Vandeputte MM, Walton SE, Shuda SA, Papsun DM, Krotulski AJ, Stove CP
Analytical and Bioanalytical ChemistryAbstract
Introduction
The emergence of structurally diverse new synthetic opioids (NSOs) has caused the opioid crisis to spiral to new depths. Little information is available about the pharmacology of most novel opioids when they first emerge. Here, using a β-arrestin 2 recruitment assay, we investigated the in vitro μ-opioid receptor (MOR) activation potential of dipyanone, desmethylmoramide, and acetoxymethylketobemidone (O-AMKD) — recent NSOs that are structurally related to the prescription opioids methadone and ketobemidone. Our findings indicate that dipyanone (EC50=39.9 nM; Emax=155% vs. hydromorphone) is about equally active as methadone (EC50=50.3 nM; Emax=152%), whereas desmethylmoramide (EC50=1335 nM; Emax=126%) is considerably less active. A close structural analogue of ketobemidone (EC50=134 nM; Emax=156%) and methylketobemidone (EC50=335 nM; Emax=117%), O-AMKD showed a lower potency (EC50=1262 nM) and efficacy (Emax=109%). Evaluation of the opioid substitution product buprenorphine and its metabolite norbuprenorphine confirmed the increased in vitro efficacy of the latter. In addition to in vitro characterization, this report details the first identification and full chemical analysis of dipyanone in a seized powder, as well as a postmortem toxicology case from the USA involving the drug. Dipyanone was quantified in blood (370 ng/mL), in which it was detected alongside other NSOs (e.g., 2-methyl AP-237) and novel benzodiazepines (e.g., flualprazolam). While dipyanone is currently not commonly encountered in forensic samples worldwide, its emergence is worrisome and representative of the dynamic NSO market.
Methods
Reference standards for morphine, fentanyl, (±)-methadone HCl, buprenorphine HCl, norbuprenorphine, dipyanone HCl, desmethylmoramide, acetoxymethylketobemidone HCl, and methylketobemidone were obtained from Cay- man Chemical (Ann Arbor, MI, USA). Ketobemidone was from Chiron AS (Trondheim, Norway) and hydromorphone HCl was purchased from Fagron (Nazareth, Belgium). All concentrations reported in this work are expressed as those of the free bases of the compounds. The human embry-onic kidney (HEK) 293T cells (passage 20) were kindly gifted by Prof. O. De Wever (Ghent University Hospital, Belgium). Dulbecco’s modified Eagle’s medium (DMEM) (GlutaMAX™), Opti-MEM® I Reduced Serum Medium, penicillin-streptomycin (10,000 U/mL and 10,000 μg/mL), and amphotericin B (250 μg/mL) were supplied by Thermo Fisher Scientific (Waltham, MA, USA). Fetal bovine serum (FBS) and poly-d-lysine were obtained from Sigma-Aldrich (Darmstadt, Germany). Promega (Madison, WI, USA) sup- plied the Nano-Glo® Live Cell Assay System, containing the Nano-Glo® Live Cell Substrate and Nano-Glo® LCS Dilu- tion Buffer. For toxicology and chemistry testing, standard reference material for dipyanone was purchased from Cay- man Chemical as a powder and prepared at a concentra- tion of 1 mg/mL in methanol. Drug-free human blood was purchased from BioIVT (Westbury, NY, USA). Sodium borate decahydrate was purchased from Sigma-Aldrich (St. Louis, MO, USA). Ethyl acetate, N-butyl chloride, liquid chromatograpy-mass spectrometry (LC-MS) grade water, and methanol were purchased from Honeywell Chemicals (Charlotte, NC, USA). Formic acid was purchased from Thermo Fisher Scientific.
Results
The intrinsic MOR activation potential of the different opioids was assessed using a βarr2 recruitment assay. All compounds were capable of activating MOR, with potencies ranging from 1.35 nM (buprenorphine) to 1.335 μM (desmethylmoramide) (Table 1). Dipyanone (EC50 = 39.9 nM) was the only NSO with a higher potency than morphine (EC50 = 142 nM), and none of the newly tested NSOs was more potent than fentanyl (EC50 = 9.35 nM). With the exception of buprenorphine, a partial agonist (Emax = 23.2%) compared to hydromorphone, the efficacy of the test drugs was comparable to or exceeding that of hydromorphone (range 98.6–162%). With EC50 values in the low nM range, buprenorphine (EC50 = 1.35 nM) and norbuprenorphine (EC50 = 2.94 nM) were the most potent compounds of the panel. While comparable in terms of potency, norbuprenorphine (Emax = 162%) was about 7 times more efficacious than buprenorphine (Emax = 23.2%) in activating MOR. Methadone (EC50 = 50.3 nM; Emax = 152%) was an order of magnitude less potent than (nor)buprenorphine, and about equally efficacious as norbuprenorphine (Fig. 2). Dipyanone (EC50 = 39.9 nM; Emax = 155%) and methadone showed a comparable opioid activity (potency and efficacy) in the employed in vitro assay. Desmethylmoramide, on the other hand, was considerably less potent and somewhat less efficacious (EC50 = 1335 nM; Emax = 126%) (Fig. 3, panel A). The concentration-response curve of a seized dipyanone powder showed an almost perfect overlap with that of the dipyanone reference standard (Fig. 3, panel B), indicating a high purity of the seized powder, as supported via chemical analysis (see “Analytical characterization”). Among the different ketobemidone analogues, ketobemidone (EC50 = 134 nM; Emax = 156%) itself was the most potent and most efficacious compound, followed by methylketobemidone (EC50 = 335 nM; Emax = 117%). O-AMKD (EC50 = 1262 nM; Emax = 109%) was about ten times less potent and had an efficacy of about 70% of that of ketobemidone (Fig. 4).
Conclusions
In summary, we investigated the in vitro MOR activation potential of NSOs that are structurally related to methadone (dipyanone and desmethylmoramide) and ketobemidone (O-AMKD). While the studied opioids are intrinsically less potent than fentanyl, their uncontrolled availability and unsupervised use are reasons for concern. The reported identification and quantification of dipyanone in a fatality (370 ng/mL) underscore this warning. This report further details the first identification and full chemical analysis (GC-MS, LC-QTOF-MS, HPLC-DAD, NMR, FTIR) of dipyanone in a seized powder. Careful monitoring is required to detect other (potentially more potent) NSOs related to prescription opioids that may emerge on recreational drug markets.
Keywords: New synthetic opioids (NSOs) · Dipyanone · Desmethylmoramide · Acetoxymethylketobemidone (O-AMKD) · μ-Opioid receptor (MOR) · Forensic toxicology
FULL ARTICLE
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