The fentanyl-specific antibody FenAb024 can shield against carfentanil effects.

The surge in opioid-related deaths, driven predominantly by fentanyl and its synthetic derivatives, has become a critical public health concern, which is particularly evident in the United States. While the situation is less severe in Europe, the European Monitoring Centre for Drugs and Drug Addiction reports a rise in drug overdose deaths, with emerging concerns about the impact of fentanyl-related molecules. Synthetic opioids, initially designed for medical use, have infiltrated illicit markets due to their low production costs and high potency, with carfentanil posing additional threats, including potential chemical weaponization. Existing overdose mitigation heavily relies on naloxone, requiring timely intervention and caregiver presence, while therapeutic prevention strategies face many access challenges. To provide an additional treatment option, we propose the use of a fentanyl-specific monoclonal antibody (mAb), as a non-opioid method of prophylaxis against fentanyl and carfentanil. This mAb shows protection from opioid effects in a pre-clinical murine model. mAbs could emerge as a versatile countermeasure in civilian and biodefense settings, offering a novel approach to combat opioid-associated mortality.

In vitro, in vivo metabolism and quantification of the novel synthetic opioid N-piperidinyl etonitazene (etonitazepipne).

OBJECTIVES: N-piperidinyl etonitazene (etonitazepipne) is a newly synthesized opioid related to the 2-benzylbenzimidazole analog class. Etonitazepipne has been formally notified and placed under intensive monitoring in Europe in January 2022. Nitazenes have high affinity at µ-opioid receptor (MOR). Etonitazepipne, specifically shows a EC50 of 2.49 nM, suggesting about 50 times higher potency combined with higher efficacy compared to morphine. Antinociceptive potency l ('hot plate test' with rats) was 192-fold greater than that of morphine. METHODS: Here we report on a post-mortem case involving etonitazepipne and its quantification using a standard addition method (SAM) through liquid chromatography tandem mass spectrometry (LC-MS/MS). In addition, characterization and identification of phase I human metabolites using in vitro assay based on pooled human liver microsomes (pHLM) was performed along with the analysis of authentic urine samples by means of high-performance liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). RESULTS: The concentration of etonitazepipne in post-mortem blood and urine was 8.3 and 11 ng/mL, respectively. SAM was validated by assessing the following parameters: intraday and interday repeatability, matrix effect and recovery rate in post-mortem blood. A total of 20 and 14 metabolites were identified after pHLM incubation and urine analysis, respectively. Most pronounced in vitro and in vivo transformations were O-deethylation, hydroxylation, ketone reduction, and combinations thereof. CONCLUSIONS: Considering small traces of the parent drug often found in real cases, the identification of metabolic biomarkers is crucial to identify exposure to this drug. O-deethylated, oxidated metabolites, and combination thereof are proposed as urinary biomarkers along with the parent compound.

Confounders of Serum Phosphatidylethanol: Role of Red Blood Cell Turnover and Cirrhosis.

Purpose: Ethyl glucuronide (EtG), ethyl sulfate (EtS) and phosphatidylethanol (PEth) are considered specific direct biomarkers for detecting alcohol consumption. However, PEth, which is produced in red blood cells (RBC), varies considerably between patients for unknown reasons. We here studied various confounders of PEth elimination including fibrosis after alcohol withdrawal. Patients and Methods: EtG, EtS and PEth together with routine laboratory and clinical parameters were studied in 100 Caucasian heavy drinkers prior and after alcohol detoxification. In addition, fibrosis stage and degree of steatosis were assessed by transient elastography (Fibroscan, Echosens, Paris). Results: All three biomarkers were highly correlated (0.61-0.72) with initial serum alcohol levels, but only PEth correlated with daily alcohol consumption. After alcohol withdrawal, PEth significantly decreased within 6.1 days from 1708 to 810 ng/mL (half-life varied from 1.6 to 15.2 days). Both levels of serum alcohol but also EtG and EtS were higher in patients with liver cirrhosis as compared to patients without fibrosis despite comparable alcohol consumption suggesting a decreased alcohol elimination in patients with cirrhosis. PEth was also elevated in cirrhosis but not significantly. In contrast, PEth elimination rate was significantly higher in patients with enhanced RBC turnover and signs of alcohol-mediated hemolytic anemia with elevated ferritin, LDH and increased mean corpuscular volume (MCV). Conclusion: We here demonstrate that alcohol elimination is decreased in patients with liver cirrhosis. In patients with cirrhosis, PEth levels are both affected in opposite directions by enhanced red blood cell turnover and elevated alcohol levels. Our data have important implications for the use and interpretation of PEth in the clinical setting.

Phospholipid metabolites of GHB as potential biomarkers in whole blood: Synthesis, analytics, and in vitro formation of homolog 16:0/18:1.

Gamma-hydroxybutyric acid (GHB) is a common drug of abuse, and the detection of a consumption or administration is a longstanding research objective in clinical and forensic toxicology. However, until now, the short detection window of GHB could not be enlarged by the use of GHB metabolites. Therefore, new biomarkers for the detection of a GHB intake are needed. In analogy to phosphatidylethanols as long-time biomarkers of ethanol, phospholipids with GHB might represent a promising compound class. While the availability of reference compounds often represents a bottleneck in clinical and forensic toxicological research, two phospholipids-phosphatidyl-GHB (16:0/18:1) and its isomer phosphatidyl beta-hydroxybutyric acid (16:0/18:1)-were successfully synthesized by a new highly versatile synthetic route. Structural characterization data, together with 1 H-, 13 C-, and 31 P-NMR and high-resolution mass spectrometry (HRMS) spectra, are reported. Subsequently, a HPLC-MS/MS method was established for the determination of both compounds (limits of detection [LOD] ≤ 2 ng/ml), and the formation of these metabolites was investigated in two in vitro experiments. The formation of phosphatidyl-GHB (16:0/18:1) was observed in an incubation experiment by converting phosphatidylcholine (16:0/18:1) and GHB with phospholipase D and in whole blood samples spiked with 50 mM GHB, respectively. Therefore, phosphatidyl-GHB (16:0/18:1) might represent a valuable new metabolite of GHB with the potential for an extension of the detection window as GHB biomarker.

Simultaneous Determination of 5- and 6-APB in Blood, Other Body Fluids, Hair and Various Tissues by HPLC--MS-MS.

5-(2-Aminopropyl)benzofuran (5-APB) and 6-(2-aminopropyl)benzofuran (6-APB) are benzofuran analogues of amphetamine and belong to the category of new psychoactive substances. Despite already published fatal 5- and 6-APB intoxication after consumption of both substances in most cases, no sensitive method for the simultaneous detection and quantification of these new psychoactive compounds in human blood samples has yet been developed. Therefore, an easy and fast sample preparation and specific high-performance liquid chromatography and tandem mass spectrometry methods for the determination of both substances in blood were established and validated. In a fatal intoxication in 2017 at the Institute of Forensic and Traffic Medicine in Heidelberg, Germany, concentrations of 850 (5-APB) and 300 ng/mL (6-APB) were determined in peripheral blood. Besides, other body fluids (central blood, urine and bile), hair and various tissues were examined to verify the presence of both compounds and to gain first insights into their distribution. In this publication, we show a method for the simultaneous determination of 5- and 6-APB in human samples by a chromatographic method and to investigate their distribution in the human body.

Development and validation of an analytical method for the simultaneous determination of the alcohol biomarkers ethyl glucuronide, ethyl sulfate, N-acetyltaurine, and 16:0/18:1-phosphatidylethanol in human blood.

As alcohol is the most common addictive substance worldwide, it is inevitable to advance the established research. New and more substantial analytical methods can be applied to reply to complex questions in legal or forensic contexts. Therefore, an analytical method for the simultaneous determination of four different alcohol biomarkers-ethyl glucuronide, ethyl sulfate, N-acetyltaurine, and 16:0/18:1-phosphatidylethanol-in human blood was developed, validated, and verified. Despite the different chemical properties of the analytes, a specific determination via HPLC-MS/MS was achieved using a novel type of a Phenomenex Luna® Omega Sugar column. Furthermore, all criteria for a successful validation were fulfilled according to forensic guidelines. The method proved to be linear and demonstrates selectivity and sufficient sensitivity for every biomarker. LODs obtained with this method of 2.6 ng/ml (EtG), 4.7 ng/ml (EtS), 12.5 ng/ml (NAcT), and 6.9 ng/ml (PEth) were in an acceptable range for routine applications, and the stability of all analytes over a range of 12 h is given. The verification of the new developed method was performed with authentic samples. Thus, whole blood and postmortem samples were analyzed to obtain information about the drinking behavior, which can answer complex questions regarding alcohol consumption.

Modified congener analysis: Quantification of cyanide in whole blood, other body fluids, and diverse beverages.

The congener analysis is routinely used for the determination of volatile compounds in body fluids and beverages for forensic investigations. Although intoxications with cyanide via smoke inhalation or ingestion of cyanide salts are frequently encountered in forensic medicine, the inclusion of hydrogen cyanide in this analysis was never studied in detail. In this work, a very simple, fast, and sensitive quantification method with headspace gas chromatography and flame ionization detection for the analysis of cyanide in whole blood-was developed and validated. In contrast to the standard sample preparation of the congener analysis, an acidification step with tartaric acid was added. A limit of detection of 50 ng/ml, good linearity (coefficient of correlation > 0.9997), high accuracy (101.5%-106.4%), and precision (relative standard deviation 1.8%-3.7%) were achieved. Authentic blood samples of 10 forensic cases were investigated with the new method. Furthermore, the method was used for the quantification of cyanide in other body fluids (serum and urine) and diverse beverages. Interferences were investigated, and the addition of aldehydes produced a clear concentration-dependent decrease of the cyanide signal. Besides, the method offers an economical use of limited sample material by the simultaneous determination of cyanide, ethanol, and congener alcohols.

Backbone modifications in peptidic inhibitors of flaviviral proteases.

The NS2B-NS3 protease is a promising target for the development of drugs against dengue virus (DENV), West Nile virus (WNV) and related flaviviruses. We report the systematic variation of the peptide backbone of the two lead compounds Bz-Arg-Lys-d-Phg-NH2 and Bz-Arg-Lys-d-Phg(OBn)-NH2. While inhibitory activity against WNV protease was generally decreased, the inhibitory potency against DENV protease could be conserved and increased in several peptidomimetics, particularly in those containing a (NMe)arginine fragment or an N-terminal α-keto amide. Methylation at the α-position of the C-terminal phenylglycine led to a 6-fold higher potency against DENV protease. Peptidomimetics with modified backbone showed increased resistance against hydrolytic attack by trypsin and α-chymotrypsin.

Synthesis and structure-activity relationships of small-molecular di-basic esters, amides and carbamates as flaviviral protease inhibitors.

Inhibitors of the flaviviral serine proteases, which are crucial for the replication of dengue and West-Nile virus, have attracted much attention over the last years. A dibasic 4-guanidinobenzoate was previously reported as inhibitor of the dengue protease with potency in the low-micromolar range. In the present study, this lead structure was modified with the intent to explore structure-activity relationships and obtain compounds with increased drug-likeness. Substitutions of the guanidine moieties, the aromatic rings, and the ester with other functionalities were evaluated. All changes were accompanied by a loss of inhibition, indicating that the 4-guanidinobenzoate scaffold is an essential element of this compound class. Further experiments indicate that the target recognition of the compounds involves the reversible formation of a covalent adduct.

Solid Phase Synthesis of C-Terminal Boronic Acid Peptides.

Peptides and peptidomimetics with a C-terminal boronic acid group have prolific applications in numerous fields of research, but their synthetic accessibility remains problematic. A convenient, high yield synthesis of peptide-boronic acids on a solid support is described here, using commercially available 1-glycerol polystyrene resin. The method is compatible with Fmoc chemistry and offers a versatile approach to aryl and alkyl aminoboronic acids without additional purification steps.

Structure-activity relationship studies on 1-heteroaryl-3-phenoxypropan-2-ones acting as inhibitors of cytosolic phospholipase A2α and fatty acid amide hydrolase: replacement of the activated ketone group by other serine traps.

Cytosolic phospholipase A2α (cPLA2α) and fatty acid amide hydrolase (FAAH) are serine hydrolases. cPLA2α is involved in the generation of pro-inflammatory lipid mediators, FAAH terminates the anti-inflammatory effects of endocannabinoids. Therefore, inhibitors of these enzymes may represent new drug candidates for the treatment of inflammation. We have reported that certain 1-heteroarylpropan-2-ones are potent inhibitors of cPLA2α and FAAH. The serine reactive ketone group of these compounds, which is crucial for enzyme inhibition, is readily metabolized resulting in inactive alcohol derivatives. In order to obtain metabolically more stable inhibitors, we replaced this moiety by α-ketoheterocyle, cyanamide and nitrile serine traps. Investigations on activity and metabolic stability of these substances revealed that in all cases an increased metabolic stability was accompanied by a loss of inhibitory potency against cPLA2α and FAAH, respectively.

1-Heteroaryl-3-phenoxypropan-2-ones as inhibitors of cytosolic phospholipase A2α and fatty acid amide hydrolase: Effect of the replacement of the ether oxygen with sulfur and nitrogen moieties on enzyme inhibition and metabolic stability.

Cytosolic phospholipase A2α (cPLA2α) and fatty acid amide hydrolase (FAAH) are enzymes, which have emerged as attractive targets for the development of analgesic and anti-inflammatory drugs. We recently reported that certain 3-phenoxy-substituted 1-heteroarylpropan-2-ones are inhibitors of cPLA2α and/or FAAH. Starting from 1-[2-oxo-3-(4-phenoxyphenoxy)propyl]indole-5-carboxylic acid (3) and 1-(1H-benzotriazol-1-yl)-3-(4-phenoxyphenoxy)propan-2-one (4), the effect of the replacement of the oxygen in position 3 of the propan-2-one scaffold by sulfur and nitrogen containing moieties on inhibition of cPLA2α and fatty acid amide hydrolase as well as on metabolic stability in rat liver S9 fractions was investigated. As a result of these structure-activity relationship studies it was found that the ether oxygen is of great importance for enzyme inhibitory potency. Replacement by sulfur led to an about 100-fold decrease of enzyme inhibition, nitrogen and substituted nitrogen atoms at this position even resulted in inactivity of the compounds. The effect of the structural variations performed on metabolic stability of the important ketone pharmacophore was partly different in the two series of compounds. While introduction of SO and SO2 significantly increased stability of the ketone against reduction in case of the indole-5-carboxylic acid 3, it had no effect in case of the benzotriazole 4. Further analysis of the metabolism of 3 and 4 in rat liver S9 fractions revealed that the major metabolite of 3 was the alcohol 53 formed by reduction of the keto group. In contrast, in case of 4 beside keto reduction an excessive hydroxylation of the terminal phenoxy group occurred leading to the dihydroxy compound 50. Experiments with enzyme inhibitors showed that the phenylhydroxylation of 4 was catalyzed by tranylcypromine sensitive cytochrome P450 isoforms, while the reduction of the ketone function of 3 and 4 was mainly caused by cytosolic short chain dehydrogenases/reductases (cSDR).

Convergent and enantioselective syntheses of cytosolic phospholipase A(2)α inhibiting N-(1-indazol-1-ylpropan-2-yl)carbamates.

Cytosolic phospholipase A2α (cPLA2α) is an important enzyme of the inflammation cascade. Therefore, inhibitors of cPLA2α are assumed to be promising drug candidates for the treatment of inflammatory disorders. Recently we have found that indole-5-carboxylic acid with a 3-(4-octylphenoxy)-2-(phenoxycarbonylamino)propyl substituent in position 1 is an inhibitor of cPLA2α. We have now synthesized a corresponding derivative with the indole heterocycle replaced by an indazole (4) employing an analogous reaction sequence as for the synthesis of the indole derivative. Besides, a more convergent synthesis for 4 was established using an aziridine as central intermediate. Furthermore, a chiral-pool based enantioselective synthesis was developed for the synthesis of (R)- and (S)-4. Starting compound for both enantiomers was the (R)-serine derived oxazolidine (R)-25. Compound 4 proved to be a moderate inhibitor of cPLA2α, with the S-enantiomer being twice as active as the R-enantiomer. The racemate 4 and the enantiomers (R)- and (S)-4 showed a high in vitro metabolic stability in rat liver S9 fractions.