that potentiates structural modifications. Here, we showed the development of novel atropisomeric fentanyl analogues that exhibit more potent antagonistic activity against MOR than naloxone, a morphinan MOR antagonist. Derivatives displaying stable axial chirality were synthesized based on the amide structure of fentanyl. The aS- and aR-enantiomers exerted antagonistic and agonistic effects on the MOR, respectively, and each atropisomer interacted with the MOR by assuming a distinct binding mode through molecular docking. These findings suggest that introducing atropisomerism into fentanyl may serve as a key feature in the molecular design of future MOR antagonists to help mitigate the opioid crisis.

Elucidating the Gas-Phase Behavior of Nitazene Analog Protomers Using Structures for Lossless Ion Manipulations Ion Mobility-Orbitrap Mass Spectrometry.

2-Benzylbenzimidazoles, or "nitazenes", are a class of novel synthetic opioids (NSOs) that are increasingly being detected alongside fentanyl analogs and other opioids in drug overdose cases. Nitazenes can be 20× more potent than fentanyl but are not routinely tested for during postmortem or clinical toxicology drug screens; thus, their prevalence in drug overdose cases may be under-reported. Traditional analytical workflows utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS) often require additional confirmation with authentic reference standards to identify a novel nitazene. However, additional analytical measurements with ion mobility spectrometry (IMS) may provide a path toward reference-free identification, which would greatly accelerate NSO identification rates in toxicology laboratories. Presented here are the first IMS and collision cross section (CCS) measurements on a set of fourteen nitazene analogs using a structures for lossless ion manipulations (SLIM)-orbitrap MS. All nitazenes exhibited two high intensity baseline-separated IMS distributions, which fentanyls and other drug and druglike compounds also exhibit. Incorporating water into the electrospray ionization (ESI) solution caused the intensities of the higher mobility IMS distributions to increase and the intensities of the lower mobility IMS distributions to decrease. Nitazenes lacking a nitro group at the R1 position exhibited the greatest shifts in signal intensities due to water. Furthermore, IMS-MS/MS experiments showed that the higher mobility IMS distributions of all nitazenes possessing a triethylamine group produced fragment ions with m/z 72, 100, and other low intensity fragments while the lower mobility IMS distributions only produced fragment ions with m/z 72 and 100. The IMS, solvent, and fragmentation studies provide experimental evidence that nitazenes potentially exhibit three gas-phase protomers. The cyclic IMS capability of SLIM was also employed to partially resolve four sets of structurally similar nitazene isomers (e.g., protonitazene/isotonitazene, butonitazene/isobutonitazene/secbutonitazene), showcasing the potential of using high-resolution IMS separations in MS-based workflows for reference-free identification of emerging nitazenes and other NSOs.

Dissecting the Role of the Hydroxyl Moiety at C14 in (+)-Opioid-Based TLR4 Antagonists via Wet-Lab Experiments and Molecular Dynamics Simulations.

Toll-like receptor 4 (TLR4) is pivotal as an innate immune receptor, playing a critical role in mediating neuropathic pain and drug addiction through its regulation of the neuroinflammatory response. The nonclassical (+)-opioid isomers represent a unique subset of TLR4 antagonists known for their effective blood-brain barrier permeability. Despite growing interest in the structure-activity relationship of these (+)-opioid-based TLR4 antagonists, the specific impact of heteroatoms on their TLR4 antagonistic activities has not been fully explored. This study investigated the influence of the hydroxyl group at C14 in six (+)-opioid TLR4 antagonists (1-6) using wet-lab experiments and in silico simulations. The corresponding C14-deoxy derivatives (7-12) were synthesized, and upon comparison with their corresponding counterparts (1-6), it was discovered that their TLR4 antagonistic activities were significantly diminished. Molecular dynamics simulations showed that the (+)-opioid TLR4 antagonists (1-6) possessed more negative binding free energies to the TLR4 coreceptor MD2, which was responsible for ligand recognition. This was primarily attributed to the formation of a hydrogen bond between the hydroxyl group at the C-14 position of the antagonists (1-6) and the R90 residue of MD2 during the binding process. Such an interaction facilitated the entry and subsequent binding of these molecules within the MD2 cavity. In contrast, the C14-deoxy derivatives (7-12), lacking the hydroxyl group at the C-14 position, missed this crucial hydrogen bond interaction with the R90 residue of MD2, leading to their egression from the MD2 cavity during simulations. This study underscores the significant role of the C14 hydroxyl moiety in enhancing the effectiveness of (+)-opioid TLR4 antagonists, which provides insightful guidance for designing future (+)-isomer opioid-derived TLR4 antagonists.

TET1-Lipid Nanoparticle Encapsulating Morphine for Specific Targeting of Peripheral Nerve for Pain Alleviation.

Background: Opioids are irreplaceable analgesics owing to the lack of alternative analgesics that offer opioid-like pain relief. However, opioids have many undesirable central side effects. Restricting opioids to peripheral opioid receptors could reduce those effects while maintaining analgesia. Methods: To achieve this goal, we developed Tet1-LNP (morphine), a neural-targeting lipid nanoparticle encapsulating morphine that could specifically activate the peripheral opioid receptor in the dorsal root ganglion (DRG) and significantly reduce the side effects caused by the activation of opioid receptors in the brain. Tet1-LNP (morphine) were successfully prepared using the thin-film hydration method. In vitro, Tet1-LNP (morphine) uptake was assessed in differentiated neuron-like PC-12 cells and dorsal root ganglion (DRG) primary cells. The uptake of Tet1-LNP (morphine) in the DRGs and the brain was assessed in vivo. Von Frey filament and Hargreaves tests were used to assess the antinociception of Tet1-LNP (morphine) in the chronic constriction injury (CCI) neuropathic pain model. Morphine concentration in blood and brain were evaluated using ELISA. Results: Tet1-LNP (morphine) had an average size of 131 nm. Tet1-LNP (morphine) showed high cellular uptake and targeted DRG in vitro. CCI mice treated with Tet1-LNP (morphine) experienced prolonged analgesia for nearly 32 h compared with 3 h with free morphine (p < 0.0001). Notably, the brain morphine concentration in the Tet1-LNP (morphine) group was eight-fold lower than that in the morphine group (p < 0.0001). Conclusion: Our study presents a targeted lipid nanoparticle system for peripheral neural delivery of morphine. We anticipate Tet1-LNP (morphine) will offer a safe formulation for chronic neuropathic pain treatment, and promise further development for clinical applications.

Decoding the κ Opioid Receptor (KOR): Advancements in Structural Understanding and Implications for Opioid Analgesic Development.

The opioid crisis in the United States is a significant public health issue, with a nearly threefold increase in opioid-related fatalities between 1999 and 2014. In response to this crisis, society has made numerous efforts to mitigate its impact. Recent advancements in understanding the structural intricacies of the κ opioid receptor (KOR) have improved our knowledge of how opioids interact with their receptors, triggering downstream signaling pathways that lead to pain relief. This review concentrates on the KOR, offering crucial structural insights into the binding mechanisms of both agonists and antagonists to the receptor. Through comparative analysis of the atomic details of the binding site, distinct interactions specific to agonists and antagonists have been identified. These insights not only enhance our understanding of ligand binding mechanisms but also shed light on potential pathways for developing new opioid analgesics with an improved risk-benefit profile.

Strategies for developing µ opioid receptor agonists with reduced adverse effects.

Opioids are currently the most effective and widely used painkillers in the world. Unfortunately, the clinical use of opioid analgesics is limited by serious adverse effects. Many researchers have been working on designing and optimizing structures in search of novel µ opioid receptor(MOR) agonists with improved analgesic activity and reduced incidence of adverse effects. There are many strategies to develop MOR drugs, mainly focusing on new low efficacy agonists (potentially G protein biased agonists), MOR agonists acting on different Gα subtype, targeting opioid receptors in the periphery, acting on multiple opioid receptor, and targeting allosteric sites of opioid receptors, and others. This review summarizes the design methods, clinical applications, and structure-activity relationships of small-molecule agonists for MOR based on these different design strategies, providing ideas for the development of safer novel opioid ligands with therapeutic potential.

High-performance fentanyl molecularly imprinted electrochemical sensing platform designed through molecular simulations.

BACKGROUND: Fentanyl and its derivatives are a type of potent opioid analgesics, with the characteristics of diverse structure, high toxicity, extremely low content, and high fatality rate. Currently, they have become one of the most serious problems in international drug abuse control due to their extensive use in drug production and use. Therefore, the development of a rapid, sensitive, and accurate method for detecting trace fentanyl is of great significance. In this study, in view of its complex structure and trace concentration, a new molecular imprinting electrochemical sensor was developed through molecular simulations followed by experimental validation to detect trace fentanyl. RESULTS: The process consisted of first obtaining the optimal functional monomer and its molar ratio through molecular simulations. The recognition sites of fentanyl-imprinted polymers were predicted to guide the synthesis of imprinted membranes with precision approach to ensure an efficient and accurate reaction process. Reduced graphene oxide (ErGO) was then deposited on glassy carbon electrode surface by electrochemical reduction to yield large numbers of active sites suitable for catalyzing reactions of fentanyl piperidine for promoted efficient electron transfer and amplified sensitivity of the sensor. Accordingly, fentanyl molecularly imprinted film was formed through one-step electropolymerization to yield greatly improved sensing selectivity due to the specific recognition of molecularly imprinted polymer. Under optimal experimental conditions, the fentanyl sensor showed an extended detection range of 3.84 × 10-9 mol L-1-1.72 × 10-6 mol L-1 and a detection limit of 1.28 × 10-9 mol L-1. SIGNIFICANCE: A distinctive feature of this sensor is its molecularly imprinted polymerized membrane, which offers excellent specific recognition, thereby boosting the sensor's selectivity. Throughout the sensor's development process, molecular simulations were employed to steer the synthesis of molecularly imprinted polymers and predict the recognition sites of fentanyl-imprinted polymers. The experimental outcomes proved to align with the simulation data. The final sensor exhibited outstanding selectivity, repeatability, stability, and high sensitivity. The sensor was effectively used to reliably track fentanyl in human serum samples, with acceptable analytical reliability, suggesting its potential for practical applications.

Amphiphilic NLC-Gel formulation loaded with Sebacoyl dinalbuphine ester and Nalbuphine for localized postoperative pain management.

Opioids are powerful analgesics; however, their significant systemic adverse effects and the need for frequent administration restrict their use. Nalbuphine (NA) is a κ-agonist narcotic with limited adverse effects, but needs to be frequently administrated due to its short elimination half-life. Whereas sebacoyl dinalbuphine ester (SDE) is a NA prodrug, which can effectively prolong the analgesic effect, but lacks immediate pain relief. Therefore, in this study, a rapid and sustained local delivery formulation to introduce NA and SDE directly into surgical sites was developed. An amphiphilic nanostructured lipid carrier (NLC) poloxamer 407 (P407) gel (NLC-Gel) was developed to permit concurrent delivery of hydrophobic SDE from the NLC core and hydrophilic NA from P407, offering a dual rapid and prolonged analgesic effect. Benefiting from the thermal-sensitive characteristic of P407, the formulation can be injected in liquid phase and instantly transit into gel at wound site. NLC-Gel properties, including particle size, drug release, rheology, and stability, were assessed. In vivo evaluation using a rat spinal surgery model highlighted the effect of the formulation through pain behavior test and hematology analysis. NLC-Gels demonstrated an analgesic effect comparable with that of commercial intramuscular injected SDE formulation (IM SDE), with only 15 % of the drug dosage. The inclusion of supplemental NA in the exterior gel (PA12-Gel + NA) provided rapid drug onset owing to swift NA dispersion, addressing acute pain within hours along with prolonged analgesic effects. Our findings suggest that this amphiphilic formulation significantly enhanced postoperative pain management in terms of safety and efficacy.

Searching for Synthetic Opioid Rescue Agents: Identification of a Potent Opioid Agonist with Reduced Respiratory Depression.

While in the process of designing more effective synthetic opioid rescue agents, we serendipitously identified a new chemotype of potent synthetic opioid. Here, we report that conformational constraint of a piperazine ring converts a mu opioid receptor (MOR) antagonist into a potent MOR agonist. The prototype of the series, which we have termed atoxifent (2), possesses potent in vitro agonist activity. In mice, atoxifent displayed long-lasting antinociception that was reversible with naltrexone. Repeated dosing of atoxifent produced antinociceptive tolerance and a level of withdrawal like that of fentanyl. In rats, while atoxifent produced complete loss of locomotor activity like fentanyl, it failed to produce deep respiratory depression associated with fentanyl-induced lethality. Assessment of brain biodistribution demonstrated ample distribution of atoxifent into the brain with a Tmax of approximately 0.25 h. These results indicate enhanced safety for atoxifent-like molecules compared to fentanyl.

Colorimetric Detection of Fentanyl Powder on Surfaces Using a Supramolecular Displacement Assay.

Fentanyl is a potent synthetic opioid with an alarmingly low lethal dosage of 2 mg. The equipment necessary to detect fentanyl in field settings (e.g., hand-held spectrometers) is restricted to highly trained, well-funded, and specialized personnel. Established point-of-need technologies, such as lateral flow immunochromatographic strips, are available; however, they often involve multiple contact-based steps (e.g., collection, mixing) that pose a higher risk to users handling unknown substances. Herein, we developed a colorimetric displacement assay capable of contactless detection of fentanyl in liquid or solid samples. The basis of our assay relies on the presence of fentanyl to displace a redox mediator, ferrocene carboxylic acid, inclusively bound in the cavity of a supramolecular host, CB[7]. The displacement is only possible in the presence of high affinity binding guests, like fentanyl (KA ∼ 106 M-1). The liberated redox guest can then react with indicator reagents that are free in solution, producing either: (i) a distinct blue color to indicate the presence of fentanyl or (ii) a pale blue tint in the absence of fentanyl. We demonstrate rapid and specific detection of fentanyl free base and fentanyl derivatives (e.g., acetyl fentanyl and furanyl fentanyl) against a panel of 9 other common drugs of abuse (e.g., morphine, cocaine, and heroin). Furthermore, we highlight the intended use of this assay by testing grains of fentanyl derivatives on a surface with a drop (i.e., 25 µL) of the assay reagent. We anticipate that this approach can be applied broadly to identify the presence of fentanyl at the point of need.

Blocking potential metabolic sites on NAT to improve its safety profile while retaining the pharmacological profile.

The number of opioid-related overdose deaths and individuals that have suffered from opioid use disorders have significantly increased over the last 30 years. FDA approved maintenance therapies to treat opioid use disorder may successfully curb drug craving and prevent relapse but harbor adverse effects that reduce patient compliance. This has created a need for new chemical entities with improved patient experience. Previously our group reported a novel lead compound, NAT, a mu-opioid receptor antagonist that potently antagonized the antinociception of morphine and showed significant blood-brain barrier permeability. However, NAT belongs to thiophene containing compounds which are known structural alerts for potential oxidative metabolism. To overcome this, 15 NAT derivatives with various substituents at the 5'-position of the thiophene ring were designed and their structure-activity relationships were studied. These derivatives were characterized for their binding affinity, selectivity, and functional activity at the mu opioid receptor and assessed for their ability to antagonize the antinociceptive effects of morphine in vivo. Compound 12 showed retention of the basic pharmacological attributes of NAT while improving the withdrawal effects that were experienced in opioid-dependent mice. Further studies will be conducted to fully characterize compound 12 to examine whether it would serve as a new lead for opioid use disorder treatment and management.

Classics in Chemical Neuroscience: Xylazine.

Xylazine (also known as "tranq") is a potent nonopioid veterinary sedative that has recently experienced a surge in use as a drug adulterant, most often combined with illicitly manufactured fentanyl. This combination may heighten the risk of fatal overdose. Xylazine has no known antidote approved for use in humans, and age-adjusted overdose deaths involving xylazine were 35 times higher in 2021 than 2018. In April 2023, the Biden Administration declared xylazine-laced fentanyl an emerging drug threat in the United States. In 2022, the Drug Enforcement Agency (DEA) reported nearly a quarter of seized fentanyl powder contained xylazine. This dramatic increase in prevalence has solidified the status of xylazine as an emerging drug of abuse and an evolving threat to public health. The following narrative review outlines the synthesis, pharmacokinetics, pharmacodynamics, and adverse effects of xylazine, as well as the role it may play in the ongoing opioid epidemic.

Multidimensional opioid abuse deterrence using a nanoparticle-polymer hybrid formulation.

Misuse of prescription opioid drugs is the leading cause of the opioid crisis and overdose-related death. Abuse deterrent formulations (ADFs) have been developed to discourage attempts to tamper with the formulation and alter the ingestion methods. However, abusers develop complex extraction strategies to circumvent the ADF technologies. For comprehensive deterrence of drug abuse, we develop tannic acid nanoparticles (NPs) that protect encapsulated opioids from solvent extraction and thermal challenge (crisping), complementing the existing formulation strategy to deter injection abuse. Here, we develop a hybrid ADF tablet (NP-Tab), consisting of iron-crosslinked tannic acid NPs encapsulating thebaine (model opioid compound), xanthan gum, and chitosan (gel-forming polymers), and evaluate its performance in common abuse conditions. NP-Tab tampered by crushing and suspended in aqueous solvents forms an instantaneous gel, which is difficult to pull or push through a 21-gauge needle. NPs insulate the drug from organic solvents, deterring solvent extraction. NPs also promote thermal destruction of the drug to make crisping less rewarding. However, NP-Tab releases thebaine in the simulated gastric fluid without delay, suggesting that its analgesic effect may be unaffected if consumed orally as prescribed. These results demonstrate that NP-Tab can provide comprehensive drug abuse deterrence, resisting aqueous/organic solvent extraction, injection, and crisping, while retaining its therapeutic effect upon regular usage.

Synthesis, characterization, and biological study of new synthetic opioid hemorphin-4 peptides containing sterically restricted nonnatural amino acids.

Some new hemorphin-4 analogs with structures of Xxx-Pro-Trp-Thr-NH2 and Tyr-Yyy-Trp-Thr-NH2, where Xxx is 2-amino-3-(4-hydroxy-2,6-dimethylphenyl)propanoic acid or 2-amino-3-(4-dibenzylamino-2,6-dimethylphenyl)propanoic acid, and Yyy is (2S,4S)-4-amino-pyrrolidine-2-carboxylic acid, were synthesized and characterized by electrochemical and spectral analyses. In vivo anticonvulsant and antinociceptive activities of peptide derivatives were studied after intracerebroventricular injection in mice. The therapeutic effects of the modified peptides on seizures and pain in mice were evaluated to provide valuable insights into the potential applications of the novel compounds. Electrochemical characterization showed that the compounds behave as weak protolytes and that they are in a soluble, stable molecular form at physiological pH values. The antioxidant activity of the peptides was evaluated with voltammetric analyses, which were confirmed by applying the 2,2-Diphenyl-1-picrylhydrazyl method. The compounds showed satisfactory results regarding their structural stability, reaching the desired centers for the manifestation of biological activity without hydrolysis processes at 37°C and physiological pH. Dm-H4 and H4-P1 exhibited 100% and 83% potency to suppress the psychomotor seizures in the 6-Hz test compared to 67% activity of H4. Notably, only the H4-P1 had efficacy in blocking the tonic component in the maximal electroshock test with a potency comparable to H4. All investigated peptides containing unnatural conformationally restricted amino acids showed antinociceptive effects. The analogs Db-H4 and H4-P1 showed the most pronounced and long-lasting effect in both experimental models of pain induced by thermal and chemical stimuli. Dm-H4 produced a dose-dependent thermal antinociception and H4-P2 inhibited only formalin-induced pain behavior.

Multitarget µ-Opioid Receptor Agonists─Neuropeptide FF Receptor Antagonists Induce Potent Antinociception with Reduced Adverse Side Effects.

The design of bifunctional compounds is a promising approach toward the development of strong analgesics with reduced side effects. We here report the optimization of the previously published lead peptide KGFF09, which contains opioid receptor agonist and neuropeptide FF receptor antagonist pharmacophores and is shown to induce potent antinociception and reduced side effects. We evaluated the novel hybrid peptides for their in vitro activity at MOP, NPFFR1, and NPFFR2 and selected four of them (DP08/14/32/50) for assessment of their acute antinociceptive activity in mice. We further selected DP32 and DP50 and observed that their antinociceptive activity is mostly peripherally mediated; they produced no respiratory depression, no hyperalgesia, significantly less tolerance, and strongly attenuated withdrawal syndrome, as compared to morphine and the recently FDA-approved TRV130. Overall, these data suggest that MOP agonist/NPFF receptor antagonist hybrids might represent an interesting strategy to develop novel analgesics with reduced side effects.

Development of a Potential-Modulated Electrochemiluminescence Measurement System for Selective and Sensitive Determination of the Controlled Drug Codeine.

Codeine is a common analgesic drug that is a pro-drug of morphine. It also has a high risk of abuse as a recreational drug because of its extensive distribution as an OTC drug. Therefore, sensitive and selective screening methods for codeine are crucial in forensic analytical chemistry. To date, a commercial analytical kit has not been developed for dedicated codeine determination, and there is a need for an analytical method to quantify codeine in the field. In the present work, potential modulation was combined with electrochemiluminescence (ECL) for sensitive determination of codeine. The potential modulated technique involved applying a signal to electrodes by superimposing an AC potential on the DC potential. When tris(2,2'-bipyridine)ruthenium(II) ([Ru(bpy)3]2+) was used as an ECL emitter, ECL activity was confirmed for codeine. A detailed investigation of the electrochemical reaction mechanism suggested a characteristic ECL reaction mechanism involving electrochemical oxidation of the opioid framework. Besides the usual ECL reaction derived from the amine framework, selective detection of codeine was possible under the measurement conditions, with clear luminescence observed in an acidic solution. The sensitivity of codeine detection by potential modulated-ECL was one order of magnitude higher than that obtained with the conventional potential sweep method. The proposed method was applied to codeine determination in actual prescription medications and OTC drug samples. Codeine was selectively determined from other compounds in medications and showed good linearity with a low detection limit (150 ng mL-1).

Morphinan Evolution: The Impact of Advances in Biochemistry and Molecular Biology.

Morphinan-based opioids, derived from natural alkaloids like morphine, codeine and thebaine, have long been pivotal in managing severe pain. However, their clinical utility is marred by significant side effects and high addiction potential. This review traces the evolution of the morphinan scaffold in light of advancements in biochemistry and molecular biology, which have expanded our understanding of opioid receptor pharmacology. We explore the development of semi-synthetic and synthetic morphinans, their receptor selectivity and the emergence of biased agonism as a strategy to dissociate analgesic properties from undesirable effects. By examining the molecular intricacies of opioid receptors and their signaling pathways, we highlight how receptor-type selectivity and signaling bias have informed the design of novel analgesics. This synthesis of historical and contemporary perspectives provides an overview of the morphinan landscape, underscoring the ongoing efforts to mitigate the problems facing opioids through smarter drug design. We also highlight that most morphinan derivatives show a preference for the G protein pathway, although detailed experimental comparisons are still necessary. This fact underscores the utility of the morphinan skeleton in future opioid drug discovery.

Assessing Abuse-Deterrent formulations utilizing Ion-Exchange resin complexation processed via Twin-Screw granulation for improved safety and effectiveness.

Opioid misuse is a public health crisis in the United States. In response, the FDA has approved drug products with abuse-deterrent features to reduce the risk of prescription opioid abuse. Abuse-deterrent formulations (ADFs) typically employ physical or chemical barriers or incorporate agonist-antagonist combinations as mechanisms to deter misuse. This study aims to assess the impact of abuse-deterrent properties, specifically ion-exchange resin complexation as a chemical barrier, on a model drug, promethazine hydrochloride (PMZ) tablets. Various formulations were developed through twin-screw wet granulation (TSWG) followed by twin-screw melt granulation (TSMG). In the TSWG process, the drug interacts with the resin through an exchange reaction, forming a drug-resin complex. Additionally, the study explored factors influencing the complex formation between the drug and resin, using the drug loading status as an indicator. DSC and ATR studies were carried out to confirm the formation of the drug-resin complex. Subsequently, hot melt granulation was employed to create a matrix tablet incorporating Kollidon® SR and Kollicoat® MAE 100P, thereby enabling sustained release properties. The drug-resin complex embedded in the matrix effectively deters abuse through methods like smoking, snorting, or parenteral injection, unless the drug can be extracted. In order to assess this, solvent extraction studies were conducted using an FDA-recommended solvents, determining the potential for abuse. Further investigations involved dissolution tests in change-over media, confirming the extended-release properties of the formulation. Results from dissolution studies comparing the ground and intact tablets provided positive evidence of the formulation's effectiveness in deterring abuse. Finally, alcohol-induced dose-dumping studies were conducted in compliance with FDA guidelines, concluding that the formulation successfully mitigates dose dumping in the presence of alcohol.

In vitro µ-opioid receptor activation potential of U10 and ß-U10, positional isomers of the synthetic opioid naphthyl U-47700.

New synthetic opioids (NSOs) with diverse chemical structures continue to appear on recreational drug markets worldwide. U-type opioids have become one of the largest groups of non-fentanyl-related NSOs. Starting in 2020, a previously unreported U-compound coined "ß-U10" (2-naphthyl U-47700; N-[2-(dimethylamino)cyclohexyl]-N-methylnaphthalene-2-carboxamide) was identified in Australia and the United States. ß-U10 is a positional isomer of α-U10 (1-naphthyl U-47700), more commonly known as "U10." Here, the first comparative in vitro pharmacological characterization of naphthyl U-47700 (U10 and ß-U10), together with the structural analogue U-47700 and fentanyl, is reported. Application of a cell-based µ-opioid receptor (MOR) activation (ß-arrestin 2 recruitment) assay demonstrated ß-U10 (EC50 = 348 nM; Emax = 150% vs. hydromorphone) to be less potent than U-47700 (EC50 = 116 nM; Emax = 154%) and fentanyl (EC50 = 9.35 nM; Emax = 146%) but considerably more active than the α-isomer (EC50 value in the µM range). For the latter, maximum receptor activation could not be reached at 100 µM. The difference in MOR activation potential for U10 and ß-U10 stresses the importance of (analytical) differentiation between closely related analytes. The emergence of ß-U10 on the recreational drug market is an example of the continuing emergence of non-fentanyl-related NSOs and further emphasizes the need to closely monitor fluctuations in the drug supply.

Design and structural validation of peptide-drug conjugate ligands of the kappa-opioid receptor.

Despite the increasing number of GPCR structures and recent advances in peptide design, the development of efficient technologies allowing rational design of high-affinity peptide ligands for single GPCRs remains an unmet challenge. Here, we develop a computational approach for designing conjugates of lariat-shaped macrocyclized peptides and a small molecule opioid ligand. We demonstrate its feasibility by discovering chemical scaffolds for the kappa-opioid receptor (KOR) with desired pharmacological activities. The designed De Novo Cyclic Peptide (DNCP)-ß-naloxamine (NalA) exhibit in vitro potent mixed KOR agonism/mu-opioid receptor (MOR) antagonism, nanomolar binding affinity, selectivity, and efficacy bias at KOR. Proof-of-concept in vivo efficacy studies demonstrate that DNCP-ß-NalA(1) induces a potent KOR-mediated antinociception in male mice. The high-resolution cryo-EM structure (2.6 Å) of the DNCP-ß-NalA-KOR-Gi1 complex and molecular dynamics simulations are harnessed to validate the computational design model. This reveals a network of residues in ECL2/3 and TM6/7 controlling the intrinsic efficacy of KOR. In general, our computational de novo platform overcomes extensive lead optimization encountered in ultra-large library docking and virtual small molecule screening campaigns and offers innovation for GPCR ligand discovery. This may drive the development of next-generation therapeutics for medical applications such as pain conditions.