Investigation of the intrinsic cannabinoid activity of hemp-derived and semisynthetic cannabinoids with ß-arrestin2 recruitment assays-and how this matters for the harm potential of seized drugs.

Cultivation of industrial low-Δ9-tetrahydrocannabinol (Δ9-THC) hemp has created an oversupply of cannabidiol (CBD)-rich products. The fact that phytocannabinoids, including CBD, can be used as precursors to synthetically produce a range of THC variants-potentially located in a legal loophole-has led to a diversification of cannabis recreational drug markets. 'Hemp-compliant', 'hemp-derived' and 'semisynthetic' cannabinoid products are emerging and being advertised as (legal) alternatives for Δ9-THC. This study included a large panel (n = 30) of THC isomers, homologs, and analogs that might be derived via semisynthetic procedures. As a proxy for the abuse potential of these compounds, we assessed their potential to activate the CB1 cannabinoid receptor with a ß-arrestin2 recruitment bioassay (picomolar-micromolar concentrations). Multiple THC homologs (tetrahydrocannabihexol, THCH; tetrahydrocannabiphorol, THCP; tetrahydrocannabinol-C8, THC-C8) and THC analogs (hexahydrocannabinol, HHC; hexahydrocannabiphorol, HHCP) were identified that showed higher potential for CB1 activation than Δ9-THC, based on either higher efficacy (Emax) or higher potency (EC50). Structure-activity relationships were assessed for Δ9-THC and Δ8-THC homologs encompassing elongated alkyl chains. Additionally, stereoisomer-specific differences in CB1 activity were established for various THC isomers (Δ7-THC, Δ10-THC) and analogs (HHC, HHCP). Evaluation of the relative abundance of 9(S)-HHC and 9(R)-HHC epimers in seized drug material revealed varying epimeric compositions between batches. Increased abundance of the less active 9(S)-HHC epimer empirically resulted in decreased potency, but sustained efficacy for the resulting diastereomeric mixture. In conclusion, monitoring of semisynthetic cannabinoids is encouraged as the dosing and the relative composition of stereoisomers can impact the harm potential of these drugs, relative to Δ9-THC products.

Fentanyl analog structure-activity relationships demonstrate determinants of diverging potencies for antinociception and respiratory depression.

Opioid overdoses, particularly those involving fentanyl-related substances (FRS), present a significant public health challenge in the United States. This structure-activity relationship (SAR) study evaluated the relationship between the chemical structure of seventeen FRS and their in vivo mu-opioid-receptor (MOR) mediated effects. SAR evaluations included fluorine substitutions on the aniline or phenethyl ring and variations in N-acyl chain length. Adult male Swiss Webster mice were administered fluorinated regioisomers of fentanyl, butyrylfentantyl and valerylfentanyl, and compared to MOR standards including morphine, buprenorphine, and fentanyl to determine if they would elicit prototypical opioid-like effects including hyperlocomotion (open-field test), antinociception (warm-water tail-withdrawal test), and hypoventilation (whole-body plethysmography test). To determine if the MOR was the pharmacological mechanism responsible for these effects, naltrexone or naloxone pretreatments were administered to evaluate their actions on FRS-induced antinociception and hypoventilation. There were three main findings. First, FRS elicited hyperlocomotion, antinociception, and hypoventilation in mice to varying degrees, similar to prototypical MOR standards. Second, the rank order of potencies for hypoventilatory effects of FRS were different for each series including FRS with increasing N-acyl chain length (i.e., acetylfentanyl, fentanyl, butyrylfentanyl, valerylfentanyl, hexanoylfentanyl), phenethyl-fluorinated regioisomers (e.g., 2'-fluorofentanyl, 3'-fluorofentanyl, 4'-fluorofentanyl), and aniline-fluorinated regioisomers (e.g., ortho-fluorofentanyl, meta-fluorofentanyl, para-fluorofentanyl). Third, the degree of separation in potencies observed for the antinociceptive and hypoventilatory effects of these drugs did not always follow that which was observed for their antinociceptive and hyperlocomotor effects. This study clarifies the in vivo activities for these FRS and elucidates a SAR for MOR-mediated effects among structural isomers.

Discovery of a series of 2-(1H-pyrazol-1-yl)pyridines as ALK5 inhibitors with potential utility in the prevention of dermal scarring.

A series of 2-(1H-pyrazol-1-yl)pyridines are described as inhibitors of ALK5 (TGFß receptor I kinase). Modeling compounds in the ALK5 kinase domain enabled some optimization of potency via substitutions on the pyrazole core. One of these compounds PF-03671148 gave a dose dependent reduction in TGFß induced fibrotic gene expression in human fibroblasts. A similar reduction in fibrotic gene expression was observed when PF-03671148 was applied topically in a rat wound repair model. Thus these compounds have potential utility for the prevention of dermal scarring.

Respiratory depressant effects of fentanyl analogs are opioid receptor-mediated.

Opioid-related fatalities involving synthetic opioids have reached unprecedented levels. This study evaluated the respiratory depressant effects of seven fentanyl analogs that have either emerged in the illicit drug supply or been identified in toxicological analyses following fatal or non-fatal intoxications. Adult male Swiss Webster mice were administered fentanyl analogs (isobutyrylfentanyl, crotonylfentanyl, para-methoxyfentanyl, para-methoxybutyrylfentanyl, 3-furanylfentanyl, thiophenefentanyl, and benzodioxolefentanyl) and their effects on minute volume as compared to mu-opioid receptor (MOR) agonist standards (fentanyl, morphine, and buprenorphine) were measured using whole body plethysmography (WBP). All drugs elicited significant (p ≤ 0.05) hypoventilation relative to vehicle for at least one dose tested: morphine (1, 3.2, 10, 32 mg/kg), buprenorphine, (0.032, 0.1, 0.32, 1, 3.2 mg/kg), fentanyl (0.0032, 0.01, 0.032, 0.1, 1, 32 mg/kg), isobutyrylfentanyl (0.1, 0.32, 1, 3.2, 10 mg/kg), crotonylfentanyl (0.1, 0.32, 1, 3.2, 10 mg/kg), para-methoxyfentanyl (0.1, 0.32, 1, 3.2, 10 mg/kg), para-methoxybutyrylfentanyl (0.32, 1, 3.2, 10 mg/kg), 3-furanylfentanyl (0.1, 0.32, 1, 3.2, 10 mg/kg), thiophenefentanyl (1, 3.2, 10, 32, 100 mg/kg), and benzodioxolefentanyl (3.2, 10, 32, 100 mg/kg). The ED50 values for hypoventilation showed a rank order of potency as follows: fentanyl (ED50 = 0.96 mg/kg) > 3-furanylfentanyl (ED50 = 2.60 mg/kg) > crotonylfentanyl (ED50 = 2.72 mg/kg) > para-methoxyfentanyl (ED50 = 3.31 mg/kg) > buprenorphine (ED50 = 10.8 mg/kg) > isobutyrylfentanyl (ED50 = 13.5 mg/kg) > para-methoxybutyrylfentanyl (ED50 = 16.1 mg/kg) > thiophenefentanyl (ED50 = 18.0 mg/kg) > morphine (ED50 = 55.3 mg/kg) > benzodioxolefentanyl (ED50 = 10,168 mg/kg). A naloxone pretreatment (10 mg/kg) attenuated the hypoventilatory effects of all drugs. These results establish that the respiratory depressant effects of these fentanyl analogs are at least in part mediated by the MOR.

Synthesis, Chemical Characterization, and µ-Opioid Receptor Activity Assessment of the Emerging Group of "Nitazene" 2-Benzylbenzimidazole Synthetic Opioids.

Several 2-benzylbenzimidazole opioids (also referred to as "nitazenes") recently emerged on the illicit market. The most frequently encountered member, isotonitazene, has been identified in multiple fatalities since its appearance in 2019. Although recent scheduling efforts targeted isotonitazene, many other analogues remain unregulated. Being structurally unrelated to fentanyl, little is known about the harm potential of these compounds. In this study, ten nitazenes and four metabolites were synthesized, analytically characterized via four different techniques, and pharmacologically evaluated using two cell-based ß-arrestin2/mini-Gi recruitment assays monitoring µ-opioid receptor (MOR) activation. On the basis of absorption spectra and retention times, high-performance liquid chromatography coupled to diode-array detection (HPLC-DAD) allowed differentiation between most analogues. Time-of-flight mass spectrometry (LC-QTOF-MS) identified a fragment with m/z 100.11 for 12/14 compounds, which could serve as a basis for MS-based nitazene screening. MOR activity determination confirmed that nitazenes are generally highly active, with potencies and efficacies of several analogues exceeding that of fentanyl. Particularly relevant is the unexpected very high potency of the N-desethylisotonitazene metabolite, rivaling the potency of etonitazene and exceeding that of isotonitazene itself. Supported by its identification in fatalities, this likely has in vivo consequences. These results improve our understanding of this emerging group of opioids by laying out an analytical framework for their detection, as well as providing important new insights into their MOR activation potential.

In vitro pharmacology of fentanyl analogs at the human mu opioid receptor and their spectroscopic analysis.

Opioids are widely misused and account for almost half of overdose deaths in the United States. The cost in terms of lives, health care, and lost productivity is significant and has been declared a national crisis. Fentanyl is a highly potent mu opioid receptor (MOR) agonist and plays a significant role in the current opioid epidemic; fentanyl and its analogs (fentalogs) are increasingly becoming one of the biggest dangers in the opioid crisis. The availability of fentalogs in the illicit market is thought to play a significant role in the recent increase in opioid-related deaths. Although there is both rodent homolog in vivo and in vitro data for some fentalogs, prior to this publication very little was known about the pharmacology of many of these illicit compounds at the human MOR (hMOR). Using gas chromatography-mass spectrometry, nuclear magnetic resonance spectroscopy, and in vitro assays, this study describes the spectral and pharmacological properties of 34 fentalogs. The reported spectra and chemical data will allow for easy identification of novel fentalogs in unknown or mixed samples. Taken together these data are useful for law enforcement and clinical workers as they will aid in the identification of fentalogs in unknown samples and can potentially be used to predict physiological effects after exposure.

Rational design and synthesis of 4-((1R,2R)-2-hydroxycyclohexyl)-2(trifluoromethyl)benzonitrile (PF-998425), a novel, nonsteroidal androgen receptor antagonist devoid of phototoxicity for dermatological indications.

4-((1 R,2 R)-2-Hydroxycyclohexyl)-2(trifluoromethyl)benzonitrile [PF-0998425, (-)- 6a] is a novel, nonsteroidal androgen receptor antagonist for sebum control and treatment of androgenetic alopecia. It is potent, selective, and active in vivo. The compound is rapidly metabolized systemically, thereby reducing the risk of unwanted systemic side effects due to its primary pharmacology. (-)- 6a was tested negative in the 3T3 NRU assay, validating our rationale that reduction of conjugation might reduce potential phototoxicity.