Medial prefrontal neuroplasticity during extended-release naltrexone treatment of opioid use disorder - a longitudinal structural magnetic resonance imaging study.

Opioid use disorder (OUD) has been linked to macroscopic structural alterations in the brain. The monthly injectable, extended-release formulation of µ-opioid antagonist naltrexone (XR-NTX) is highly effective in reducing opioid craving and preventing opioid relapse. Here, we investigated the neuroanatomical effects of XR-NTX by examining changes in cortical thickness during treatment for OUD. Forty-seven OUD patients underwent structural magnetic resonance imaging and subjectively rated their opioid craving ≤1 day before (pre-treatment) and 11 ± 3 days after (on-treatment) the first XR-NTX injection. A sample of fifty-six non-OUD individuals completed a single imaging session and served as the comparison group. A publicly available [¹¹C]carfentanil positron emission tomography dataset was used to assess the relationship between changes in cortical thickness and µ-opioid receptor (MOR) binding potential across brain regions. We found that the thickness of the medial prefrontal and anterior cingulate cortices (mPFC/aCC; regions with high MOR binding potential) was comparable between the non-OUD individuals and the OUD patients at pre-treatment. However, among the OUD patients, mPFC/aCC thickness significantly decreased from pre-treatment to on-treatment. A greater reduction in mPFC/aCC thickness was associated with a greater reduction in opioid craving. Taken together, our study suggests XR-NTX-induced cortical thickness reduction in the mPFC/aCC regions in OUD patients. The reduction in thickness does not appear to indicate a restoration to the non-OUD level but rather reflects XR-NTX's distinct therapeutic impact on an MOR-rich brain structure. Our findings highlight the neuroplastic effects of XR-NTX that may inform the development of novel OUD interventions.

CARDIORESPIRATORY EFFECTS OF VATINOXAN IN BLESBOK (DAMALISCUS PYGARGUS PHILLIPSI) IMMOBILIZED WITH THIAFENTANIL-MEDETOMIDINE.

Combinations of a low dose of opioid, such as thiafentanil, and a high dose of medetomidine, are increasingly being used for immobilization of African ungulates. Both drugs can have undesirable cardiorespiratory effects. In this study we assessed whether vatinoxan, a peripherally acting alpha2-adrenergic receptor antagonist, can be used to alleviate some of these effects without affecting the immobilization quality. Eight healthy, female, boma-confined blesbok (Damaliscus pygargus phillipsi), weighing a mean (SDtion) of 56.8 (4.4) kg, were immobilized twice in a randomized cross-over study with a 2-wk washout period using (1) 0.5 mg thiafentanil + 1.5 mg medetomidine (TM), (2) TM + vatinoxan: 0.5 mg thiafentanil + 1.5 mg medetomidine + 15 mg vatinoxan per milligram medetomidine (total of 22.5 mg, administered intramuscularly at 10 min post recumbency). Heart rate, respiratory rate, rectal temperature, oxygen saturation (SpO2), arterial blood pressure, and sedation scores from 1 to 5 (1 = limited effect; 5 = excessively deep) were measured every 5 min. Arterial blood gases (PaO2 and PaCO2) were measured at 10, 15, 25, and 35 min postrecumbency and the alveolar--arterial oxygen gradient (P[A-a]O2) was calculated. Induction times and immobilization quality did not differ between groups. The heart rate was significantly higher and the mean arterial pressure significantly lower in blesbok after receiving vatinoxan. All animals were hypoxemic and there were no significant differences in the respiratory rates, PaO2, PaCO2, SpO2, or P(A-a)O2 gradients at any time point. Although vatinoxan did not improve respiratory variables and blood oxygenation in these animals, the change in cardiovascular variables may suggest that it improves tissue perfusion, a positive outcome that requires further investigation.

Structure-Activity Relationships of the Fentanyl Scaffold: Identification of Antagonists as Potential Opioid Overdose Reversal Agents.

Opioid-related overdoses account for almost half of all drug overdose deaths in the United States and cause more preventable deaths every year than car crashes. Fentanyl, a highly potent mu opioid receptor (MOR) agonist and its analogues (fentalogues) are increasingly found in illicit drug samples, both where the primary drug of abuse is an opioid and where it is not. The prevalence of fentalogues in the illicit drug market is thought to be the primary driver of the increased number of opioid-related overdose deaths since 2016. In fact, fentanyl and its analogues are involved in more than 70% of opioid-related overdoses. The standard opioid overdose rescue therapy naloxone is often insufficient to reverse opioid overdoses caused by fentalogue agonists under current treatment paradigms. However, the pharmacology of many fentalogues is unknown. Moreover, within the fentalogue series of compounds, it is possible that antagonists could be identified that might be superior to naloxone as opioid overdose reversal agents. In this report, we explore the pharmacology of 70 fentalogues and identify compounds that behave as MOR antagonists in vitro and demonstrate with one of these reversals of fentanyl-induced respiratory depression in the mouse. Such compounds could provide leads for the development of effective agents for the reversal of opioid overdose.

Investigation of [11C]carfentanil for mu opioid receptor quantification in the rat brain.

[11C]Carfentanil ([11C]CFN) is the only selective carbon-11 labeled radiotracer currently available for positron emission tomography (PET) imaging of mu opioid receptors (MORs). Though used extensively in clinical research, [11C]CFN has not been thoroughly characterized as a tool for preclinical PET imaging. As we were occasionally observing severe vital sign instability in rat [11C]CFN studies, we set out to investigate physiological effects of CFN mass and to explore its influence on MOR quantification. In anesthetized rats (n = 15), significant dose-dependent PCO2 increases and heart rate decreases were observed at a conventional tracer dose range (IV, > 100 ng/kg). Next, we conducted baseline and retest [11C]CFN PET scans over a wide range of molar activities. Baseline [11C]CFN PET studies (n = 27) found that nondisplaceable binding potential (BPND) in the thalamus was positively correlated to CFN injected mass, demonstrating increase of MOR availability at higher injected CFN mass. Consistently, when CFN injected mass was constrained < 40 ng/kg (~ 10% MOR occupancy in rats), baseline MOR availability was significantly decreased. For test-retest variability (TRTV), better reproducibility was achieved by controlling CFN injected mass to limit the difference between scans. Taken together, we report significant cardiorespiratory depression and a paradoxical influence on baseline MOR availability at conventional tracer doses in rats. Our findings might reflect changes in cerebral blood flow, changes in receptor affinity, or receptor internalization, and merits further mechanistic investigation. In conclusion, rat [11C]CFN PET requires stringent quality assurance of radiotracer synthesis and mass injected to avoid pharmacological effects and limit potential influences on MOR quantification and reproducibility.

Combining surface-enhanced Raman spectroscopy (SERS) and paper spray mass spectrometry (PS-MS) for illicit drug detection.

There is an ongoing effort in the US illicit drug market to make new psychoactive compounds more potent and addictive. Due to continuous chemical modifications, many fentanyl analogs are developed and mixed with more traditional illicit drugs, such as cocaine and heroin. Detecting fentanyl and fentanyl analogs in these illicit drug mixtures has become more crucial because of the increased potency and associated health risks. Most confirmatory procedures require time-consuming and expensive, highly sophisticated laboratory equipment and experimental procedures, which can delay critical information that might save a victim or find a suspect. In this study, we propose miniaturizing and accelerating this process by combining surface-enhanced Raman spectroscopy (SERS) analysis and paper spray mass spectrometry (PS-MS). For this aim, dual-purposed paper substrates were developed through soaking in Au/Ag nanostars suspensions. These novel, in-house prepared paper SERS substrates showed stability for up to four weeks with and without the presence of drug compounds. Fentanyl analogs with similar SERS spectra were differentiated by coupling with PS-MS. The limit of detection (LOD) for fentanyl on the paper substrates is 34 µg/mL and 0.32 µg/mL for SERS and PS-MS, respectively. Fentanyl and fentanyl analogs show selective SERS enhancement that helped to detect trace amounts of these opioids in heroin and cocaine street samples. In short, we propose the combination of SERS/PS-MS by using modified paper substrates to develop cost-effective, sensitive, rapid, portable, reliable, and reproducible methods to detect illicit drugs, especially trace amounts of fentanyl and fentanyl analogs in illicit drug mixtures. The combination of these two category A techniques allows for the identification of illicit drugs according to the SWGDRUG guidelines.

Aberrant type 2 dopamine receptor availability in violent offenders with psychopathy.

Psychopathy is characterized by antisocial behavior, poor behavioral control and lacking empathy, and structural alterations in the corresponding neural circuits. Molecular brain basis of psychopathy remains poorly characterized. Here we studied type 2 dopamine receptor (D2R) and mu-opioid receptor (MOR) availability in convicted violent offenders with high psychopathic traits (n = 11) and healthy matched controls (n = 17) using positron emission tomography (PET). D2R were measured with radioligand [11C]raclopride and MORs with radioligand [11C]carfentanil. Psychopathic subjects had lowered D2R availability in caudate and putamen, and striatal D2R availability was also associated with degree of psychopathic traits in this prisoner sample. No group differences were found in MOR availability, although in the prisoner sample, psychopathic traits were negatively correlated with MOR availability in the amygdala and nucleus accumbens. We conclude that D2R signaling could be the putative neuromolecular pathway for psychopathy, whereas evidence for alterations in the MOR system is more limited.

Temperature and pH-dependent stability of fentanyl analogs: Degradation pathways and potential biomarkers.

The collection, storage, and transport of samples prior to and during analysis is of utmost importance, especially for highly potent analogs that may not be present in high concentrations and are susceptible to pH or thermally mediated degradation. An accelerated stability study was performed on 17 fentanyl analogs (fentalogs) over a wide range of pH (2-10) and temperature (20-60°C) conditions over 24 h. Dilute aqueous systems were used to investigate temperature and pH-dependent kinetics using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Liquid chromatography-quadrupole/time-of-flight-mass spectrometry (LC-Q/TOF-MS) was used for structural elucidation of degradants. With the exception of remifentanil, all fentalogs evaluated were stable at pH 6 or lower. Fentalogs were generally unstable in strongly alkaline environments and at elevated temperatures. Remifentanil was the least stable drug and N-dealkylated fentalogs were the most stable. Fentanyl degraded to acetylfentanyl, norfentanyl, fentanyl N-oxide, and 1-phenethylpyridinium salt (1-PEP). A total of 26 unique breakdown products were observed for 15 of the fentanyl derivatives studied. Common degradation pathways involved N-dealkylation, oxidation of the piperidine nitrogen, and ß-elimination of N-phenylpropanamide followed by oxidation/dehydration of the piperidine ring. Ester and amide hydrolysis, demethylation at the propanamide, and O-demethylation were observed for selected fentalogs only. The potential for analyte loss should be considered during the pre-analytical phase (i.e., shipping and transport) where environmental conditions may not be controlled, as well as during the analysis itself.

Performance Evaluation of 2 FDA-Approved Fentanyl Immunoassays against LC-MS/MS Reference.

BACKGROUND: Fentanyl, a synthetic opioid, has caused many recent overdose deaths. Diagnosis of fentanyl abuse is not served by traditional opiate assays due to differences in chemical structure between synthetics and natural opioids. To our knowledge, this is the first study that uses liquid chromatography-tandem mass spectrometry (LC-MS/MS) as the reference method to evaluate and compare the ARK Fentanyl II Assay (ARK II) and the Fentanyl II Enzyme Immunoassay by Roche (FEN2). The ARK II is designed to detect fentanyl in urine samples, whereas the FEN2 is designed to detect norfentanyl, which is the major metabolite. METHODS: Two hundred patient urine samples including 100 positive and 100 negative samples according to an in-house LC-MS/MS assay were selected for the study. These samples were tested using the ARK II and the FEN2 to determine their performances relative to LC-MS/MS results. RESULTS: The FEN2 showed a positive and negative predictive value of 100% and 97% and a concordance with LC-MS/MS of 98.5% (kappa 0.97). The ARK II showed a positive and negative predictive value of 100% and 95% and a concordance with LC-MS/MS of 97.5% (kappa 0.95). Additionally, the FEN2 accurately identified 9 positive samples with a range of fentanyl concentrations from 0 to 18 ng/mL for which norfentanyl levels were less than the cutoff of 5 ng/mL, indicating potentially greater sensitivity than otherwise stated. CONCLUSIONS: The FEN2 and the ARK II were evaluated to be similar in terms of positive and negative predictive value during the analysis of 200 patient samples, as well as equally concordant with the LC-MS/MS reference, despite differences in design.

Dependency of fentanyl analogue protomer ratios on solvent conditions as measured by ion mobility-mass spectrometry.

Recently, our group has shown that fentanyl and many of its analogues form prototropic isomers ("protomers") during electrospray ionization. These different protomers can be resolved using ion mobility spectrometry and annotated using mobility-aligned tandem mass spectrometry fragmentation. However, their formation and the extent to which experimental variables contribute to their relative ratio remain poorly understood. In the present study, we systematically investigated the effects of mixtures of common chromatographic solvents (water, methanol, and acetonitrile) and pH on the ratio of previously observed protomers for 23 fentanyl analogues. Interestingly, these ratios (N-piperidine protonation vs. secondary amine/O = protonation) decreased significantly for many analogues (e.g., despropionyl ortho-, meta-, and para-methyl fentanyl), increased significantly for others (e.g., cis-isofentanyl), and remained relatively constant for the others as solvent conditions changed from 100% organic solvent (methanol or acetonitrile) to 100% water. Interestingly, pH also had significant effects on this ratio, causing the change in ratio to switch in many cases. Lastly, increasing conditions to pH ≥ 4.0 also prompted the appearance of new mobility peaks for ortho- and para-methyl acetyl fentanyl, where all previous studies had only showed one single distribution. Because these ratios have promise to be used qualitatively for identification of these (and emerging) fentanyl analogues, understanding how various conditions (i.e., mobile phase selection and/or chromatographic gradient) affect their ratios is critically important to the development of advanced ion mobility and mass spectrometry methodologies to identify fentanyl analogues.

Intoxications involving methoxyacetylfentanyl and U-47700: a study of 3 polydrug fatalities.

Novel synthetic opioids (NSOs) represent an emerging group of novel psychoactive substances, acting as agonists at the opioid receptors. NSOs include fentanyl-related compounds, e.g. methoxyacetylfentanyl (MeACF), and non-fentanyl analogs, e.g. "U compounds" including U-47700. Here we present three cases of death involving MeACF and U-47700, with particular reference to preliminary data on pharmacokinetics and tissue distribution.After a complete post-mortem examination, general unknown screenings and analysis of drugs of abuse were performed on postmortem samples by immunoassays, gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. To quantify the analytes of interest in post-mortem blood and tissues, the standard addition method was used. A toxicological significance score (TSS), weighing the role of the NSO in each death case, was assigned.Case 1 died at the hospital after consumption of U-47700, methadone (serum levels: 2,600 ng/ml and 37 ng/ml), tilidine and benzodiazepines. In case 2, U-47700 (204 ng/ml) together with methadone (290 ng/ml), flubromazepam (480 ng/ml) and diazepam (300 ng/ml) were detected in peripheral blood. In case 3, methoxyacetylfentanyl (266 ng/ml), furanylfentanyl (4.3 ng/ml) 4-ANPP (15 ng/ml) and alprazolam (69 ng/ml) were quantified in femoral blood. In all cases, the NSO likely contributed to the death (TSS = 3).NSOs appear to be often consumed in the setting of polydrug intoxications, especially in combination with other opioids and benzodiazepines, which often exert synergistic effects. The standard addition method remains the most reliable in post-mortem analysis and toxicological results should always be evaluated together with circumstantial and autopsy data.

Validation of a sampling method and liquid chromatography mass spectrometry analysis method for measurement of fentanyl and five other illicit drugs.

With the increased provision of services by health authorities and community organizations allowing supervised inhalation of illicit substances comes concerns about the potential for secondhand exposure to the substances being used, whether in the adjacent community or to workers at the sites. In order to address community concerns surrounding secondhand illicit substance exposure and better protect harm reduction workers, a validated sampling and LC-MS/MS analysis method was developed for 6 illicit drugs: fentanyl, heroin, methamphetamine, cocaine, etizolam, and bromazolam. It was found that the filter used needed to be silanized to be made more inert and avoid loss of analyte due to degradation. Using the silanized filters, recoveries were good (>90%) and the collected samples were found to be stable at room temperature for 2 wk. The sampling volume validated was up to 960 L. The sensitivity and range of the method make it appropriate for short-term (15 min), full shift (8 h), or environmental sampling.

Fentanyl Deaths in Infants and Children: A Case Series and Literature Review.

ABSTRACT: Death due to fentanyl and its various analogs has resulted in an exponential rise in deaths throughout the United States, overwhelming many medical examiner offices for over a decade. Its potency and prevalence have caused fentanyl to become the most reported substance in overdose fatalities, with an accompanying increase in exposure of the most vulnerable, infants and children. This report provides information about fentanyl in the pediatric population, including case examples, proposed investigative practices, published therapeutic and lethal blood concentrations, and available resources for future cases. Nine cases of pediatric death between 2013 and 2023 due to fentanyl were reviewed. Five case summaries are presented that highlight classic features of fentanyl deaths in infants, children, and teenagers. Deaths due to fentanyl have continued to rise year after year. Infants and children, most of whom are opioid naive, are at ever increased risk for exposure to high levels of fentanyl. The legal ramifications of a positive fentanyl level in a child increase the need for caution on the part of the forensic pathologist. Understanding what can and cannot be proven by autopsy as well as what resources are available to strengthen one's justification for fentanyl being the primary cause of death is critical.

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.

Total-body imaging of mu-opioid receptors with [11C]carfentanil in non-human primates.

PURPOSE: Mu-opioid receptors (MORs) are widely expressed in the central nervous system (CNS), peripheral organs, and immune system. This study measured the whole body distribution of MORs in rhesus macaques using the MOR selective radioligand [11C]carfentanil ([11C]CFN) on the PennPET Explorer. Both baseline and blocking studies were conducted using either naloxone or GSK1521498 to measure the effect of the antagonists on MOR binding in both CNS and peripheral organs. METHODS: The PennPET Explorer was used for MOR total-body PET imaging in four rhesus macaques using [11C]CFN under baseline, naloxone pretreatment, and naloxone or GSK1521498 displacement conditions. Logan distribution volume ratio (DVR) was calculated by using a reference model to quantitate brain regions, and the standard uptake value ratios (SUVRs) were calculated for peripheral organs. The percent receptor occupancy (%RO) was calculated to establish the blocking effect of 0.14 mg/kg naloxone or GSK1521498. RESULTS: The %RO in MOR-abundant brain regions was 75-90% for naloxone and 72-84% for GSK1521498 in blocking studies. A higher than 90% of %RO were observed in cervical spinal cord for both naloxone and GSK1521498. It took approximately 4-6 min for naloxone or GSK1521498 to distribute to CNS and displace [11C]CFN from the MOR. A smaller effect was observed in heart wall in the naloxone and GSK1521498 blocking studies. CONCLUSION: [11C]CFN total-body PET scans could be a useful approach for studying mechanism of action of MOR drugs used in the treatment of acute and chronic opioid use disorder and their effect on the biodistribution of synthetic opioids such as CFN. GSK1521498 could be a potential naloxone alternative to reverse opioid overdose.

Assessment of the antinociceptive, respiratory-depressant, and reinforcing effects of the low pKa fluorinated fentanyl analogs, FF3 and NFEPP.

RATIONALE: Recent studies report that fentanyl analogs with relatively low pKa values produce antinociception in rodents without other mu opioid-typical side effects due to the restriction of their activity to injured tissue with relatively low pH values. However, it is unclear if and to what degree these compounds may produce mu opioid-typical side effects (respiratory depression, reinforcing effects) at doses higher than those required to produce antinociception. OBJECTIVES: The present study compared the inflammatory antinociceptive, respiratory-depressant, and reinforcing effects of fentanyl and two analogs of intermediate (FF3) and low (NFEPP) pKa values in terms of potency and efficacy in male and female Sprague-Dawley rats. METHODS: Nociception was produced by administration of Complete Freund's Adjuvant into the hind paw of subjects, and antinociception was measured using an electronic Von Frey test. Respiratory depression was measured using whole-body plethysmography. Reinforcing effects were measured in self-administration using a progressive-ratio schedule of reinforcement. The dose ranges tested for each drug encompassed no effect to maximal effects. RESULTS: All compounds produced full effects in all measures but varied in potency. FF3 and fentanyl were equipotent in antinociception and self-administration, but FF3 was less potent than fentanyl in respiratory depression. NFEPP was less potent than fentanyl in every measure. The magnitude of potency difference between antinociception and other effects was greater for FF3 than for NFEPP or fentanyl, indicating that FF3 had the widest margin of safety when relating antinociception to respiratory-depressant and reinforcing effects. CONCLUSIONS: Low pKa fentanyl analogs possess potential as safer analgesics, but determining the optimal degree of difference for pKa relative to fentanyl will require further study due to some differences between the current results and findings from prior work with these analogs.

Case Series: Hepatic and Splenic Titanium Dioxide Deposition in Association With Intravenous Drug Use.

ABSTRACT: Titanium dioxide is a versatile compound that is found in a variety of consumer products, medical hardware, and pharmaceuticals. Although oral and topical ingestion of this compound is common, intravenous introduction is much less common. We present three cases where significant titanium dioxide deposits were identified in liver and splenic tissue of three decedents, all of whom died of illicit drug overdose in the same geographic area and had fentanyl and its metabolites in blood on postmortem toxicologic testing. At autopsy, liver sections had a granular texture with fine white stippling grossly, and histologic examination of hepatic and splenic tissues showed scattered patches of black granular material with pink birefringence. Energy-dispersive x-ray spectroscopy performed on these tissues revealed the presences of clusters of titanium dioxide. Immunohistochemical staining of both the liver and spleen with CD68 confirmed the titanium dioxide clusters were within macrophages. Intravenous titanium dioxide nanoparticle elimination studies in rats suggest a time sensitive period for this elimination, with a transient period of pigment deposition between 1-58 days following injection. If a time-dependent link between titanium dioxide pigment deposition within tissues and intravenous drug use can be shown, this could be a valuable tool for Pathologists.

Fiber Laser-Generated Silver-109 Nanoparticles for Laser Desorption/Ionization Mass Spectrometry of Illicit Drugs.

Cannabinoids and opioids are the most prominently used drugs in the world, with fentanyl being the main cause of drug overdose-related deaths. Monitoring drug use in groups as well as in individuals is an important forensic concern. Analytical methods, such as mass spectrometry (MS), have been found most useful for the identification of drug abuse on a small and large scale. Pulsed fiber laser 2D galvoscanner laser-generated nanomaterial (PFL 2D GS LGN) was obtained from monoisotopic silver-109. Nanomaterial was used for laser desorption/ionization mass spectrometry of selected illicit drug standards with standard high-resolution reflectron-based time-of-flight MALDI apparatus. Δ9-THC, 11-OH-THC, 11-COOH-THC, fentanyl, codeine, 6-monoacetylmorphine (6-MAM), heroin, tramadol, and methadone were chosen as test compounds. Illicit drugs were tested in a concentration range from 100 µg/mL to 10 pg/mL, equating to 50 µg to 50 fg per measurement spot. For all analyzed compounds, identification and quantification by silver-109-assisted laser desorption/ionization (LDI) MS was possible, with uncommon [M + 109Ag3]+ and [M - H]+ ions present for certain structures. The results of the quantitative analysis of drugs using silver-109 PFL 2D GS LGN for LDI MS are presented. Laser-generated NPs are proven to be useful for the analysis of selected drugs, with exceptionally good results for fentanyl monitoring in a broad range of concentrations.

Exploring Novel Fentanyl Analogues Using a Graph-Based Transformer Model.

The structures of fentanyl and its analogues are easy to be modified and few types have been included in database so far, which allow criminals to avoid the supervision of relevant departments. This paper introduces a molecular graph-based transformer model, which is combined with a data augmentation method based on substructure replacement to generate novel fentanyl analogues. 140,000 molecules were generated, and after a set of screening, 36,799 potential fentanyl analogues were finally obtained. We calculated the molecular properties of 36,799 potential fentanyl analogues. The results showed that the model could learn some properties of original fentanyl molecules. We compared the generated molecules from transformer model and data augmentation method based on substructure replacement with those generated by the other two molecular generation models based on deep learning, and found that the model in this paper can generate more novel potential fentanyl analogues. Finally, the findings of the paper indicate that transformer model based on molecular graph helps us explore the structure of potential fentanyl molecules as well as understand distribution of original molecules of fentanyl.

Evaluation of in-house drug evidence testing by King County Medical Examiner's Office in "real-time" fatal overdose surveillance.

With the escalating overdose epidemic, many surveillance efforts have appeared. In 2018, King County Medical Examiner's Office (KCMEO) initiated a fatal overdose surveillance project aimed at expediting death certification and disseminating timely information. In this project, KCMEO investigators collected items of evidence of drug use from overdose death scenes, which were tested by five in-house methods, four using handheld devices: TruNarc Raman spectrometer, with and without the manufacture's H-Kit, Rigaku ResQ Raman spectrometer, and MX908 mass spectrometer. The fifth in-house method used fentanyl-specific urine test strips. Results from in-house testing were compared with results from Washington State Patrol (WSP) Materials Analysis Laboratory. From 2019 to 2022, there were 4244 evidence items of drugs and paraphernalia collected from 1777 deaths scenes. A total of 7526 in-house tests were performed on collected specimens, and 2153 tests were performed by the WSP laboratory using standard analytical methods. The WSP results served as reference standards to calculate performance metrics of the in-house methods. Sensitivities, specificities, and predictive values ranged from good to poor depending on the method, drug, and evidence type. Certain drugs were often associated with specific evidence types. Acetaminophen was frequently found in combination with fentanyl. Fentanyl test strips gave good scores for detecting fentanyl; otherwise, in-house methods using handheld devices had poor performance scores with novel drugs and drugs diluted in mixtures. The results showed that in-house testing of drug evidence has value for medical examiner overdose surveillance, but it is resource intensive, and success depends on collaboration with forensic laboratories.

Differentiating Structurally Similar Fentanyl Analogs by Comparing Density Functional Theory (DFT) Calculations and Surface-Enhanced Raman Spectroscopy (SERS) Results.

Fentanyl and fentanyl analogs are the main cause of recent overdose deaths in the United States. The presence of fentanyl analogs in illicit drugs makes it difficult to estimate their potencies. This makes the detection and differentiation of fentanyl analogs critically significant. Surface-enhanced Raman spectroscopy (SERS) can differentiate structurally similar fentanyl analogs by yielding spectroscopic fingerprints for the detected molecules. In previous years, five fentanyl analogs, carfentanil, furanyl fentanyl, acetyl fentanyl, 4-fluoroisobutyryl fentanyl (4-FIBF), and cyclopropyl fentanyl (CPrF), gained popularity and were found in 76.4% of the fentanyl analogs trafficked. In this study, we focused on 4-FIBF, CPrF, and structurally similar fentanyl analogs. We developed methods to differentiate these fentanyl analogs using theoretical and experimental methods. To do this, a set of fentanyl analogs were examined using density functional theory (DFT) calculations. The DFT results obtained in this project permitted the assignment of spectral bands. These results were then compared with normal Raman and SERS techniques. Structurally similar fentanyl analogs show important differences in their spectra, and they have been visually differentiated from each other both theoretically and experimentally. Additional results using principal component analysis and soft independent modeling of class analogy show they can be distinguished using this technique. The limit of detection values for FIBF and CPrF were determined to be 0.35 ng/mL and 4.4 ng/mL, respectively, using SERS. Experimental results obtained in this project can be readily implemented in field applications and smaller laboratories, where inexpensive portable Raman spectrometers are often present and used in drug analysis.