No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Hexahydrocannabinol Pharmacology, Toxicology, and Analysis: The First Evidence for a Recent New Psychoactive Substance
Abstract
Background:
During the last two years, hexahydrocannabinol (HHC), the hydrogenated derivative of tetrahydrocannabinol has been freely sold by internet websites as a "legal" replacement to THC and cannabis in a range of highly attractive branded and unbranded products, some of which are sold as "legal highs". Potentially, there could be a large demand for HHC products by individuals in Europe and internationally.
Methods:
Studies reporting HHC pharmacology, toxicology and analysis were identified from Pub- med and Scopus databases, and official international organizations' websites were considered.
Results:
HHC showed the effects of the typical cannabinoid on the central nervous system, with lower potency than Δ 9-THC. A few studies highlighted that 9(R)-HHC is more potent than 9(S)-HHC. This molecule showed an affinity for cannabinoid receptor CB1 both in vitro and in vivo, suggesting a pos- sible therapeutic effect in several pathologies. However, the affinity for the CB1 receptor suggests a possible addiction potential, inducing the users to misuse it. Since actual intoxication cases have not yet been reported, the HHC harmful potential was not described, probably due to the lack of effective analytical methods to detect HHC in biological matrices. Conversely, different analytical assays were developed and validated to separate HHC epimers in natural and non-natural sources.
Conclusion:
Similarly to other NPS, the HHC represents a cheaper alternative to the controlled Δ 9-THC. Its monitoring is a crucial challenge for toxicological and forensic purposes. To this concern, it is essential to further investigate HHC to support health providers in the identification of related intoxications.
Supplementary resource (1)
... The European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) reported a significant increase in seizures of a low-THC herbal cannabis material containing The European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) re ported a significant increase in seizures of a low-THC herbal cannabis material containin SCRAs, reaching 242 kg, which is 6.5 times higher than in 2020 and 1210 times higher tha 2019 [1,2,4]. More recently, the subclass of semi-synthetic cannabinoids has raised con cerns due to the growing popularity of new analogs like Δ8-THC and hexahydrocanna binol (HHC) [5,6]. Although HHC was discovered in 1940, its emergence in the Unite States' drug market happened in late 2021, with its identification as a drug of abuse oc curring in May 2022. ...
... Following the seizure of HHC-containing products in 20 EU Membe States, the EMCDDA placed strict controls on HHC as a new NPS by March 2023. HHC can be easily synthesized from cannabidiol (CBD), extracted from low-THC cannabis [6] A hexahydro cyclohexyl ring structure characterizes HHC, which naturally occurs a two different epimers: 9(R)-HHC and 9(S)-HHC ( Figure 1). According to preliminar studies, the epimers show different pharmacological activities. ...
... According to preliminar studies, the epimers show different pharmacological activities. While 9(R)-HHC exhibit stronger "cannabis" effects, the 9(S)-epimer shows no activity even at high doses [5,6 Furthermore, preliminary evidence suggested different pharmacokinetics for HHC ep mers. Recently, a few studies have described their metabolism in human hepatocytes i vitro and their metabolite excretion in urine, revealing important features about th Recently, a few studies have described their metabolism in human hepatocytes in vitro and their metabolite excretion in urine, revealing important features about the different metabolic fates of the two epimers. ...
In 2023, hexahydrocannabinol (HHC) attracted the attention of international agencies due to its rapid spread in the illegal market. Although it was discovered in 1940, less is known about the pharmacology of its two naturally occurring epimers, 9(R)-HHC and 9(S)-HHC. Thus, we aimed to investigate the disposition of hexahydrocannabinol epimers and their metabolites in whole blood, urine and oral fluid following a single controlled administration of a 50:50 mixture of 9(R)-HHC and 9(S)-HHC smoked with tobacco. To this end, six non-user volunteers smoked 25 mg of the HHC mixture in 500 mg of tobacco. Blood and oral fluid were sampled at different time points up to 3 h after the intake, while urine was collected between 0 and 2 h and between 2 and 6 h. The samples were analyzed with a validated HPLC-MS/MS method to quantify 9(R)-HHC, 9(S)-HHC and eight metabolites. 9(R)-HHC showed the highest C max and AUC 0-3h in all the investigated matrices, with an average concentration 3-fold higher than that of 9(S)-HHC. In oral fluid, no metabolites were detected, while they were observed as glucuronides in urine and blood, but with different profiles. Indeed, 11nor-9(R)-HHC was the most abundant metabolite in blood, while 8(R)OH-9(R) HHC was the most prevalent in urine. Interestingly, 11nor 9(S) COOH HHC was detected only in blood, whereas 8(S)OH-9(S) HHC was detected only in urine.
... The first total stereoselective synthesis of natural (6aR,9R,10aR)-6,6,9-trimethyl-3-pentyl-6a,7,8,9,10,10ahexahydro-6H-benzo[c]chromen-1-ol (1) and its unnatural 6aR,9S,10aR)-6,6,9-trimethyl-3-pentyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol (7) diastereomer was developed by Tietze [18] starting with 5-pentylcyclohexane-1,3-dione (8) and optically pure citronellal (9a or 9b) via a intramolecular Diels-Alder reaction and aldol condensation followed by aromatization and elimination along a two-step reaction (Scheme 1). Using this procedure, Anderson et al. [24] synthesized HHC homologs such as one lacking the C-11 methyl group (6aR,10aR)-6,6-dimethyl-3-pentyl-6a,7,8,9,10,10ahexahydro-6H-benzo[c]chromen-1-ol (13) and the C-9 geminal dimethyl analog of HHC (6aR,10aR)-6,6,9,9-tetramethyl-3-pentyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol (14) with 52% and 71% of yield, respectively ( Figure 2). They reported an action Using this procedure, Anderson et al. [24] synthesized HHC homologs such as one lacking the C-11 methyl group (6aR,10aR)-6,6-dimethyl-3-pentyl-6a, 7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol (13) and the C-9 geminal dimethyl analog of HHC (6aR,10aR)- 6,6,9,9-tetramethyl-3-pentyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol (14) with 52% and 71% of yield, respectively ( Figure 2). ...
... Using this procedure, Anderson et al. [24] synthesized HHC homologs such as one lacking the C-11 methyl group (6aR,10aR)-6,6-dimethyl-3-pentyl-6a,7,8,9,10,10ahexahydro-6H-benzo[c]chromen-1-ol (13) and the C-9 geminal dimethyl analog of HHC (6aR,10aR)-6,6,9,9-tetramethyl-3-pentyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol (14) with 52% and 71% of yield, respectively ( Figure 2). They reported an action Using this procedure, Anderson et al. [24] synthesized HHC homologs such as one lacking the C-11 methyl group (6aR,10aR)-6,6-dimethyl-3-pentyl-6a, 7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol (13) and the C-9 geminal dimethyl analog of HHC (6aR,10aR)- 6,6,9,9-tetramethyl-3-pentyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol (14) with 52% and 71% of yield, respectively ( Figure 2). They reported an action mechanism for the non-electrophilic tetrahydrocannabinol derivatives (13 and 14) through the production of spinal antinociception mediated by TRPA1, which demonstrates that the stimulation of this ion channel could be a new approach to relieve pain. ...
... Using this procedure, Anderson et al. [24] synthesized HHC homologs such as one lacking the C-11 methyl group (6aR,10aR)-6,6-dimethyl-3-pentyl-6a, 7,8,9,10,10ahexahydro-6H-benzo[c]chromen-1-ol (13) and the C-9 geminal dimethyl analog of HHC (6aR,10aR)-6, 6,9,9-tetramethyl-3-pentyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol (14) with 52% and 71% of yield, respectively ( Figure 2). They reported an action mechanism for the non-electrophilic tetrahydrocannabinol derivatives (13 and 14) through the production of spinal antinociception mediated by TRPA1, which demonstrates that the stimulation of this ion channel could be a new approach to relieve pain. ...
Natural and non-natural hexahydrocannabinols (HHC) were first described in 1940 by Adam and in late 2021 arose on the drug market in the United States and in some European countries. A background on the discovery, synthesis, and pharmacology studies of hydrogenated and saturated cannabinoids is described. This is harmonized with a summary and comparison of the cannabinoid receptor affinities of various classical, hybrid, and non-classical saturated cannabinoids. A discussion of structure–activity relationships with the four different pharmacophores found in the cannabinoid scaffold is added to this review. According to laboratory studies in vitro, and in several animal species in vivo, HHC is reported to have broadly similar effects to Δ9-tetrahydrocannabinol (Δ9-THC), the main psychoactive substance in cannabis, as demonstrated both in vitro and in several animal species in vivo. However, the effects of HHC treatment have not been studied in humans, and thus a biological profile has not been established.
... HHC exists in two enantiomeric forms, (-)-and (+)-HHC, whereby (+)-HHC does not occur naturally. (-)-HHC can occur as (9R) or (9S) diastereomer, whereby (9R)-HHC shows a significantly higher potency and efficacy than the (9S) diastereomer [12][13][14][15] . The metabolism of HHC appears to be similar to that of THC, resulting in the main metabolites 11-hydroxy-HHC (11-OH-HHC), 8-hydroxy-HHC (8-OH-HHC), and 11-nor-9carboxy-HHC (HHC-COOH) 2,8,16 . ...
The semi-synthetic cannabinoid hexahydrocannabinol (HHC) has become a highly discussed topic in forensic toxicology since 2022 due to its legal availability at this time and its psychoactive effects. This study aimed to investigate the pharmacokinetics, effects, and immunological detectability of HHC after oral (25 mg HHC fruit gum) and inhalative (three puffs from HHC vape) consumption with three participants per group. Serum (up to 48 h), urine (up to five days), and saliva (up to 48 h) samples were collected at different relevant time points and analyzed by HPLC-MS/MS for (9R)/(9S)-HHC, 11-hydroxy-HHC, and (9R)/(9S)-HHC carboxylic acid with a fully validated method. Additionally, immunological detectability was investigated with three different commercially available tests. To address the psychoactive effects, the subjective “high” feeling (scale 0–10) was monitored and different psychophysical tests (e.g. modified Romberg test, walk and turn) were conducted. Overall, the pharmacokinetics and effects of HHC were comparable to tetrahydrocannabinol (THC). However, the route of administration as well as inter-individual factors played a crucial role regarding maximum concentrations, pharmacokinetic profiles, and psychoactive effects.
... The interaction between cannabinoids and pulmonary immune function, for instance, is complex and not fully understood, making it crucial to study the effects of these compounds on lung cells. 3,[8][9][10][11][12][13][14] Additionally, potential liver toxicity from ingested cannabinoids is another area requiring close examination. ...
Background: The surge in the popularity of cannabis has led to an increase in the number of companies producing hemp-derived consumable cannabinoid products. Despite extensive exploration of cannabinoid efficacy, safety remains underreported. Any contaminants that are not deemed analytes of interest are ignored, leaving their identities and safety profiles a mystery. The unregulated nature of the cannabinoid market places the onus on reputable companies to set industry standards for product cleanliness. Objective: This study aimed to address this gap by assessing high and low potency forms of three popular hemp-derived cannabinoids – Delta 8-tetrahydrocannabinol (Δ8-THC), Hexahydrocannabinol (HHC), and Delta 9-tetrahydrocannabiphorol (Δ9-THCP). Methods: After identifying contaminants, the products were evaluated for toxicity in vitro using one liver and two lung cell lines in an effort to simulate the effects of oral consumption and inhalation. Results: Our study revealed that none of the compounds exhibited toxicity in the liver cell line, while all of the compounds exhibited toxicity in both of the lung cell lines – with the exception of one high-potency HHC sample. Conclusion: These findings highlight the critical need for stringent quality control in the cannabinoid industry, emphasizing the importance for both companies and consumers to prioritize clean, well-tested products to ensure safety in an increasingly unregulated market.
... Among this series of cannabinoids, HHC may be seen as an exotic compound in the cannabis consumer market but it is not exactly a new cannabinoid as it was discovered in 1940 by Adams and Todd in their laboratories while exploring with the hydrogenation reaction on the THC molecule in marijuana (Adams et al. 1940). Therefore, during the last two years, HHC has been openly sold on the internet websites as a "legal" and cheaper alternative to THC and cannabis (Graziano et al. 2023). ...
Since its discovery as one of the main components of cannabis and its affinity towards the cannabinoid receptor CB1, serving as a means to exert its psychoactivity, Δ⁹-tetrahydrocannabinol (Δ⁹-THC) has inspired medicinal chemists throughout history to create more potent derivatives. Initially, the goal was to synthesize chemical probes for investigating the molecular mechanisms behind the pharmacology of Δ⁹-THC and finding potential medical applications. The unintended consequence of this noble intent has been the proliferation of these compounds for recreational use. This review comprehensively covers the most exhaustive number of THC-like cannabinoids circulating on the recreational market. It provides information on the chemistry, synthesis, pharmacology, analytical assessment, and experiences related to the psychoactive effects reported by recreational users on online forums. Some of these compounds can be found in natural cannabis, albeit in trace amounts, while others are entirely artificial. Moreover, to circumvent legal issues, many manufacturers resort to semi-synthetic processes starting from legal products extracted from hemp, such as cannabidiol (CBD). Despite the aim to encompass all known THC-like molecules, new species emerge on the drug users’ pipeline each month. Beyond posing a significantly high public health risk due to unpredictable and unknown side effects, scientific research consistently lags behind the rapidly evolving recreational market.
... Thus, the current legal status of HHC would appear to be based on it being classified as non-'psychoactive substance' under the Criminal Justice (Psychoactive Substances) Act. Although there is a lack of scientific literature HHC's acute effects on humans (Graziano et al. 2023), we contend that this is a misclassification. Many online accounts, and accounts communicated to the author, outline its psychoactive effects, and indeed stores themselves often advertise the product as intoxicating. ...
Use of both cannabis and synthetic cannabinoids has been regularly linked to the development of psychotic illness. Thus, semisynthetic cannabinoids such as hexahydrocannabinol (HHC), which have a similar neurobiological profile to delta-9-THC, may also be expected to lead to psychotic illness. However, no such relationship has yet been reported in scientific literature. HHC is readily available online and in many vape shops in Ireland. Here, we present two cases of psychotic illness which appear to have been precipitated by use of legally purchased HHC and discuss its psychotogenic role and factors linked to its current widespread availability.
... Marketed as a legal substitute for THC and cannabis, HHC is featured in a diverse array of both branded and unbranded products. This trend suggests a potential surge in demand for HHC products among individuals in the USA and Europe [12]. HHC exhibits usual cannabinoid effects on the central nervous system, albeit with lower potency compared to D9-THC. ...
The surge in the popularity of cannabinoids has led to a proliferation of companies catering to the demand for such products. As the number of suppliers rises, so does the availability of consumable cannabinoid products. While some products undergo testing to meet acceptable standards, many companies opt for minimal testing that overlooks uncommon contaminants potentially harmful during smoking or inhalation. The unregulated cannabinoid market relies on reputable companies to establish standards ensuring the cleanliness of cannabinoid products. Although numerous reports explore the efficacy of cannabinoids, safety remains less extensively documented. Misconceptions about recreational cannabis use and variations in study methodologies, indications, dosing, and administration protocols hinder the overall assessment of the safety of cannabinoid-based medicines. The similarity in retention times and UV absorbance among many cannabinoids adds complexity to distinguishing isomers. Alternative techniques such as LC/MS, GC/MS, and NMR can aid in characterizing cannabinoids. Our study involved testing both high-purity cannabinoids and products from various companies, including crude and distilled THC, HHC, and THCP products which are popular among consumers. These tests were conducted against in-vitro lung cell lines to simulate the absorption of these products during inhalation. Considering the unregulated nature of the markets and the presence of both high-quality and low-quality products, our findings emphasize the importance for companies and consumers to prioritize clean products to remain competitive.
... Although the pharmacological potency of semi-synthetic cannabinoids appears to be lower than that of SCRAs, their misuse potential is very high due to the ease with which they can be manufactured and their legal status. However, little is known about their pharmacological profiles, representing a new challenge for forensic and clinical toxicologists [4,5]. ...
The New Psychoactive Substances (NPS) phenomenon represents an ever-changing global issue, with a number of new molecules entering the illicit market every year in response to international banning laws [...]
... HHC has 3 chiral carbon atoms. In the semi-synthetic HHC produced from CBD of these 3 chiral centers the stereochemistry of 2 in positions 6a and 10a is fixed (as R) and the chiral carbon in position 9a is present in both stereochemical configurations (R or S) [6]. Thus, 2 epimers exist. ...
... Since its discovery in 1964, tetrahydrocannabinol (THC) and related analogs such as cannabidiol (CBD), natural and non-natural saturated cannabinoids have caught the attention of research groups all over the world [12][13][14][15]. Hexahydrocannbinol (HHC) is a newer cannabinoid to hit the cannabis consumer market, but it is not exactly a new cannabinoid. ...
Natural and non-natural hexahydrocannabinols (HHC) were first described in 1940 by Adam and in late 2021 arose on the drug market in the United States and in some European countries. A background on the discovery, synthesis, and pharmacology studies of hydrogenated and saturated cannabinoids is described. This is harmonized with a summary and comparison of the cannabinoid receptor affinities of various classical, hybrid, and non-classical saturated cannabinoids. A discussion of structure-activity relationships with the four different pharmacophores found in the cannabinoid scaffold is added to this review. According to laboratory studies in vitro, and in several animal species in vivo, HHC is reported to have broadly similar effects to Δ9-tetrahydrocannabinol (Δ9-THC), the main psychoactive substance in cannabis, as demonstrated both in vitro and in several animal species in vivo. However, the effects of HHC treatment have not been studied in humans, and thus a biological profile has not been established.
Cannabinoids relieve pain, nausea, anorexia and anxiety, and improve quality of life in several cancer patients. The immunotherapy with checkpoint inhibitors (ICIs), although very successful in a subset of patients, is accompanied by moderate to severe immune-related adverse events (ir-AE) that often necessitate its discontinuation. Because of their role in symptomatic relief, cannabinoids have been used in combination with immune checkpoint inhibitor (ICI) immunotherapy. A few studies strongly suggest that the use of medicinal cannabis in cancer patients attenuates many of the ir-AE associated with the use of ICI immunotherapy and increase its tolerability. However, no significant beneficial effects on overall survival, progression free survival or cancer relapses were observed; rather, some of the studies noted adverse effects of concurrent administration of cannabinoids with ICI immunotherapy on the clinical benefits of the latter. Because of cannabinoids’ well documented immunosuppressive effects mediated through the cannabinoid recptor-2 (CB2), we propose considering this receptor as an inhibitory immune checkpoint per se. A simultaneous neutralization of CB2, concurrent with cannabinoid treatment, may lead to better clinical outcomes in cancer patients receiving ICI immunotherapy. In this regard, cannabinoids such as cannabidiol (CBD) and cannabigerol (CBG), with little agonism for CB2, may be better therapeutic choices. Additional strategies e.g., the use of monoacylglycerol lipase (MAGL) inhibitors that degrade some endocannabinoids as well as lipogenesis and formation of lipid bilayers in cancer cells may also be explored. Future studies should take into consideration gut microbiota, CYP450 polymorphism and haplotypes, cannabinoid-drug interactions as well as genetic and somatic variations occurring in the cannabinoid receptors and their signaling pathways in cancer cells for personalized cannabis-based therapies in cancer patients receiving ICIs. This may lead to rational knowledge-based regimens tailored to individual cancer patients.
Recently, tetrahydrocannabinol (THC) isomers and other semi-synthetic cannabinoids have been introduced into the consumer market as alternatives to botanical cannabis. To assess the prevalence of these potential new analytical targets, a liquid chromatography-tandem mass spectrometry confirmation method was developed for the quantitation of seven cannabinoid metabolites and the qualitative identification of four others in urine. The validated method was applied to authentic urine specimens that screened positive by immunoassay (50 ng/mL cutoff; n=1300). The most commonly observed analytes were 11-nor-9-carboxy-Δ8- and Δ9-THC (Δ8- and Δ9-THCCOOH), with the combination of the two seen as the most prominent analyte combination found. In addition to these metabolites, Δ1°-THCCOOH was observed in 77 specimens. This is the first study to report Δ1°-THCCOOH in authentic urine specimens, with this analyte always appearing in combination with Δ9-THCCOOH. Cross-reactivity studies were performed for (6aR,9R)-Δ1°-THCCOOH using the Beckman Coulter Emit® II Plus Cannabinoid immunoassay and demonstrated cross reactivity equivalent to the Δ9-THCCOOH cutoff, providing added confidence in the reported prevalence and detection patterns. Additionally, 11-nor-9(R)-carboxy-hexahydrocannabinol (9(R)-HHCCOOH) was the most abundant stereoisomer (n=12) in specimens containing HHC metabolites alone (n=14). This is in contrast to 9(S)-HHCCOOH, which was the predominant stereoisomer in specimens containing Δ8- and/or Δ9-THCCOOH. Although HHC and Δ1°-THC metabolites are emerging toxicology findings, based on these specimens collected between April 2022 and May 2024, an analytical panel containing Δ8- and Δ9-THCCOOH appears to be sufficient for revealing cannabinoid exposure within workplace monitoring and deterrence programs.
Recently, hexahydrocannabinol (HHC) was posed under strict control in Europe due to the increasing HHC-containing material seizures. The lack of analytical methods in clinical laboratories to detect HHC and its metabolites in biological matrices may result in related intoxication underreporting. We developed and validated a comprehensive GC-MS/MS method to quantify 9(R)-HHC, 9(S)-HHC, 9αOH-HHC, 9βOH-HHC, 8(R)OH-9(R)-HHC, 8(S)OH-9(S)HHC, 11OH-9(R)HHC, 11OH-9(S)HHC, 11nor-carboxy-9(R)-HHC, and 11nor-carboxy-9(S)-HHC in whole blood, urine, and oral fluid. A novel QuEChERS extraction protocol was optimized selecting the best extraction conditions suitable for all the three matrices. Urine and blood were incubated with β-glucuronidase at 60 °C for 2 h. QuEChERS extraction was developed assessing different ratios of Na2SO4:NaCl (4:1, 2:1, 1:1, w/w) to be added to 200 µL of any matrix added with acetonitrile. The chromatographic separation was achieved on a 7890B GC with an HP-5ms column, (30 m, 0.25 mm × 0.25 µm) in 12.50 min. The analytes were detected with a triple-quadrupole mass spectrometer in the MRM mode. The method was fully validated following OSAC guidelines. The method showed good validation parameters in all the matrices. The method was applied to ten real samples of whole blood (n = 4), urine (n = 3), and oral fluid (n = 3). 9(R)-HHC was the prevalent epimer in all the samples (9(R)/9(S) = 2.26). As reported, hydroxylated metabolites are proposed as urinary biomarkers, while carboxylated metabolites are hematic biomarkers. Furthermore, 8(R)OH-9(R)HHC was confirmed as the most abundant metabolite in all urine samples.
With wider availability of synthetic and semi-synthetic cannabinoids in the consumer space, there is a growing impact on public health and safety. Forensic toxicology laboratories should keep these compounds in mind as they attempt to remain effective in screening for potential sources of human performance impairment. Enzyme-linked immunosorbent assay (ELISA) is a commonly utilized tool in forensic toxicology, as its efficiency and sensitivity make it useful for rapid and easy screening for a large number of drugs. This screening technique has lower specificity, which allows for broad cross-reactivity among structurally similar compounds. In this study, the Cannabinoids Direct ELISA kit from Immunalysis was utilized to assess the cross-reactivities of 24 cannabinoids and metabolites in whole blood. The assay was calibrated with 5 ng/mL of 11-nor-9-carboxy-Δ9-tetrahydrocannabinol and the analytes of interest were evaluated at concentrations ranging from 5 to 500 ng/mL. Most parent compounds demonstrated cross-reactivity ≥20 ng/mL, with increasing alkyl side-chain length relative to Δ9-tetrahydrocannabinol resulting in decreased cross-reactivity. Of the 24 analytes, only the carboxylic acid metabolites, 11-nor-9-carboxy-Δ8-tetrahydrocannabinol, 11-nor-9(R)-carboxy-hexahydrocannabinol and 11-nor-9(S)-carboxy-hexahydrocannabinol, were cross-reactive at levels ≤10 ng/mL. Interestingly, 11-nor-9(R)-carboxy-hexahydrocannabinol demonstrated cross-reactivity at 5 ng/mL, where its stereoisomer 11-nor-9(S)-carboxy-hexahydrocannabinol, did not. As more information emerges about the prevalence of these analytes in blood specimens, it is important to understand and characterize their impact on current testing paradigms.
This study investigated the effects of hexahydrocannabinol (HHC) and other unclassified cannabinoids, which were recently introduced to the recreational drug market, on cannabis drug testing in urine and oral fluid samples. After the appearance of HHC in Sweden in 2022, the number of posts about HHC on an online drug discussion forum increased significantly in the spring of 2023, indicating increased interest and use. In parallel, the frequency of false positive screening tests for tetrahydrocannabinol (THC) in oral fluid, and for its carboxy metabolite (THC-COOH) in urine, rose from <2% to >10%. This suggested that HHC cross-reacted with the antibodies in the immunoassay screening, which was confirmed in spiking experiments with HHC, HHC-COOH, HHC acetate (HHC-O), hexahydrocannabihexol (HHC-H), hexahydrocannabiphorol (HHC-P), and THC-P. When HHC and HHC-P were classified as narcotics in Sweden on 11 July 2023, they disappeared from the online and street shops market and were replaced by other unregulated variants (e.g. HHC-O and THC-P). In urine samples submitted for routine cannabis drug testing, HHC-COOH concentrations up to 205 (mean 60, median 27) µg/L were observed. To conclude, cannabis drug testing cannot rely on results from immunoassay screening, as it cannot distinguish between different tetra- and hexahydrocannabinols, some being classified but others unregulated. The current trend for increased use of unregulated cannabinols will likely increase the proportion of positive cannabis screening results that need to be confirmed with mass spectrometric methods. However, the observed cross-reactivity also means a way to pick up use of new cannabinoids that otherwise risk going undetected.
Context
Hexahydrocannabinol (HHC), a compound derived from synthetic production using cannabidiol (CBD) or delta‐9‐tetrahydrocannabinol (Δ ⁹ ‐THC), has gained recent attention due to its presence in seized materials across Europe. Sold legally in various forms, HHC poses potential health risks, particularly as a legal alternative to THC in some countries. Despite its historical description in the 1940s, limited toxicology data, pharmacological understanding, and analytical methods for HHC exist.
Method
This study proposes analytical techniques using mass spectrometry to detect, identify, and quantify (9 R )‐HHC and (9 S )‐HHC, concurrently with THC and CBD in various matrices, including oral fluid, whole blood, and seized material. Three distinct methods were employed for different matrices: GC/MS for seized material, GC/MS/MS for whole blood, and UHPLC/MS/MS for oral fluid. Methods were validated qualitatively for oral fluid with a FLOQSwab® device and quantitatively in whole blood and seized material according to Peters et al's recommendations and ICH guidelines.
Results
Validated methods were considered reliable in detecting and quantifying HHC isomers in terms of repeatability, reproducibility, and linearity with r ² systematically >0.992. These methods were applied to authentic cases, including seized materials and biological samples from traffic control (whole blood and oral fluid). In seized materials, (9 R )‐HHC levels ranged from 2.09% to 8.85% and (9 R )‐HHC/(9 S )‐HHC ratios varied from 1.36 to 2.68. In whole blood sample, (9 R )‐HHC and (9 S )‐HHC concentrations were, respectively, 2.38 and 1.39 ng/mL. For all analyzed samples, cannabinoids such as THC and CBD were also detected.
Conclusion
This research contributes analytical insights into differentiating and simultaneously analyzing (9 R )‐HHC and (9 S )‐HHC, using widely applicable mass spectrometric methods. The study emphasizes the need for vigilance among toxicologists, as new semisynthetic cannabinoids continue to emerge in Europe, with potential health implications. The findings underscore the importance of reliable analytical methods for monitoring these compounds in forensic and clinical settings.
We report studies pertaining to two isomeric hexahydrocannabinols (HHCs), (9R)-HHC and (9S)-HHC, which are derivatives of the psychoactive cannabinoids Δ9- and Δ8-THC. HHCs have been known since the 1940s, but have become increasingly available to the public in the United States and are typically sold as a mixture of isomers. We show that (9R)-HHC and (9S)-HHC can be prepared using hydrogen-atom transfer reduction, with (9R)-HHC being accessed as the major diastereomer. In addition, we report the results of cannabinoid receptor studies for (9R)-HHC and (9S)-HHC. The binding affinity and activity of isomer (9R)-HHC are similar to that of Δ9-THC, whereas (9S)-HHC binds strongly in cannabinoid receptor studies but displays diminished activity in functional assays. This is notable, as our examination of the certificates of analysis for >60 commercially available HHC products show wide variability in HHC isomer ratios (from 0.2:1 to 2.4:1 of (9R)-HHC to (9S)-HHC). These studies suggest the need for greater research and systematic testing of new cannabinoids. Such efforts would help inform cannabis-based policies, ensure the safety of cannabinoids, and potentially lead to the discovery of new medicines.
The characterization of any compound is important in the field of chemistry. As the field of cannabinoid chemistry grows so does the need for the characterization of new cannabinoids or rare cannabinoids that gain popularity within the consumer and research fields. Hexahydrocannabinol (HHC) a hydrogenated analogue of Δ9-tetrahydrocannabinol (THC), also found in trace amounts naturally within the Cannabis sativa plant, has been gaining attention and popularity within the cannabis industry. Hexahydrocannabidiol (H4CBD) is a synthetic hydrogenated analogue to cannabidiol (CBD). Identifying the diastereomers of the cannabinoids with instrumentation plays a huge role within the chemistry field adding valuable information of the structure and the parameters for others to identify such cannabinoids. Elucidation and characterization of HHC and H4CBD were performed using current analytical techniques such as 1D and 2D nuclear magnetic resonance (NMR), high performance liquid chromatography (HPLC), and gas chromatography-mass spectrometry (GC-MS), effectively characterizing both the diastereomers of HHC and H4CBD.
Cannabinoid-induced tetrad is a preclinical model commonly used to evaluate if a pharmacological compound is an agonist of the central type-1 cannabinoid (CB1) receptor in rodents. The tetrad is characterized by hypolocomotion, hypothermia, catalepsy, and analgesia, four phenotypes that are induced by acute administration of CB1 agonists exemplified by the prototypic cannabinoid delta-9-tetrahydrocannabinol (THC). This unit describes a standard protocol in mice to induce tetrad phenotypes with THC as reference cannabinoid. We provide typical results obtained with this procedure showing a dose effect of THC in different mouse strains. The effect of the CB1 antagonist rimonabant is also shown. This tetrad protocol is well adapted to reveal new compounds acting on CB1 receptors in vivo.
Behavioral studies of chronic CB(1) receptor activation may provide a pharmacological approach to understanding efficacy-related differences among CB(1) ligands and, as well, mechanistic commonalities between cannabinoid and non-cannabinoid drugs. In the present studies, the effects of CB(1) agonists (AM411, AM4054, WIN55,212.2, Δ(9)-THC, methanandamide), CB(1) antagonists (SR141716A, AM4113), and of dopamine (DA)-related [methamphetamine, SKF82958, SCH23390, R-(-)-NPA, haloperidol) and opioid (morphine, naltrexone) drugs on scheduled-controlled responding under a 30-response fixed-ratio (FR30) schedule of stimulus-shock termination in squirrel monkeys were compared before and during chronic treatment with the long-acting CB(1) agonist AM411 (1.0 mg/kg/day, i.m.). Pre-chronic treatment with all drugs except naltrexone (1-10 mg/kg) produced dose-related decreases in responses rates. Dose-response re-determinations during chronic treatment revealed: a) >250 fold (AM411, methanandamide) and >45-fold (AM4054, WIN55,212.2, Δ(9)-THC) rightward shifts in the ED(50) values for CB(1) agonists; b) >100-fold and >20-fold leftward shifts in the ED(50) values for SR141716A and AM4113, respectively; and c) approximately 4.8-and 10-fold rightward shifts in the ED(50) values for methamphetamine and the DA D(2) agonist R-(-)-NPA, respectively. Dose-response relationships for other DA-related and opioid drugs were unchanged by chronic CB(1) agonist treatment. Differences in the magnitude of tolerance among CB(1) agonists during chronic treatment may be indicative of differences in their pharmacological efficacy, whereas the enhanced sensitivity to behaviorally disruptive effects of CB(1) antagonists may provide evidence for CB(1)-related behavioral and/or physical dependence. Finally, the development of cross-tolerance to methamphetamine and R-(-)-NPA bolsters previous evidence of interplay between CB(1) and DA D(2) signaling mechanisms.
Δ-Tetrahydrocannabinol (THC) has been characterized as a partial agonist at cannabinoid CB1 receptors in vitro; however, it often produces the same maximum effects in vivo as other cannabinoid agonists. This study was carried out to determine whether THC would antagonize the hypothermic effects of another cannabinoid agonist, AM2389, in mice. Male mice were injected with 1-100 mg/kg THC, 0.01-0.1 mg/kg AM2389, or a combination of 30 mg/kg THC and 0.1-1.0 mg/kg AM2389, and rectal temperature was recorded for up to 12 h after injection. THC reduced the temperature by 5.6°C at a dose of 30 mg/kg; further increases in the dose did not produce larger effects, indicating a plateau in the THC dose-effect function. AM2389 reduced temperature by 9.0°C at a dose of 0.1 mg/kg. One hour pretreatment with 30 mg/kg THC attenuated the hypothermic effects of 0.1 mg/kg AM2389; a 10-fold higher dose, 1.0 mg/kg AM2389, was required to further decrease temperature, reflecting a five-fold rightward shift of the lower portion of the AM2389 dose-effect function following THC pretreatment. These results indicate that, in an assay of mouse hypothermia, THC exerts both agonist and antagonist effects following acute administration, and mark the first demonstration of partial agonist/antagonist effects of THC in vivo.
Marijuana and many of its constituent cannabinoids influence the central nervous system (CNS) in a complex and dose-dependent manner. Although CNS depression and analgesia are well documented effects of the cannabinoids, the mechanisms responsible for these and other cannabinoid-induced effects are not so far known. The hydrophobic nature of these substances has suggested that cannabinoids resemble anaesthetic agents in their action, that is, they nonspecifically disrupt cellular membranes. Recent evidence, however, has supported a mechanism involving a G protein-coupled receptor found in brain and neural cell lines, and which inhibits adenylate cyclase activity in a dose-dependent, stereoselective and pertussis toxin-sensitive manner. Also, the receptor is more responsive to psychoactive cannabinoids than to non-psychoactive cannabinoids. Here we report the cloning and expression of a complementary DNA that encodes a G protein-coupled receptor with all of these properties. Its messenger RNA is found in cell lines and regions of the brain that have cannabinoid receptors. These findings suggest that this protein is involved in cannabinoid-induced CNS effects (including alterations in mood and cognition) experienced by users of marijuana.
The major active ingredient of marijuana, delta 9-tetrahydrocannabinol (delta 9-THC), has been used as a psychoactive agent for thousands of years. Marijuana, and delta 9-THC, also exert a wide range of other effects including analgesia, anti-inflammation, immunosuppression, anticonvulsion, alleviation of intraocular pressure in glaucoma, and attenuation of vomiting. The clinical application of cannabinoids has, however, been limited by their psychoactive effects, and this has led to interest in the biochemical bases of their action. Progress stemmed initially from the synthesis of potent derivatives of delta 9-THC, and more recently from the cloning of a gene encoding a G-protein-coupled receptor for cannabinoids. This receptor is expressed in the brain but not in the periphery, except for a low level in testes. It has been proposed that the nonpsychoactive effects of cannabinoids are either mediated centrally or through direct interaction with other, non-receptor proteins. Here we report the cloning of a receptor for cannabinoids that is not expressed in the brain but rather in macrophages in the marginal zone of spleen.
The enteric nervous system of several species, including the mouse, rat, guinea pig and humans, contains cannabinoid CB1 receptors that depress gastrointestinal motility, mainly by inhibiting ongoing contractile transmitter release. Signs of this depressant effect are, in the whole organism, delayed gastric emptying and inhibition of the transit of non-absorbable markers through the small intestine and, in isolated strips of ileal tissue, inhibition of evoked acetylcholine release, peristalsis, and cholinergic and non-adrenergic non-cholinergic (NANC) contractions of longitudinal or circular smooth muscle. These are contractions evoked electrically or by agents that are thought to stimulate contractile transmitter release either in tissue taken from morphine pretreated animals (naloxone) or in unpretreated tissue (gamma-aminobutyric acid and 5-hydroxytryptamine). The inhibitory effects of cannabinoid receptor agonists on gastric emptying and intestinal transit are mediated to some extent by CB1 receptors in the brain as well as by enteric CB1 receptors. Gastric acid secretion is also inhibited in response to CB1 receptor activation, although the detailed underlying mechanism has yet to be elucidated. Cannabinoid receptor agonists delay gastric emptying in humans as well as in rodents and probably also inhibit human gastric acid secretion. Cannabinoid pretreatment induces tolerance to the inhibitory effects of cannabinoid receptor agonists on gastrointestinal motility. Findings that the CB1 selective antagonist/inverse agonist SR141716A produces in vivo and in vitro signs of increased motility of rodent small intestine probably reflect the presence in the enteric nervous system of a population of CB1 receptors that are precoupled to their effector mechanisms. SR141716A has been reported not to behave in this manner in the myenteric plexus-longitudinal muscle preparation (MPLM) of human ileum unless this has first been rendered cannabinoid tolerant. Nor has it been found to induce "withdrawal" contractions in cannabinoid tolerant guinea pig ileal MPLM. Further research is required to investigate the role both of endogenous cannabinoid receptor agonists and of non-CB1 cannabinoid receptors in the gastrointestinal tract. The extent to which the effects on gastrointestinal function of cannabinoid receptor agonists or antagonists/inverse agonists can be exploited therapeutically has yet to be investigated as has the extent to which these drugs can provoke unwanted effects in the gastrointestinal tract when used for other therapeutic purposes.
Cannabis sativa L. preparations have been used in medicine for millenia. However, concern over the dangers of abuse led to the banning of the medicinal use of marijuana in most countries in the 1930s. Only recently, marijuana and individual natural and synthetic cannabinoid receptor agonists and antagonists, as well as chemically related compounds, whose mechanism of action is still obscure, have come back to being considered of therapeutic value. However, their use is highly restricted. Despite the mild addiction to cannabis and the possible enhancement of addiction to other substances of abuse, when combined with cannabis, the therapeutic value of cannabinoids is too high to be put aside. Numerous diseases, such as anorexia, emesis, pain, inflammation, multiple sclerosis, neurodegenerative disorders (Parkinson's disease, Huntington's disease, Tourette's syndrome, Alzheimer's disease), epilepsy, glaucoma, osteoporosis, schizophrenia, cardiovascular disorders, cancer, obesity, and metabolic syndrome-related disorders, to name just a few, are being treated or have the potential to be treated by cannabinoid agonists/antagonists/cannabinoid-related compounds. In view of the very low toxicity and the generally benign side effects of this group of compounds, neglecting or denying their clinical potential is unacceptable - instead, we need to work on the development of more selective cannabinoid receptor agonists/antagonists and related compounds, as well as on novel drugs of this family with better selectivity, distribution patterns, and pharmacokinetics, and - in cases where it is impossible to separate the desired clinical action and the psychoactivity - just to monitor these side effects carefully.
Since the early 2000s, there has been a turmoil on the global illicit cannabinoid market. Parallel to legislative changes in some jurisdictions regarding herbal cannabis, unregulated and cheap synthetic cannabinoids with astonishing structural diversity have emerged. Recently, semi-synthetic cannabinoids manufactured from hemp extracts by simple chemical transformations have also appeared as recreational drugs. The burst of these semi-synthetic cannabinoids into the market was sparked by legislative changes in the United States, where cultivation of industrial hemp restarted. By now, hemp-derived cannabidiol (CBD), initially a blockbuster product on its own, became a "precursor" to semi-synthetic cannabinoids such as hexahydrocannabinol (HHC), which appeared on the drug market in 2021. The synthesis and cannabimimetic activity of HHC were first reported eight decades ago in quest for the psychoactive principles of marijuana and hashish. Current large-scale manufacture of HHC is based on hemp-derived CBD extract, which is converted first by cyclization into a Δ8 /Δ9 -THC mixture, followed by catalytic hydrogenation to afford a mixture of (9R)-HHC and (9S)-HHC epimers. Preclinical studies indicate that (9R)-HHC has THC-like pharmacological properties. The animal metabolism of HHC is partially clarified. The human pharmacology including metabolism of HHC is yet to be investigated, and (immuno)analytical methods for the rapid detection of HHC or its metabolites in urine are lacking. Herein, the legal background for the revitalization of hemp cultivation, and available information on the chemistry, analysis, and pharmacology of HHC and related analogs, including HHC acetate (HHC-O) is reviewed.
Qualitative analysis of several commercial products containing Δ8-tetrahydrocannabinol (Δ8-THC) as a major component using GC-MS resulted in the identification of several impurities along with Δ8-THC. In an attempt to isolate and identify these impurities, a commercial Δ8-THC distillate was selected for the isolation work. Eleven impurities were isolated using a variety of chromatographic techniques, and their chemical structures were determined. These include Δ4,8-iso-tetrahydrocannabinol (1), Δ4-iso-tetrahydrocannabinol (2), Δ8-cis-iso-tetrahydrocannabinol (3), 4,8-epoxy-iso-tetrahydrocannabinol (4), 8-hydroxy-iso-tetrahydrocannabinol (5), 9β-hydroxyhexahydrocannabinol (6), 9α-hydroxyhexa-hydrocannabinol (7), iso-tetrahydrocannabifuran (8), cannabicitran (CBT, 9), olivetol (10), and Δ9-THC (11). The chemical structures of the purified compounds were determined using several spectroscopic methods, including 1D (1H, 13C, and DEPT-135) and 2D (COSY, HMQC, HMBC, and NOESY) NMR, LC-MS, and GC-MS. Other naturally occurring cannabinoids and impurities were also identified in GC-MS chromatograms but were not isolated. These were cannabidiol (CBD, 12), cannabinol (CBN, 13), hexahydrocannabinol (HHC, 14), and Δ8-tetrahydrocannabivarin (Δ8-THCV, 15). The chemical structure of Δ8-THCV (15), for which a standard was not available, was confirmed by partial synthesis and NMR analysis. This is the first report for many of the above compounds as well as Δ8-THCV as impurities in Δ8-THC products.
Introduction: Hexahydrocannabinols (HHCs), referred to as (9R)-HHC and (9S)-HHC diastereoisomers, are poorly studied cannabinoids naturally found in small concentrations in the pollen and the seeds of the hemp plants. Aim: In this study, for the first time, we describe the finding of (9R)-HHC and (9S)-HHC in two commercialized hemp derived products. Methods: The achievement of reference standards by semisynthetic or isolation approach allows us to develop and validate a gas chromatography mass spectrometry method for the identification and quantification of HHCs in hemp-derived resin. Results: The two analyzed samples showed percentage of 42.5 and 41.5 for (9R)-HHC and of 23.6 and 23.6 for (9S)-HHC. Conclusions: Despite the lack of in-depth studies about HHCs activity, potency, toxicity, and safety, these cannabinoids are emerging on the light-cannabis (hemp) market probably because legislations still do not clearly regulate them. Since analytical assay for hemp-derived products usually include only Δ9-THC, THC-A, CBD, and CBD-A, a thorough investigation could be carried out to reveal the possible addition of "new" compounds that might be a matter of safety.
We report the development of novel cannabinergic probes that can stabilize the cannabinoid receptors (CBRs) through tight binding interactions. Ligand design involves the introduction of select groups at a judiciously chosen position within the classical hexahydrocannabinol template (monofunctionalized probes). Such groups include the electrophilic isothiocyanato, the photoactivatable azido, and the polar cyano moieties. These groups can also be combined to produce bifunctionalized probes potentially capable of interacting at two distinct sites within the CBR-binding domains. These novel compounds display remarkably high binding affinities for CBRs and are exceptionally potent agonists. A key ligand (27a, AM11245) exhibits exceptionally high potency in both in vitro and in vivo assays and was designated as "megagonist," a property attributed to its tight binding profile. By acting both centrally and peripherally, 27a distinguishes itself from our previously reported "megagonist" AM841, whose functions are restricted to the periphery.
The first total synthesis of potent cannabinoid, 9β-11-hydroxyhexahydrocannabinol is achieved through proline catalyzed inverse-electron-demand Diels-Alder reaction. Using this asymmetric catalysis, the cyclohexane ring is constructed with two chiral centers as a single diastereomer with 97% ee. The creation of the third chiral center and benzopyran ring is demonstrated with the elegant synthetic strategies. This mild and efficient synthetic methodology provides a new route for the asymmetric synthesis of the other potent hexahy-drocannabinols.
The cannabinoid receptor 1 (CB1) is the principal target of the psychoactive constituent of marijuana, the partial agonist Δ(9)-tetrahydrocannabinol (Δ(9)-THC). Here we report two agonist-bound crystal structures of human CB1 in complex with a tetrahydrocannabinol (AM11542) and a hexahydrocannabinol (AM841) at 2.80 Å and 2.95 Å resolution, respectively. The two CB1-agonist complexes reveal important conformational changes in the overall structure, relative to the antagonist-bound state, including a 53% reduction in the volume of the ligand-binding pocket and an increase in the surface area of the G-protein-binding region. In addition, a 'twin toggle switch' of Phe200(3.36) and Trp356(6.48) (superscripts denote Ballesteros-Weinstein numbering) is experimentally observed and appears to be essential for receptor activation. The structures reveal important insights into the activation mechanism of CB1 and provide a molecular basis for predicting the binding modes of Δ(9)-THC, and endogenous and synthetic cannabinoids. The plasticity of the binding pocket of CB1 seems to be a common feature among certain class A G-protein-coupled receptors. These findings should inspire the design of chemically diverse ligands with distinct pharmacological properties.
The title compounds have been prepared in high optical and diastereoisomeric purity by diethylaluminium chloride-assisted condensation of suitable phenols with (R)-(+)- or (S)-(–)-citronellal; the X-ray crystal structure of a hexahydrocannabinol analogue is reported.
A series of tetrahydro- and hexahydrocanrab'nol derivatives was prepared in which the substituents at position 9 were varied. These compounds were evaluated in mice for their effects on locomotor activity, body temperature, muscle tone, and analgesia. Depression of body temperature and locomotor function was demonstrated by several compounds, but all were devoid of any significant analgesic activity.
Administration of pure 1-δ9-tetrahydrocannabinol to mice had the following dose-dependent nzeurochemical and behavioral effects: a slight but significant
increase in concentrations of 5-hydroxytryptamine in whole brain; a decrease in concentration of norepinephrine in brain after
administration of low doses and an increase after high doses; diminished spontaneous activity, mloderate hypothermnia, hypersetisitivity
to tactile and auditory stimiuli, and ataxia after low doses; and sedation, pronounced hypothermia, and markedly diminished
spon taneous activity and reactivity after high doses. The duration of the effects on body temperature and spontaneous activity
correlated generally with the changes in brain amines. The characteristic changes in brain amines do not correspond exactly
to those observed with other psychotropic drugs.
This Letter reports new and efficient synthetic approaches for biol. interesting cannabinoid analogs. The key strategies involve ethylenediamine diacetate/triethylamine-catalyzed cyclization. As an application of this methodol., one-step synthesis of biol. active natural (-)-hexahydrocannabinol (I) and its unnatural enantiomer (+)-hexahydrocannabinol was carried out. [on SciFinder(R)]
Both natural and synthetic cannabinoids have been shown to suppress the growth of tumor cells in culture and in animal models by affecting key signaling pathways including angiogenesis, a pivotal step in tumor growth, invasion, and metastasis. In our search for cannabinoid-like anticancer agents devoid of psychoactive side effects, we synthesized and evaluated the anti-angiogenic effects of a novel series of hexahydrocannabinol analogs. Among these, two analogs LYR-7 [(9S)-3,6,6,9-tetramethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol] and LYR-8 [(1-((9S)-1-hydroxy-6,6,9-trimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-2-yl)ethanone)] were selected based on their anti-angiogenic activity and lack of binding affinity for cannabinoid receptors. Both LYR-7 and LYR-8 inhibited VEGF-induced proliferation, migration, and capillary-like tube formation of HUVECs in a concentration-dependent manner. The inhibitory effect of the compounds on cell proliferation was more selective in endothelial cells than in breast cancer cells (MCF-7 and tamoxifen-resistant MCF-7). We also noted effective inhibition of VEGF-induced new blood vessel formation by the compounds in the in vivo chick chorioallantoic membrane (CAM) assay. Furthermore, both LYR analogs potently inhibited VEGF production and NF-κB transcriptional activity in cancer cells. Additionally, LYR-7 or LYR-8 strongly inhibited breast cancer cell-induced angiogenesis and tumor growth. Together, these results suggest that novel synthetic hexahydrocannabinol analogs, LYR-7 and LYR-8, inhibit tumor growth by targeting VEGF-mediated angiogenesis signaling in endothelial cells and suppressing VEGF production and cancer cell growth.
The effects of a number of cannabinoids in squirrel monkeys trained to respond on a chain fixed-interval fixed-ratio schedule of food presentation were determined after intraperitoneal (i.p.) and intraventricular (i.v.t.) administration. The order of potency was (+/-)-9-nor-9 beta-OH hexahydrocannabinol, 11-OH-delta 9-tetrahydrocannabinol, delta 9-tetrahydrocannabinol (delta 9-THC), cannabinol and cannabidiol. (+/-)-9-Nor-9 alpha-OH-hexahydrocannabinol was inactive at doses up to 3 mg/kg i.p. and 0.1 mg/kg i.v.t. Although the order of potency was the same by both routes of administration, the i.v.t./i.p. potency ratio differed markedly. This demonstrates the importance of route of administration in assessing structure-activity relationships of cannabinoids and suggests that differences in penetration to the central nervous system may be an important determinant of behavioral activity. Although 11-OH-delta 9-THC was more potent than the parent compound delta 9-THC by both routes, the potency difference was less after i.v.t. administration. It was also demonstrated that metabolic conversion of [3H]delta 9-THC does not take place in squirrel monkey brain when administered i.v.t. which could account for the direct i.v.t. effects of delta 9-THC. These observations suggest that metabolic conversion of delta 9-THC in the liver is not necessary for its behavioral effects.
The 1,1-dimethylheptyl (DMH) homologue of 7-hydroxy-delta 6-tetrahydrocannabinol (3) is the most potent cannabimimetic substance reported so far. Hydrogenation of 3 leads to a mixture of the epimers of 5'-(1,1-dimethylheptyl)-7-hydroxyhexahydrocannabinol or to either the equatorial (7) or to the axial epimer (8), depending on the catalysts and conditions used. Compound 7 discriminates for delta 1-THC (2) in pigeons (ED50 = 0.002 mg/kg, after 4.5 h), at the potency level of 3, and binds to the cannabinoid receptor with a KD of 45 pM, considerably lower than the Ki of 180 pM measured for compound 3 and the Ki of 2.0 nM measured for CP-55940 (1), a widely employed ligand. Tritiated 7 was used as a novel probe for the cannabinoid receptor.
The compound 9-beta-hydroxy-hexahydrocannabinol [(-)-9 beta-OH-HHC] was designed to fit a combined theoretical profile of an analgesic cannabinoid (equatorial alcohol at C-9, phenol at C-1 and a C-3 side chain) with reduced psychoactivity (axial C-9 substituent which protrudes into the alpha face). (-)-9 beta-OH-HHC was synthesized by the addition of methyl Grignard to 9-oxo-11-nor-HHC. Its alpha epimer was obtained by the regiospecific epoxide ring opening of 9 alpha, 10 alpha-epoxy-HHC acetate. (-)-9 beta-OH-HHC and (-)-9 alpha-OH-HHC were each evaluated in a battery of tests in mice and were found to be 10-25 times less potent than (-)-trans-delta 9-tetrahydrocannabinol (delta 9-THC) in all tests including the tail flick test for antinociception (analgesia). Molecular mechanics calculations [MMP2(85)] revealed that, in the global minimum energy conformation of (-)-9 beta-OH-HHC, the axial methyl at C-9 protrudes into the alpha face of the molecule, while the axial hydroxyl at C-9 in (-)-9 alpha-OH-HHC protrudes into this same face. These calculations also identified a higher energy carbocyclic ring (twist) conformer of each in which there is no protrusion of a C-9 substituent of the carbocyclic ring into the alpha face. The minimal activity of both compounds is attributed to these higher energy forms.(ABSTRACT TRUNCATED AT 250 WORDS)
We have found a correlation between cannabinoid psychopharmacological activity and the orientation of the C9 substituent in one class of cannabinoid derivatives. We report here a study of the active cannabinoids delta 9-tetra-hydrocannabinol (delta 9-THC), delta 8-tetrahydrocannabinol (delta 8-THC), and 11 beta-hexahydrocannabinol (11 beta-HHC); the minimally active cannabinoid 11 alpha-hexahydrocannabinol (11 alpha-HHC); and the inactive cannabinoids delta 7-tetrahydrocannabinol (delta 7-THC) and delta 9,11-tetrahydrocannabinol (delta 9,11-THC). Our working hypothesis is that there are two components of cannabinoid structure which confer upon these compounds reactivity characteristics crucial to activity: the directionality of the lone pairs of electrons of the phenyl group hydroxyl oxygen and the orientation of the carbocyclic ring relative to this oxygen. The structures of these six molecules were optimized by using the method of molecular mechanics as encoded in the MMP2(85) program. Other possible minimum-energy conformations of the carbocyclic ring were calculated by driving one torsion angle in this ring by use of the dihedral driver option in MMP2(85). The rotational energy behavior of the phenyl group hydroxyl in each molecule was studied also by using the dihedral driver option in MMP2(85). We found that the carbocyclic ring in 11 alpha-HHC can exist in either a chair or a twist conformation. The carbocyclic ring in delta 9-THC, in delta 8-THC, and in delta 7-THC was found to exist only in a half-chair conformation, while the carbocyclic ring in 11 beta-HHC and in delta 9,11-THC was found to exist only in a chair form. The results of the rotational energy profiles indicated that the minimum-energy positions of the phenyl group hydroxyls are nearly identical in all molecules. These molecules, then, were found to differ only in the conformation of the carbocyclic ring in each. This conformation, in turn, determines the orientation of this ring and its C9 substituent relative to the oxygen of the phenyl group hydroxyl. In order to assess the orientation of the carbocyclic ring with respect to the phenyl group hydroxyl oxygen in each optimized structure, the following nonbonded torsion angles were measured: C10-C10a-C1-O, C8-C7-C1-O, C11-C9-C1-O, and C9-Q-C1-O (where Q is a dummy atom placed midway between C8 and C10).(ABSTRACT TRUNCATED AT 400 WORDS)
The binding of [3H]-5'-trimethylammonium delta 8-tetrahydrocannabinol (THC) [( 3H]TMA) to rat neuronal membranes was studied. TMA is a positively charged analog of delta 8THC modified on the 5' carbon, a portion of the molecule not important for its psychoactivity. Unlabeled TMA inhibits field-stimulated contractions of the guinea-pig ileum (IC50 = 1 microM) in the same presynaptic manner as delta 9THC. [3H]TMA binds saturably and reversibly to brain membranes with high affinity (KD = 89 nM) to apparently one class of site (Hill coefficient, 1.1). Highest binding site density occurs in the brain, but several peripheral organs also display specific binding. Detergent solubilizes the sites without affecting their pharmacological properties. Molecular sieve chromatography reveals a bimodal peak of [3H]TMA binding activity of approximately 60,000 daltons apparent molecular weight. delta 9THC competitively inhibits [3H]TMA binding potently (Ki = 27 nM) and stereoselectively. For some cannabinoids potency in behavioral and physiological tests parallels their affinity for the [3H]TMA binding site. However, several nonpsychotropic cannabinoids are active at the binding site.
The interaction of delta 9-tetrahydrocannabinol (delta 9-THC) and related cannabinoids with opioid receptors of neuronal membranes has been investigated. Treatment of membranes with delta 9-THC consistently decreased specific in vitro binding of [3H]dihydromorphine (mu opioid) in a dose-dependent fashion. Similar dose-dependent changes were elicited by cannabidiol and (+/-)-hexahydrocannabinol. Equilibrium binding studies in which brain membranes were titrated with [3H]dihydromorphine in the presence of delta 9-THC demonstrated that the decrease in [3H]dihydromorphine binding is due to a reduction in the number of binding sites, with no significant alteration in receptor affinity. This result suggests that the interaction of delta 9-THC with opioid receptors is a noncompetitive one. Delta 9-THC also inhibited the binding of the delta opioid [3H]D-Pen2, D-Pen5-enkephalin and the opioid antagonist [3H]naloxone (Ki = 16 and 19 microM, respectively) but failed to inhibit the binding of the kappa opioid [3H]ethylketocyclazocine (after suppression of mu and delta receptor binding), the phencyclidine analog [3H]N-(1-[2-theinyl]cyclohexyl)piperidine, the dopamine antagonist [3H]spiroperidol or the muscarinic antagonist [3H]quinuclidinyl benzilate. Moreover, delta 9-THC inhibited the binding of [3H]etorphine (potent opioid agonist) to solubilized, partially purified opioid receptors with a Ki value similar to that observed for the membrane-bound receptors. This finding indicates that the allosteric modulation of the opioid receptor by delta 9-THC is the result of a direct interaction with the receptor protein or with a specific protein-lipid complex and not merely the result of a perturbation of the lipid bilayer of the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)
Using the genetically unique tetrahydrocannabinol-seizure susceptible (THC-SS) rabbit, the behavioral effect of 14 cannabinoids or related structures were determined and compared to the effects of 11 previously tested cannabinoids. Relative potencies of the cannabinoid-induced convulsions in THC-SS rabbits were generally comparable to reported relative potencies of cannabinoid-produced psychoactivity in humans and other behavioral activity in monkeys or other species. These data suggest that the THC-SS rabbit may represent an experimentally convenient and reliable animal model for studies of structure--psychoactivity relationships of marijuana-like compounds.
Several pairs of cannabinoid isomers were synthesized and tested for psychotropic activity in rhesus monkeys. Two regularities were observed: (a) In the absence of the other substituents, the equatorial stereochemistry of the substituent at C-1 determines activity. (b) Two groups of THC-type cannabinoids which differ only in that the chemical groupings in one of them at C-1, C-2 are situated at C-1, C-6 in the other (but retain their stereochemistry) have almost equivalent psychotropic activity.
11-Hydroxy-3-(1',1'-dimethylheptyl)hexahydrocannabinol (1) was synthesized from the known cannabimimetic analog (+/-)-nabilone. Racemic 1 was resolved by HPLC on a semipreparative CHIRALCEL OD column (Daicel, Inc.), and pharmacological activities of the individual enantiomers were evaluated in the mouse model. The (-)-enantiomer was found to be much more potent than the (+)-enantiomer in all the four measures with the potency ratios in the production of catalepsy (RI), hypoactivity (SA), hypothermia (RT), and antinociception (TF) being 93, 143, 186, and 322, respectively. The racemic 11 alpha-OH diastereomer (2), a reaction side product, was also evaluated in the mouse model. Only small differences in the pharmacological activity of racemic 1 and 2 were found in the above four measures.
Cannabinoid receptors were named because they have affinity for the agonist delta9-tetrahydrocannabinol (delta9-THC), a ligand found in organic extracts from Cannabis sativa. The two types of cannabinoid receptors, CB1 and CB2. are G protein coupled receptors that are coupled through the Gi/o family of proteins to signal transduction mechanisms that include inhibition of adenylyl cyclase, activation of mitogen-activated protein kinase, regulation of calcium and potassium channels (CB1 only), and other signal transduction pathways. A class of the eicosanoid ligands are relevant to lipid-mediated cellular signaling because they serve as endogenous agonists for cannabinoid receptors, and are thus referred to as endocannabinoids. Those compounds identified to date include the eicosanoids arachidonoylethanolamide (anandamide), 2-arachidonoylglycerol and 2-arachidonylglyceryl ether (noladin ether). Several excellent reviews on endocannabinoids and their synthesis, metabolism and function have appeared in recent years. This paper will describe the biological activities, pharmacology, and signal transduction mechanisms for the cannabinoid receptors, with particular emphasis on the responses to the eicosanoid ligands.
Med Cannabis Cannabinoids
- I Ujváry
- Hhc
Ujváry, I. HHC and related cannabinoids. Med Cannabis Cannabinoids, 2022, 5, 159-198.
http://dx.doi.org/10.1159/000527113
Hexahydrocannabinol: review of the chemistry and pharmacology of an understudied cannabinoid
- I Ujvàry
Ujvàry, I. Hexahydrocannabinol: review of the chemistry and
pharmacology of an understudied cannabinoid. Med Cannabis
Cannabinoids, 2023, 5(1), 165.
http://dx.doi.org/10.1159/000527113
9-trimethyl-3-pentyl-6H[1]dibenzo[b,d]pyran-1-ol (hexahydrocannabinol; HHC) by Denmark as a new psychoactive substance under the terms of Regulation (EC) No 1920/2006 and Council Framework Decision
EMCDDA (2022c), Formal notification of 6a,7,8,9,10,10a-hexahydro-6,6,9-trimethyl-3-pentyl-6H[1]dibenzo[b,d]pyran-1-ol
(hexahydrocannabinol; HHC) by Denmark as a new psychoactive
substance under the terms of Regulation (EC) No 1920/2006 and
Council Framework Decision 2004/757/JHA. EU-EWS-RCS-FN-2022-0031, EMCDDA, Lisbon, 21 October 2022.
1-delta9-tetrahydrocannabinol: neurochemical and behavioral effects in the mouse
- D Holtzman
- R A Lovell
- J H Jaffe
- D X Freedman
Holtzman, D.; Lovell, R.A.; Jaffe, J.H.; Freedman, D.X. 1-delta9-tetrahydrocannabinol: neurochemical and behavioral effects in the
mouse. Science, 1969, 163(3874), 1464-1467.
http://dx.doi.org/10.1126/science.163.3874.1464 PMID: 5773112
Hexahydrocannabinol (HHC) and related substances
EMCDDA, Technical report: Hexahydrocannabinol (HHC)
and
related
substances.
2023.
Available
from:
https://www.emcdda.europa.eu/publications/technical-reports/hhcand-related-substances_en
Separation and quantitation of natural and unnatural THC isomers and analogues by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS)
- K Karin
- A Holt
- E R Smith
- S Orlowicz
- A M Taylor
- L Prengman
- C E Wolf
- G Williams
- M R Peace
- J L Poklis
Karin, K.; Holt, A.; Smith, E.R.; Orlowicz, S.; Taylor, A.M.;
Prengman, L.; Wolf, C.E.; Williams, G.; Peace, M.R.; Poklis, J.L.
Separation and quantitation of natural and unnatural THC isomers
and analogues by high-performance liquid chromatography tandem
mass spectrometry (HPLC-MS/MS). 52nd Annu Meet Soc Forensic
Toxicol Clevel OH, 2022, p. 163.
A qualitative analysis of THC isomers and derivatives in e-liquids
- E R Smith
- K N Karin
- J L Poklis
- M R Peace
Smith, E.R., Karin, K.N., Poklis, J.L. and Peace, M.R. (2022), 'A
qualitative analysis of THC isomers and derivatives in e-liquids',
Poster #12, presented at the SOFT 2022 Annual Meeting in Cleveland, OH, October 30 -November 4, 2022. Available from:
https://soft.memberclicks.net/assets/Programs/2022%20Program%
20Book%20for%20Website%20101922.pdf
Analysis of Hexahydrocannabinols: Eliminating Uncertainty in its Identification
- R A Sams
Sams RA. Analysis of Hexahydrocannabinols: Eliminating Uncertainty
in
its
Identification.
2022.
Available
from:
https://forgehemp.com/wp-content/uploads/2022/03/Analysis-of-Hexahydrocannabinols-280222.pdf
Characterization of Hexahydrocannabinol Diastereomers by
- A I Stothard
- N K Layle
- M J Martin
- J Liu
- J R Bassman
- J B Williams
- K W Hering
- H H Diastereomers
- Nmr
- Hplc
- Tlc Gc-Ms
Stothard, A.I.; Layle, N.K.; Martin, M.J.; Liu, J.; Bassman, J.R.;
Williams, J.B.; Hering, K.W.; Diastereomers, H.H.C. Characterization of Hexahydrocannabinol Diastereomers by NMR, HPLC,
GC-MS,
and
TLC,
2020,
19.
Available
from:
https://cdn2.caymanchem.com/cdn/cms/caymanchem/LiteratureCM
S/Characterization of HHC Diastereomers.pdf
HHC and related cannabinoids
- I Ujváry
- Ujváry I.
Tetrahydrocannabinol homologs and analogs with marihuana activity X1
- Adams R Smith
- C M Loewe
- Adams R.
Structure of cannabidiol. VIII. Position of the double bonds in cannabidiol. Marihuana activity of tetrahydrocannabinols
- Adams R Loewe
- S Pease
- D C Cain
- C K Wearn
- R B Baker
- R B Wolff
- Adams R.