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.

Investigating the chemical impurity profiles of fentanyl preparations and precursors to identify chemical attribution signatures for synthetic method attribution.

From an analytical chemistry standpoint, determining the chemical attribution signatures (CAS) of synthetic reaction mixtures is an impurity profiling exercise. Identifying and understanding the impurity profile and CAS of these chemical agents would allow them to be exploited for chemical forensic information, such as how a particular chemical agent was synthesised. Being able to determine the synthetic route used to make a chemical agent allows for the possibility of batches of the agent, and individual incidents using that agent, to be forensically linked. This information is of particular benefit to agencies investigating the nefarious and illicit use of chemical agents. One such chemical agent of interest to law enforcement and national security agencies is fentanyl. In this study two acylation methods for the final step of fentanyl production, herein termed the Janssen and Siegfried methods, were investigated by liquid chromatography- high resolution mass spectrometry (LC-HRMS) and multivariate statistical analysis (MVA). From these data, fifty-five chemical impurities were identified. Of these, ten were specific CAS for the Janssen method, and five for the Siegfried method. Additionally, analytical data from four different literature methods for production of the fentanyl precursor 4-anilino-N-phenethylpiperidine (ANPP), were compared to the results obtained from the method of production (Valdez) used in this study. Comparison of the LC-HRMS data for these five methods allowed for four Valdez specific impurities to be identified. These may be useful CAS for the Valdez method of ANPP production.

Towards compound identification of synthetic opioids in nontargeted screening using machine learning techniques.

The constant evolution of the illicit drug market makes the identification of unknown compounds problematic. Obtaining certified reference materials for a broad array of new analogues can be difficult and cost prohibitive. Machine learning provides a promising avenue to putatively identify a compound before confirmation against a standard. In this study, machine learning approaches were used to develop class prediction and retention time prediction models. The developed class prediction model used a naïve Bayes architecture to classify opioids as belonging to either the fentanyl analogues, AH series or U series, with an accuracy of 89.5%. The model was most accurate for the fentanyl analogues, most likely due to their greater number in the training data. This classification model can provide guidance to an analyst when determining a suspected structure. A retention time prediction model was also trained for a wide array of synthetic opioids. This model utilised Gaussian process regression to predict the retention time of analytes based on multiple generated molecular features with 79.7% of the samples predicted within ±0.1 min of their experimental retention time. Once the suspected structure of an unknown compound is determined, molecular features can be generated and input for the prediction model to compare with experimental retention time. The incorporation of machine learning prediction models into a compound identification workflow can assist putative identifications with greater confidence and ultimately save time and money in the purchase and/or production of superfluous certified reference materials.

Synthesis and Pharmacology of a Novel µ-δ Opioid Receptor Heteromer-Selective Agonist Based on the Carfentanyl Template.

In this work, we studied a series of carfentanyl amide-based opioid derivatives targeting the mu opioid receptor (µOR) and the delta opioid receptor (δOR) heteromer as a credible novel target in pain management therapy. We identified a lead compound named MP135 that exhibits high G-protein activity at µ-δ heteromers compared to the homomeric δOR or µOR and low ß-arrestin2 recruitment activity at all three. Furthermore, MP135 exhibits distinct signaling profile, as compared to the previously identified agonist targeting µ-δ heteromers, CYM51010. Pharmacological characterization of MP135 supports the utility of this compound as a molecule that could be developed as an antinociceptive agent similar to morphine in rodents. In vivo characterization reveals that MP135 maintains untoward side effects such as respiratory depression and reward behavior; together, these results suggest that optimization of MP135 is necessary for the development of therapeutics that suppress the classical side effects associated with conventional clinical opioids.

Synthesis and pharmacological evaluation of novel cis and trans 3-substituted anilidopiperidines.

BACKGROUND: 4-Anilidopiperidine class of synthetic opioid analgesics, with it's representative fentanyl, are by far the most potent and clinically significant for the treatment of the severe chronic and surgical pain. However, side effects of µ-opioids are often quite serious. In order to improve the pharmacological profile of this class of opioid analgesics, a novel fentanyl analogs were designed, synthesized and evaluated in vivo for their antinociceptive activity. METHODS: The title compounds were prepared using known synthetic transformations, including N-bromoacetamide mediated Hofmann rearrangement, highly selective carbamate cleavage with trimethylsilyl iodide and dehydration of carboxamide group to nitrile in the presence of SOCl2. The antinociceptive activity of the synthesized compounds was determined by tail-immersion and formalin test. RESULTS: The scalable synthetic route towards novel fentanyl analogs bearing nitrogen groups in position C3 of piperidine ring is designed. In addition, Hofmann rearrangement was substantially improved for the more efficient synthesis of previously published 3-substituted fentanyl analogs. The series of ten fentanyl analogs was tested in vivo for their antinociceptive activity. The most potent compound of the series was found to be cis-4, based on the determined ED50 values in tail-immersion test. CONCLUSION: Of ten compounds tested for their antinociceptive activity, compound cis-4 is characterized by high potency, rapid beginning and short duration of action and due to this might be incorporated in different pharmaceutical forms.

An unusual detection of tert-butyl-4-anilinopiperidine-1-carboxylate in seizures of falsified 'Xanax' tablets and in items in a suspected heroin seizure submitted by Irish law enforcement.

The identification of tert-butyl-4-anilinopiperidine-1-carboxylate (4-anilinopiperdine-t-BOC or 4-AP-t-BOC) in many seized falsified 'Xanax' tablets has been reported after being encountered in forensic casework in late 2019 and early 2020 in Ireland. This substance was also detected in a pink powder submitted for analysis in March 2020. The pink powder was part of a larger seizure comprising brown powders which contained morphine or diamorphine (heroin) or a type of counterfeit heroin or heroin adulterant (known as 'bash'). Novel benzodiazepines and other substances are being detected as ingredients in falsified benzodiazepine tablets more frequently on the illicit market. The detection of 4-AP-t-BOC in benzodiazepine tablets is noteworthy and 4-AP-t-BOC is added to the list of adulterants found in benzodiazepine tablets emerging in Europe. The presence of 4-AP-t-BOC in both falsified 'Xanax' and powdered seizures is unusual, and analytical data are presented to assist with the identification of this compound in suspected illicit substances. The presence of 4-AP-t-BOC in the tablets was confirmed using gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry analyses, and spectral fragmentation pathways were suggested. To the authors' best knowledge, information about the biological activity of 4-AP-t-BOC is not available. The removal of the t-BOC protecting group yields 4-anilinopiperidine which has been reported to be involved in the synthesis of fentanyl.

Naloxone Dosing After Opioid Overdose in the Era of Illicitly Manufactured Fentanyl.

INTRODUCTION: Illicitly manufactured fentanyl (IMF) is responsible for a growing number of deaths. Some case series have suggested that IMF overdoses require significantly higher naloxone doses than heroin overdoses. Our objective was to determine if the naloxone dose required to treat an opioid overdose is associated with the finding of fentanyl, opiates, or both on urine drug screen (UDS). METHODS: A retrospective chart review was conducted at a single emergency department and its affiliated emergency medical services (EMS) agency. The charts of all patients who received naloxone through this EMS from 1/1/2017 to 6/15/2018 were reviewed. The study included patients diagnosed with a non-suicidal opioid overdose whose UDS was positive for opiates, fentanyl, or both. Data collected included demographics, vital signs, initial GCS, EMS and ED naloxone administrations, response to treatment, laboratory findings, and ED disposition. The fentanyl-only and fentanyl + opiate groups were compared to the opiate-only group using the stratified (by ED provider) variant of the Mann-Whitney U test. RESULTS: Eight hundred and thirty-seven charts were reviewed, and 121 subjects were included in the final analysis. The median age of included subjects was 38 years and 75% were male. In the naloxone dose analysis, neither the fentanyl-only (median 0.8 mg, IQR 0.4-1.6; p = 0.68) nor the fentanyl + opiate (median 0.8 mg, IQR 0.4-1.2; p = 0.56) groups differed from the opiate-only group (median 0.58 mg, IQR 0.4-1.6). CONCLUSION: Our findings refute the notion that high potency synthetic opioids like illicitly manufactured fentanyl require increased doses of naloxone to successfully treat an overdose. There were no significant differences in the dose of naloxone required to treat opioid overdose patients with UDS evidence of exposure to fentanyl, opiates, or both. Further evaluation of naloxone stocking and dosing protocols is needed.

Application of the fentanyl analog screening kit toward the identification of emerging synthetic opioids in human plasma and urine by LC-QTOF.

Human exposures to fentanyl analogs, which significantly contribute to the ongoing U.S. opioid overdose epidemic, can be confirmed through the analysis of clinical samples. Our laboratory has developed and evaluated a qualitative approach coupling liquid chromatography and quadrupole time-of-flight mass spectrometry (LC-QTOF) to address novel fentanyl analogs and related compounds using untargeted, data-dependent acquisition. Compound identification was accomplished by searching against a locally-established mass spectral library of 174 fentanyl analogs and metabolites. Currently, our library can identify 150 fentanyl-related compounds from the Fentanyl Analog Screening (FAS) Kit), plus an additional 25 fentanyl-related compounds from individual purchases. Plasma and urine samples fortified with fentanyl-related compounds were assessed to confirm the capabilities and intended use of this LC-QTOF method. For fentanyl, 8 fentanyl-related compounds and naloxone, lower reportable limits (LRL100), defined as the lowest concentration with 100 % true positive rate (n = 12) within clinical samples, were evaluated and range from 0.5 ng/mL to 5.0 ng/mL for urine and 0.25 ng/mL to 2.5 ng/mL in plasma. The application of this high resolution mass spectrometry (HRMS) method enables the real-time detection of known and emerging synthetic opioids present in clinical samples.

International Control Efforts to Curb the Global Production and Trafficking of Fentanyl and Other Synthetic Opioids.

The purpose of this Policy Watch column is to provide a brief overview of the global problems associated with the illicit production and trafficking of synthetic opioids as well as international efforts and policy approaches designed to curb them. An in-depth evaluation of drug control efforts of many different nations is important for a comprehensive analysis. However, because of the vast amount of information available, this column is limited to cooperative global control efforts, not efforts specific to any one nation. A great deal of information about production methods, clandestine laboratories, international trafficking methods, and online sales over the dark Web has been omitted. It is important to understand key issues regarding the illicit cultivation and distribution of plant-based opioids derived from natural compounds found in opium poppies (heroin, opium, morphine, codeine) or other drugs (cocaine, methamphetamines, etc.), but the scope of this column is limited to discussion about synthetic opioids including fentanyl, fentanyl analogues, and fentanyl precursor chemicals. Issues about human rights and ethical considerations for nurses and other addictions professionals are also discussed.

Non-Medical Use of Novel Synthetic Opioids: A New Challenge to Public Health.

Background: In the last decade there has been a progressive increase in the use of new psychoactive substances (NPSs) that are not yet under international control. In particular, novel synthetic opioids (NSOs) have reappeared on the recreational drug market in the last few years. As a result, the use of NSOs has increased rapidly. This poses an emerging and demanding challenge to public health. Aim: To raise awareness among clinicians and other professionals about NPSs, especially NSOs, to summarize current knowledge about pharmacological properties, forms of NSO on the market, pattern of use, effects and consequences of use. Methods: An electronic search was carried out on the Medline/PubMed and Google Scholar databases to find selected search terms. Results: Some NPSs are already controlled, while others can be legally sold directly on the drug market (mainly via internet, less so by drug dealers) or be used as precursors for the synthesis of other designer drugs that mimic the psychoactive effects of controlled substances. Potential side-effects of NSOs include miosis, sedation, respiratory depression, hypothermia, inhibition of gastrointestinal propulsion, death (from opioid overdose). Conclusions: The severity of the opioid crisis has intensified with the introduction of highly potent NSOs on the drug market. As long as addicts are dying from overdose or similar causes, there is something more constructive to do than waiting for addicts to overdose on heroin at a place located near a remedy, as if to say, within reach of naloxone.

Synthesis and Biological Evaluation of Fentanyl Analogues Modified at Phenyl Groups with Alkyls.

A series of fentanyl analogues modified at the phenyl group of the phenethyl with alkyl and/or hydroxyl and alkoxy, and the phenyl group in the anilido moiety replaced with benzyl or substituted benzyl, were synthesized. The in vitro opioid receptor functional activity of these compounds was evaluated by assessment of their ability to modulate forskolin-stimulated cAMP accumulation and by their ability to induce ß-arrestin2 recruitment. Compound 12 is a potent µ-opioid (MOP) receptor agonist, a potent κ-opioid (KOP) receptor antagonist with weak ß-arrestin2 recruitment activity. Compounds 10 and 11 are potent MOP receptor agonists with weak δ-opioid (DOP) receptor antagonist activity and moderate KOP receptor antagonist activity as well as weak ß-arrestin2 recruitment activity at the MOP receptor. These compounds are promising leads for discovery of potent opioid analgesics with reduced side effects relative to clinically available strong opioid analgesics.

Determination of Fentanyl Analog Exposure Using Dried Blood Spots with LC-MS-MS.

Fentanyl, and the numerous drugs derived from it, are contributing to the opioid overdose epidemic currently underway in the USA. To identify human exposure to these growing public health threats, an LC-MS-MS method for 5 µL dried blood spots (DBS) was developed. This method was developed to detect exposure to 3-methylfentanyl, alfentanil, α-methylfentanyl, carfentanil, fentanyl, lofentanil, sufentanil, norcarfentanil, norfentanyl, norlofentanil, norsufentanil, and using a separate LC-MS-MS injection, cyclopropylfentanyl, acrylfentanyl, 2-furanylfentanyl, isobutyrylfentanyl, ocfentanil and methoxyacetylfentanyl. Preparation of materials into groups of compounds was used to accommodate an ever increasing need to incorporate newly identified fentanyls. This protocol was validated within a linear range of 1.00-100 ng/mL, with precision ≤12% CV and accuracy ≥93%, as reported for the pooled blood QC samples, and limits of detection as low as 0.10 ng/mL. The use of DBS to assess fentanyl analog exposures can facilitate rapid sample collection, transport, and preparation for analysis that could enhance surveillance and response efforts in the ongoing opioid overdose epidemic.

Detection of Fentanyl Analogs and Synthetic Opioids in Real Hair Samples.

Novel synthetic opioids include various analogs of fentanyl and emerging non-fentanyl compounds with different chemical structures, such as AH-7921, MT-45 and U-47700. In recent years, these drugs have rapidly emerged on the drug market, and their abuse has been increasing worldwide. The motivations for use of these new compounds include their legal status, ready availability, low cost, users' curiosity or preference for their particular pharmacological properties and the intention to avoid detection. Furthermore, more common drugs like heroin are now increasingly being replaced or cut with fentanyl or new designer opioids; thus, many drug users are unintentionally or unknowingly using synthetic fentanyl analogs. In this scenario, the detection of new psychoactive substances in hair can provide insight into their current diffusion among the population and social characteristics of these synthetic drug users. In this manuscript, we describe a simple, fast, specific and sensitive UHPLC-MS-MS method able to detect 13 synthetic opioids (including fentanyl analogs) and metabolites in hair samples. Furthermore, the method includes the detection of 4-anilino-N-phenethyl-piperidine (4-ANPP), which is considered both a precursor and a metabolite of several fentanyl analogs. The method was applied to 34 real hair samples collected in New York City from subjects who had reported past-year non-medical opioid and/or heroin use. In total, 17 samples tested positive for at least one target analyte, with oxycodone (nine samples) and tramadol (eight samples) being the most common. Among these, the method was able to quantify furanyl-fentanyl and fentanyl in the pg/mg range in two samples. Simultaneously, also 4-ANPP was detected, giving evidence for the first time that this compound can be selected as a marker of fentanyl analogs use via hair testing. In conclusion, this study confirmed the increasing diffusion of new synthetic opioids and "fentalogs" with high potency among non-medical opioid users.

Fentanilo: una molécula y múltiples formulaciones galénicas de trascendencia clínica en el tratamiento del dolor irruptivo oncológico / Fentanyl: a molecule and multiple dose formulations of clinical transcendence in the in the treatment of breakthrough cancer pain

El manejo del dolor irruptivo oncológico exige tener prevista una medicación 'de rescate' con opioides de liberación rápida. Sin embargo, pese a la existencia de medios para su control, este es muchas veces inadecuado. La mayoría de los autores señalan que el inicio y la duración de la acción de los opiáceos orales no son las adecuadas para el tratamiento del dolor irruptivo. Los opioides de acción rápida, especialmente el fentanilo, son los fármacos de elección para el tratamiento del dolor irruptivo. En este trabajo se revisan las características de las diferentes formulaciones transmucosas bucales, sublinguales e intranasales de fentanilo que, pese a ser siempre la misma molécula, tienen matices diferenciales de importancia. De hecho, estas presentaciones no son intercambiables. La elección del tratamiento más adecuado para cada paciente debe tener en consideración los perfiles farmacocinéticos de las distintas formulaciones, ya que estas definen cuándo se inicia el efecto, cuánto dura la analgesia y cuál es el nivel del pico plasmático máximo (Cmax), muchas veces responsable de los efectos adversos. La bibliografía general y diversos consensos de expertos recomiendan individualizar el tratamiento tras una cuidadosa evaluación del dolor. Las dosis de productos de fentanilo administradas deben tener en cuenta el nivel de tolerancia opioide de los pacientes, el perfil farmacocinético de cada preparado y las condiciones de cada paciente en relación con las vías de administración. En determinadas circunstancias clínicas, las presentaciones de fentanilo intranasal parece tener un inicio de acción más rápido y ser mejor toleradas desde el punto digestivo que las presentaciones transmucosas orales. La preferencia del paciente debe también tenerse en consideración (AU)

The breakthrough cancer pain management requires planned a 'rescue' medication with quick-release opioids. However, despite the existence of media for their control, this is often inappropriate. Most of the authors point out that the onset and duration of action of oral opioids are not appropriate for the treatment of breakthrough pain. Fast-acting opioid, notably fentanyl, are the drugs of choice for the treatment of breakthrough pain. This paper reviews the characteristics of the different transmucosal formulations of fentanyl that, despite the same molecule, have differential degrees of importance. In fact, these presentations are not interchangeable. The choice of the most appropriate treatment for each patient should take into consideration the different Pharmacokinetic profiles, since they define when the effect starts, how long lasting analgesia and what is the level of the plasma peak (Cmax), often responsible for the adverse effects. General bibliography and various consensus of experts recommended to individualize the treatment after a carefully evaluation of the pain. Administered doses of different fentanyl formulations must take into account patient’s opioid tolerance, the pharmacokinetic profile of each products and conditions of each patient in relation to the routes of administration. In certain clinical circumstances, intranasal fentanyl appears to have a faster onset of action and be better tolerated at the gastrointestinal level that buccal presentations. The preference of the patient must also be taken into consideration (AU)

An updated synthesis of [11 C]carfentanil for positron emission tomography (PET) imaging of the µ-opioid receptor.

[11 C]Carfentanil ([11 C]CFN) is a selective radiotracer for in vivo positron emission tomography imaging studies of the µ-opioid system that, in our laboratories, is synthesized by methylation of the corresponding carboxylate precursor with [11 C]MeOTf, and purified using a C2 solid-phase extraction cartridge. Changes in the commercial availability of common C2 cartridges have necessitated future proofing the synthesis of [11 C]CFN to maintain reliable delivery of the radiotracer for clinical imaging studies. An updated synthesis of [11 C]CFN is reported that replaces a now obsolete purification cartridge with a new commercially available version and also substitutes the organic solvents used in traditional production methods with ethanol.

Chemical Attribution of Fentanyl Using Multivariate Statistical Analysis of Orthogonal Mass Spectral Data.

Attribution of the origin of an illicit drug relies on identification of compounds indicative of its clandestine production and is a key component of many modern forensic investigations. The results of these studies can yield detailed information on method of manufacture, starting material source, and final product, all critical forensic evidence. In the present work, chemical attribution signatures (CAS) associated with the synthesis of the analgesic fentanyl, N-(1-phenylethylpiperidin-4-yl)-N-phenylpropanamide, were investigated. Six synthesis methods, all previously published fentanyl synthetic routes or hybrid versions thereof, were studied in an effort to identify and classify route-specific signatures. A total of 160 distinct compounds and inorganic species were identified using gas and liquid chromatographies combined with mass spectrometric methods (gas chromatography/mass spectrometry (GC/MS) and liquid chromatography-tandem mass spectrometry-time of-flight (LC-MS/MS-TOF)) in conjunction with inductively coupled plasma mass spectrometry (ICPMS). The complexity of the resultant data matrix urged the use of multivariate statistical analysis. Using partial least-squares-discriminant analysis (PLS-DA), 87 route-specific CAS were classified and a statistical model capable of predicting the method of fentanyl synthesis was validated and tested against CAS profiles from crude fentanyl products deposited and later extracted from two operationally relevant surfaces: stainless steel and vinyl tile. This work provides the most detailed fentanyl CAS investigation to date by using orthogonal mass spectral data to identify CAS of forensic significance for illicit drug detection, profiling, and attribution.