Liquid chromatography coupled with photodiode array detection (HPLC-PDA) is commonly used for the analysis of Δ⁸-THC in samples from herbal cannabis and/or cannabis-derived products (extracts, oils, concentrates, and hash). In contrast, liquid chromatography coupled to mass spectrometry (LC-MS) is the preferred method for the determination of Δ⁸-THC and its metabolites in biological fluids. Additional analytical techniques, including gas chromatography-mass spectrometry (GC-MS), gas chromatography with flame ionization detector (GC/FID), and quantitative nuclear magnetic resonance (QNMR) were also reported for Δ⁸-THC analysis.

HPLC-PDA methods are very often utilized in association with the quantification of Δ⁸-THC in complex extracts from C. Sativa. In 1979, Masoud et al. reported the very first method for the analysis of Δ⁸-THC in Mexican cannabis extracts using HPLC with electrochemical detection [50]. More recently, Correia et al. (2023) developed and validated an HPLC-PDA method for the quantification of Δ⁸-THC, in addition to Δ⁹-THC, CBD, CBN, Δ⁹-THCA, and CBDA in cannabis products, following an ultrasound-assisted solid–liquid extraction protocol [51].

Recently, an HPLC method was validated for the analysis of Δ⁸-THC and other 14 cannabinoids in eight cannabis plant material samples of four chemovars (high THC, high CBD, THC/CBD, and high CBG) and two e-cigarettes. Δ⁸-THC content (%w/w) was in the range of 0.036–0.60 in high THC, high CBD, THC/CBD chemovars, while it was not detected in both CBG chemovar and cannabis e-cigarettes [52].

An HPLC-PDA method was developed by Duffy et al. in response to the e-cigarette or Vaping Product Use-Associated Lung Injury (EVALI) outbreak in New York State. The analyzed samples from cannabis vaporizer cartridges contained abnormally high amounts of Δ⁸-THC, whereas medical marijuana products typically do not. Δ⁸-THC amounts ranged from 9.6 to 98% w/w in the analyzed vaping products. Moreover, untargeted analysis by GC-MS and LC-HRMS was performed for the determination of diluents and other cartridge components in order to further understand the occurrence of EVALI [53].

Using standards in the validation phase, Δ⁸-THC could be baseline separated from ten other cannabinoids using acetonitrile (with 0.05% formic acid) and water (with 0.05% formic acid) as a mobile phase in a gradient mode [54]. This study used an Ultrahigh-Performance Liquid Chromatograph coupled with a Photodiode Array and single quadrupole Mass Spectrometry detectors (UPLC-PDA-MS) for its analysis in different C. sativa samples including leaves, flower buds, and hashish. However, only a negligible amount of Δ⁸-THC was found in one hashish sample [54].

Different analytical techniques have been employed for the evaluation of cannabinoid content, including Δ⁸-THC in hemp seed oil and hemp distillate. In 2019, Križman et al. developed an HPLC-PDA method, applying isocratic elution at 37 °C for the analysis of hemp plant samples. However, only one plant sample analyzed contained a detectable amount of Δ⁸-THC [55]. Based on the author’s claim, the use of 275 nm detection wavelength gave a significantly better sensitivity (signal-to-noise ratio) than the conventional 228 nm wavelength commonly used for cannabinoid analysis. In the total of four hemp samples, Δ⁸-THC was detected and quantified in only one sample at a concentration of 0.06% w/w [55].

A UPLC-PDA method was developed and validated by Song et al. (2022) for the determination of 16 cannabinoids, including Δ⁸-THC in different hemp-derived concentrates. The mobile phase composition was optimized by studying different percentages of acetonitrile and formic acid concentrations in water. The best resolution between Δ⁹-THC and Δ⁸-THC peaks was achieved using 0.028% formic acid and 73% acetonitrile [56]. Method specificity was validated by quadrupole time-of-flight (Q-TOF) mass spectrometry. Only two out of nine hemp concentrates analyzed contained Δ⁸-THC, namely one type of Δ⁸-THC distillate and one type of Δ⁸-THC hemp shatter, where Δ⁸-THC contents were 70.96% and 72.32% (w/w), respectively [56].

Generally, LC-MS/MS techniques are used for analyzing Δ⁸-THC in complex cannabis matrices, such as blood samples or food products. The technique cannot distinguish Δ⁸-THC from interfering cannabinoids such as Δ⁹-THC, CBD, CBC, and CBL, based on the analyte’s mass-to-charge ratio (m/z), as they exhibit nearly identical m/z values and share matching fragmentation spectra [57]. As a result, for accurate analysis of Δ⁸-THC, a full chromatographic separation has to be achieved.

Christinat et al. (2020) developed and validated an LC-MS/MS method for the quantification of 15 phytocannabinoids, including Δ⁸-THC in different food products. The mobile phase consisted of 0.1% formic acid solution in water (solvent A) and acetonitrile (solvent B) in a gradient elution mode. In 14 out of 23 hemp-based food products, Δ⁸-THC amounts were <1 mg/kg, while one CBD oil sample contained as much as 4.96 mg/kg [58].

An ESI-LC/MS method was reported for the metabolic profiling of phytocannabinoids in cannabis, where cannabinoid concentration from ethanolic extraction was determined using deuterated cannabinoids (Δ⁸-THC-d₉, Δ⁹-THC-d₉, CBD-d₃, and CBN-d₃) as internal standards. The identified Δ⁸-THC amount was below the limit of quantification of the reported method (1.25 ng/mL) [59].

In 2012, Trofin et al. developed a GC-MS method for the determination of cannabinoids with baseline separation of Δ⁸-THC and Δ⁹-THC, in different types of herbal cannabis. The analysis was performed using a fused silica capillary column and a temperature program from 150 °C to 280 °C [60].

In the United States, commercial products containing Δ⁸-THC in high concentrations are gaining popularity, since the 2018 Farm Bill made tremendous changes to the regulation of hemp products [61]. A wide variety of Δ⁸-THC products are being sold on the shelves of shops, gas stations, or from online sources.

These products are manufactured from CBD derived from CBD-rich hemp flowers, mainly based on the acid cyclization of cannabidiol (CBD) as the reactant (Figure 4). The cyclization reaction categorizes the resulting Δ⁸-THC by some scientists as a synthetic cannabinoid, thereby designating it as a controlled substance [46]. However, the discussion on the legality of Δ⁸-THC is ongoing for now. According to the stated arguments, Δ⁸-THC in commercial products is typically a synthetic product; consequently, there are valid concerns regarding the presence of impurities in these products with unknown effects on the human body. A recent FDA consumer warning update (2022) addressed several concerns with the chemical manufacturing process mentioned above [38]. Some manufacturers may use potentially unsafe household chemicals to make Δ⁸-THC through this chemical synthesis process. Additional undisclosed chemicals may be used to change the color of the final product or to increase shelf life. The final Δ⁸-THC product may have potentially harmful by-products (contaminants), and there is uncertainty with respect to other potential contaminants that may be present or produced depending on the composition of the raw starting materials. When consumed or inhaled, contaminants from the synthetic starting materials, as well as byproducts of the synthetic products of Δ⁸-THC, can be harmful.

Δ⁸-THC marketed products were found to contain unnatural cannabinoids suggesting these were of synthetic origin [37].

As a result of the synthetic production methods and the lack of regulatory requirements, hemp-derived products containing Δ⁸-THC and other isomers are at greater risk of contamination and adulteration than herbal cannabis products sourced from licensed cannabis producers for medical or adult use. By the year 2020, the Interim Final Rule (2020 IFR) issued by the Drug Enforcement Administration (DEA) confirmed that hemp-derived THC products are not legalized by the 2018 Farm Bill [62]. As a result, the increased proportion of Δ⁸-THC and impurities present in various non-traditional marijuana-containing products, such as vape cartridges, extracts, and edibles, has become an important analytical challenge.

In order to address this emerging class of Δ⁸-THC containing consumer products, different analytical techniques have been reported in the literature. Radwan et al. (2023) isolated and characterized impurities found in synthesized Δ⁸-THC raw material in the USA using GC-MS, one Dimension and two Dimension Nuclear Magnetic Resonance spectroscopic (1D and 2D NMR) analysis, as well as High-Resolution Electron Spray Ionization Mass Spectrometry (HR-ESI-MS) [37]. The identified compounds included olivetol, Δ^4,8-iso-tetrahydrocannabinol (Δ^4,8-iso-THC), iso-tetrahydrocannabifuran (iso-THCBF), cannabidiol (CBD), Δ⁸-iso-tetrahydrocannabinol (Δ⁸-iso-THC), Δ⁸-cis-iso-tetrahydrocannabinol (Δ⁸-cis-iso-THC), 4,8-epoxy-iso-tetrahydrocannabinol (4,8-epoxy-iso-THC), Δ⁹-THC, 8-hydroxy-iso-THC (8-OH-iso-THC), 9α-hydroxyhexahydrocannabinol (9α-OH-HHC), and 9β-hydroxyhexahydrocannabinol (9β-OH-HHC) (Figure 5).

The presence of the precursor molecule olivetol suggests a fully synthetic Δ⁸-THC in these products. This hypothesis is supported by some studies that reported the synthesis of tetrahydrocannabinols from olivetol as a precursor [63,64]. The presence of olivetol was also reported by Meehan-Atrash and Rahman (2021) as a contaminant in 27 Δ⁸-THC products [65]. The study found that none of the analyzed products had accurate labeling of Δ⁸-THC content (within a range of ±20% of the concentration on the label). Moreover, all the products contained reaction by-products in addition to olivetol, including Δ^4,8-iso-THC, 9-ethoxyhexahydrocannabinol (9-EtO-HHC), Δ⁹-THC, and a previously unidentified cannabinoid (iso-THCBF, Figure 5) in addition to heavy metals [65]. Quantitative NMR and GC-MS methodology were also used to analyze the major components of 27 e-cigarette products advertising Δ⁸-THC. All products were found to contain at least some undisclosed diluents and/or reaction side-products from the conversion of CBD to Δ⁸-THC. Among others, triethyl citrate (diluent; present in 7 out of 27 samples), olivetol (likely the starting material of the chemical synthesis in 22/27 samples), and Δ⁸-THC -iso-THC (a byproduct of the acid-catalyzed CBD cyclization; 27/27 samples) were identified (Figure 5).

In addition, the previously unknown cannabinoid (5aR,9aS)-5a-isopropyl-8-methyl-3-pentyl-5a,6,7,9a-tetrahydrodibenzo[b,d]furan-1-ol or iso-THCBF was found (Figure 5). It was present in nearly all products tested, but was not quantifiable in products containing the diluent triethyl citrate due to spectral overlap. Analysis of heavy metals by ICP-MS confirmed detectable levels of magnesium, chromium, nickel, copper, zinc, mercury, lead, and silicon [65]. Moreover, QNMR indicated that Δ⁸-THC levels can vary as much as 40% from the labeled value, suggestive of poor testing capabilities or perhaps even falsified results. For one of the brands tested, the average of the sums of Δ⁸-THC and Δ⁸-iso-THC for each product was not significantly different from the average reported Δ⁸-THC content, suggesting the analysis method (HPLC-UV as stated in the certificate of analysis) cannot discriminate between the two compounds [65].

Ray et al. (2022) used ¹H-NMR, HPLC-UV, and HPLC-MS to determine unknown impurities in Δ⁸-THC consumer products purchased online or from local retailers from the USA [66]. Ten Δ⁸-THC products, including distillates and vaporizers, were analyzed during the study. The results indicated that the tested samples contain a range of impurities in concentrations far beyond what is declared on certificates of analysis for these products. Impurities were identified as compounds resulting from low-quality CBD (extracted from hemp used as starting material), as well as known side reaction products from the cyclization reaction used to convert CBD into Δ⁸-THC. The identified compounds included cannabidivarin (CBDV) and cannabidihexol (CBDH). The study concluded that the problem with these products is threefold: impure CBD starting material, poor post-reaction purification, and inadequate separation of closely related cannabinoids during laboratory analysis of the final product [66].

In one study, in addition to Δ⁸-THC, the cannabinoids Δ⁹-THC, CBD, 9(S/R)-Δ^6a,10a-THC, and (6aR,9R)-Δ¹⁰-THC were identified in an e-cigarette cartridge. All of these are isomers of Δ⁹-THC. The identification was based on retention time and fragmentation patterns compared with reference materials. Moreover, some unknown components were also detected within the vape oil of the same e-cigarette [44].

The main cause of the presence of THC isomers is probably due to exposure of cannabis source materials, such as cannabis concentrates or converted hemp materials, to chemical and thermal treatments during the manufacturing process. Using GC-MS and HPLC-PDA analyses, some isomers have been identified in many of the vaping liquids and distillates including Δ⁸-THC, Δ⁹-THC, 9R-Δ^6a,10a-THC, 9S-Δ^6a,10a-THC, 6aR,9R-Δ¹⁰-THC, 6aR,9S-Δ¹⁰-THC, Δ^6a,10a-THC, exo-THC, Δ^6a,10a-tetrahydrocannabivarin (Δ^6a,10-THCV), THCA, Δ⁹-THCV, Δ⁸-THCV, CBD, CBDV, CBN, CBG, and CBC [67]. Significant amounts of Δ⁸-THC were found in the analyzed vaping liquids and distillates, ranging from 0.4 to 79% w/w, while Δ⁹-THC amounts ranged from 1.4 to 93.1% w/w as determined by GC-MS analysis. In addition, low amounts of the Δ⁸-THCV, Δ⁹-THCV, and CBDV were determined. The chemical structures of THC isomers are depicted in Figure 6.

In human liver microsomes, Δ⁸-THC is enzymatically metabolized similar to Δ⁹-THC. In brief, Δ⁸-THC is oxidized to the pharmacologically active, 11-hydroxy-Δ⁸-tetrahydrocannabinol (11-OH-Δ⁸-THC) [68,69], and then further to the non-psychoactive 9-carboxy-11-nor-Δ⁸-tetrahydrocannabinol (Δ⁸-THC-COOH), which is finally conjugated to form its corresponding glucuronide before being excreted, mainly in urine [70,71,72]. Figure 7 shows the metabolic scheme for Δ⁸-THC and Δ⁹-THC.

The terminal elimination half-life of Δ⁸-THC has not yet been reported in humans. The terminal half-life of Δ⁹-THC is up to 4 days in chronic users [70]. This long half-life is attributed to the slow release of the highly lipophilic Δ⁹-THC from fat tissue to plasma. Given the similar distribution to fat tissue, it is expected that Δ⁸-THC will have a similarly long terminal half-life, which means that Δ⁸-THC metabolites may be detected for days or even weeks after the last consumption [70]. Although human data on the metabolism of Δ⁸-THC is limited, some information has been learned from cases involving accidental overdose [73].

At the Karolinska University Laboratory in Sweden, and during routine LC-MS/MS analysis of the human Δ⁹-THC metabolite Δ⁹-THC-COOH in urine, an unidentified peak was detected with a slightly different retention time than Δ⁹-THC-COOH. Using an optimized LC-HRMS method with better separation between the two peaks, the unknown peak was confirmed to be Δ⁸-THC COOH [74].

Pharmacokinetic studies were conducted using the LC-MS method for the determination of Δ⁸-THC and its metabolite Δ⁸-THC-COOH in guinea pig plasma. A liquid–liquid extraction protocol was developed to extract the target analytes from guinea pig plasma using acetonitrile: ethyl acetate (50:50, v/v) [75].

Rzeppa et al. (2021) used HPLC-MS/MS for the analysis of Δ⁸-THC-COOH as a less common metabolite compared to Δ⁹-THC-COOH. The authors also carried out a GC-MS/MS for simultaneous analysis of Δ⁸-THC-COOH. The content of Δ⁸-THC-COOH in doping samples ranged from 0.05 to 2.83% [43]. These findings are in line with those made by in vitro experiments using human liver microsomes [70].

LC-QTOF/MS was used for the analysis of Δ⁹-THC and Δ⁸-THC in the serum and urine of a 2-year-old girl accidentally intoxicated with Δ⁸-THC-infused gummies [76]. The analysis confirmed the presence of Δ⁸-THC and the absence of Δ⁹-THC in the gummy. Plasma concentrations of Δ⁸-THC and Δ⁸-THC-COOH were 107.6 ng/mL and 746.5 ng/mL, respectively, while urine analysis exhibited 1550 ng/mL of Δ⁸-THC-COOH. The study revealed that the symptoms observed in individuals exposed to Δ⁸-THC closely resemble the established effects of Δ⁹-THC. These resemblances pose challenges for clinicians attempting to pinpoint the exact substance that has been ingested.

Another LC-MS/MS method was developed for the analysis of Δ⁸-THC and Δ⁹-THC and their metabolites in blood. Analyte extraction was carried out using 10 mM phosphate-buffered saline, 1% phosphoric acid, and mixed solvent (80:10:10 hexane/ethyl acetate/methyl-tert-butyl-ether) [44].

Analysis of 1504 urine specimens with a positive immunoassay Δ⁹-THC initial test using a liquid chromatography-tandem mass spectrometry (LC-MS-MS) showed the presence of Δ⁸-THC-COOH in 378 samples (15 ng/mL, cutoff), compared to 1144 specimens containing Δ⁹-THC-COOH [77].

Isomeric separation was achieved between Δ⁸-THC-COOH and Δ⁹-THC-COOH by using an automated online μSPE-LC-MS/MS method. Δ⁸-THC-COOH were detected in 54 out of 78 urine samples. An Acquity UPLC HSS T3 (2.1 × 100 mm, 1.8 µm, Waters, Milford, MA, USA) column was used for chromatographic separation. The mobile phase consisted of 0.05% acetic acid in water and methanol with gradient elution [78].

For many years, oral fluid has typically been used for drug testing. Different extraction methods were employed for Δ⁸-THC analysis in oral fluid. In order to avoid strong acidic conditions preventing CBD conversion to Δ⁸-THC, LC-MS/MS methods using solid phase extraction and solid phase microextraction methods were optimized for the measurement of cannabinoids including Δ⁸-THC in oral fluid (Table 1) [79,80,81].

An HPLC-MS/MS method was developed for the analysis of Δ⁸-THC, Δ⁹-THC, CBD, and 10 additional cannabinoids and their metabolites in oral fluid (including 11-OH-Δ⁹-THC, Δ⁹-THC-COOH, THCV, CBDV, cannabidiorcol (CBD-C₁), CBC, CBN, and CBG [79]. Baseline separation between Δ⁸-THC and Δ⁹-THC peaks was achieved using a CORTECS^® Cl8 analytical column at 26 °C under a gradient elution of the mobile phase (A) 0.1% formic acid in water/acetonitrile (95:5, v/v) and (B) 0.1% formic acid in acetonitrile. In 11 out of the 200 oral fluid samples, Δ⁸-THC was detected at concentrations ranging from 0.2 to 339.5 ng/mL. In the same study, two multiple reaction monitoring transitions (MRMs) were monitored for Δ⁸-THC; a quantifier; 315.1 → 123.0 (m/z) and a qualifier 315.1 → 135.1 (m/z), and only one MRM transition was monitored for the deuterated internal standard of Δ⁸-THC (Δ⁸-THC-d₉); 324.21 → 123.l (m/z) for the analysis of Δ⁸-THC. The results showed that only one sample contained Δ⁸-THC and its concentration was as high as 925.7 ng/mL, suggesting possible mucosa saturation [79].

A 2D Ultra-Fast Liquid Chromatography tandem mass spectrometry (UFLC-MS-MS) method was developed and validated to separate Δ⁸-THC and Δ⁹-THC isomers and their metabolites in human blood. A liquid–liquid extraction protocol was used for extracting the target cannabinoids from whole blood. Complete chromatographic separation of Δ⁸-THC-COOH from Δ⁹-THC-COOH was achieved over a run time of 10 min. The authors claimed that “this is the first report of a method that successfully quantitates these primary cannabinoids in blood specimens where significant concentrations of both Δ⁸-THC and Δ⁹-THC isomers are present” [44].

Due to structural similarities, the existence of Δ⁸-THC-COOH in a urine specimen could potentially negatively interfere with the testing process for Δ⁹-THC-COOH. Consequently, LC-MS-based analytical methods may experience identification problems with the two isomers, unless they are chromatographically well separated. This is because they have the same molecular mass, and Δ⁸-THC-COOH also showed the same most abundant MS transitions, thereby often meeting the MS acceptance requirements for a positive identification as Δ⁹-THC-COOH. There have been several studies demonstrating interfering peaks during the detection and/or quantitation of Δ⁹-THC-COOH by LC-MS/MS or GC-MS [79].

Hadener et al. (2017) expressed special concern about the potential overestimation of Δ⁹-THC-COOH blood levels due to the existence of its Δ⁸ isomer, using LC-ESI-MS/MS. This could contribute to inconsistencies in Δ⁹-THC-COOH concentrations reported by different laboratories [82].

A drug screening by immunoassay is typically the first step in drug testing for both forensic and clinical samples. The cross-reactivity immunoassay of Δ⁸-THC-COOH in the EMIT II phase was assessed at a cutoff of 20 ng/mL. The authors carried out a confirmatory GC-MS method to prevent false positive results of overestimation of Δ⁹-THC-COOH. Moreover, the reported GC-MS method succeeded in the separation and quantification of both Δ⁹-THC-COOH and Δ⁸-THC-COOH as TMS derivatives using relative retention time compared to Δ⁹-THC-COOH-d₉ as the internal standard [83].

A baseline separation of Δ⁸-THC-COOH and Δ⁹-THC-COOH peaks has been achieved using GC-MS analysis. Although Δ⁸-THC-COOH and Δ⁹-THC-COOH show common fragmentation ions, they were eluted 0.05 min apart, resulting in baseline resolution. The developed method was then applied for the analysis of Δ⁸-THC-COOH and Δ⁹-THC-COOH in postmortem urine samples. A total of 26 out of 194 postmortem urine samples were positive for Δ⁸-THC-COOH, with six samples only positive for Δ⁸-THC-COOH without the presence of Δ⁹-THC-COOH [84]. A summary of the analysis of Δ⁸-THC and its metabolites in different biological matrices is displayed in Table 1.

A recent study on the stability of Δ⁸-THC and its metabolites; 11-OH-Δ⁸-THC and Δ⁸-THC-COOH in drug-free urine was reported. The study was carried out at different pHs (4.5, 7, and 9) and temperature conditions (4 °C, 20 °C, and 45 °C) for 28 days using an Abbott Architect Plus c4000 Autoanalyzer. The Lin-Zhi enzymatic immunoassay method at the cut-off concentration of 25 ng/mL was used for the analysis. Samples were analyzed daily in the first week and once a week for the remainder of the study or until the target analyte was below the cut-off concentration of 25 ng/mL [85].

The study revealed that the response of Δ⁸-THC varied significantly depending on pH and temperature conditions. At 4 °C, Δ⁸-THC instrument response decreased by about 30% over 7 days, irrespective of pH. Furthermore, no notable declines in response were observed for Δ⁸-THC at 4 °C, regardless of pH, for the remainder of the study. However, at 20 °C, regardless of pH, Δ⁸-THC instrument response decreased by more than 70% over 7 days. By day 14, Δ⁸-THC was undetectable at 20 °C, regardless of pH. Additionally, Δ⁸-THC was not detectable after a single day at 45 °C, regardless of pH [85].

During the period of 7 days, the instrument responses for 11-OH-Δ⁸-THC decreased by 10–15%, 15–20%, and 40–60% at 4 °C, 20 °C, and 45 °C, respectively, regardless of pH. However, at 4 °C, there was no significant decline in the 11-OH-Δ⁸-THC instrument response for the remainder of the study, regardless of pH. By day 14, 11-OH-Δ⁸-THC was not detectable at pH 4.5 and 45 °C. However, at 20 °C and 45 °C, the instrument responses for 11-OH-Δ⁸-THC declined by 10–20% and 40–60%, respectively, for the remainder of the study, regardless of pH. As for Δ⁸-THC-COOH, at pH = 4.5, the instrument response decreased by 40–70% while at pH = 7 and pH = 9, the response decreased by 0–5% regardless of temperature. For the remainder of the study and at pH = 7 and pH = 9, no significant decline in Δ⁸-THC-COOH response was observed [85].

In another stability study, it was suggested that the effectiveness of behavioral modification in rats might depend on the stability of Δ⁸-THC. Specifically, when pure Δ⁸-THC was used, it led to the fastest reduction in avoidance behavior. On the other hand, when partially deteriorated Δ⁸-THC was employed, it did not have a significant impact on avoidance extinction. However, it is important to note that this observation could be influenced by factors like tolerance and the possibility of withdrawal symptoms arising [86]. Inside the human body, Δ⁸-THC was rapidly and extensively metabolized by the liver into the l1-hydroxy metabolite, shedding light on the assumption that the active form of Δ⁸-THC may be the 11-hydroxy metabolites [87].

The endocannabinoid system comprises the endocannabinoids, cannabinoid receptors, and enzymes responsible for the synthesis and degradation of endocannabinoids [88].

Both cannabinoid binding receptors, CB1 and CB2, are G protein-coupled receptors that are part of the endocannabinoid system interacting and responding to cannabinoids and endocannabinoids [89]. CB1 receptors are highly concentrated in specific brain regions and are less abundant in a more widespread manner, primarily influencing the psychoactive effects of cannabinoids. CB1 receptors affect functions like mood and appetite. Their activation is associated with the psychoactive effects of cannabis. CB2 receptors have a more limited distribution, being located in various immune cells and a small number of neurons. CB2 receptors mainly modulate inflammation and immune cell activity without causing psychoactive effects [88]. Both CB1 and CB2 receptors predominantly couple to inhibitory G proteins, sharing pharmacological influences with other GPCRs. Consequently, the cellular response to specific cannabinoid receptor ligands is intricately shaped by factors such as partial agonism, functional selectivity, and inverse agonism [90]. Both CB receptors play a role in the body’s response to cannabinoids, whether they are produced by the body (endocannabinoids) or those from external sources. The identified eicosanoids such as anandamide (arachidonoylethanolamide), 2-arachidonoylglycerol, and 2-arachidonylglyceryl ether (noladin ether) [91].

Interestingly, Δ⁸-THC exhibits the ability to competitively attach to the orthosteric sites of both CB1 and CB2 receptors, with Ki values falling within the nanomolar (nM) range. Notably, the Δ⁸-THC intriguing effects are not restricted to a specific species, as evidenced by consistent impacts observed across human, rat, and mouse receptors. In detail, Govaerts and colleagues examined binding affinities of Δ⁸-THC in both murine tissues and cultured cells, that were genetically modified to express human cannabinoid receptors. They utilized the GTPγS functional assay to measure the ability of Δ8-THC to activate the G protein-coupled receptors documented binding affinities of 251 nM and 417 nM against CB1 and CB2 receptors, respectively [92]. Husni et al. [32] utilized also the GTPγS functional assay. He and Radwan et al. [27] independently reported affinity values of 78 nM and 12 nM against CB1 and CB2 receptors in human-transfected cells (HEK293), respectively. On the other hand, Nadipuram and colleagues reported close affinity values of 28.5 nM and 25.0 nM against the same receptors in human-transfected cells (HEK293), respectively [93]. Similarly, in their study focused on the synthesis of specific ligands for the CB2 receptor in rats’ brain cortex tissue, Huffman’s research team reported an affinity value for Δ⁸-THC of 44 nM [94]. In contrast, when considering mouse CB1 and CB2 receptors, Δ⁸-THC exhibited binding affinities, as measured by Ki values, within a range of 39.3 nM to 179 nM [92,95,96,97,98,99,100]. The antiemetic effects of 5 mg/kg and 10 mg/kg Δ⁸-THC could be counteracted by a CB1 antagonist, but not by a CB2 antagonist. It is important to highlight that Δ⁸-THC exhibited superior effectiveness in preventing vomiting compared to Δ⁹-THC, despite its lower affinity for CB1 and CB2 receptors [101]. In human-origin stably transfected CHO-K1 cell lines, Δ⁸-THC displayed a dose-dependent inhibition of forskolin-induced cyclic AMP accumulation, demonstrating an EC₅₀ of 82 nM [102]. A separate investigation utilizing Rat CHO-K1 (transfected) cell lines observed a similar dose-dependent inhibition of forskolin-induced cyclic AMP accumulation by Δ⁸-THC, revealing an EC₅₀ of 27.4 nM [103]. Furthermore, an additional study involving whole brain synaptosomes revealed a distinct U-shaped response in Δ⁸-THC -mediated inhibition of forskolin-induced cyclic AMP accumulation, reaching an Emax of 13% inhibition at 1 μM [104]. These comprehensive findings depict Δ⁸-THC as a versatile modulator of cannabinoid receptors, exerting intricate effects at molecular and cellular levels across diverse biological systems. Its competitive binding to CB1 and CB2 receptors, demonstrated with nanomolar affinity across species, showcases a nuanced molecular engagement.

Several manuscripts have investigated how Δ⁸-THC influences the levels of acetylcholine in order to elucidate its impact on the central nervous system. The effects of Δ⁸-THC on the hippocampus were found to be dose-dependent, and this was linked to alterations in acetylcholine turnover in the striatum through a stereospecificity-affected turnover [105,106]. Conversely, another study did not find convincing proof to suggest any intrinsic pharmacological impact of the Δ⁸-THC on monoamine oxidase (MAO) activity [107]. Noticeable reductions in dopamine and serotonin concentrations were observed in both the hypothalamus and the hippocampus, respectively. Meanwhile, norepinephrine levels tended to rise in both the hypothalamus and hippocampus [108]. Observable reductions in serotonin concentration were also detected in another study upon the uptake of Δ⁸-THC [109]. Δ⁸-THC exposure and stressful environment may lead to increased dopamine reuptake in the striatum, potentially influencing cannabis-induced behaviors under stress [110]. Furthermore, Δ⁸-THC induced a dual-phase impact on the absorption and discharge of dopamine and norepinephrine in both the corpus striatum and hypothalamic areas of the brain [111]. Sagratella and colleagues undertook several investigations on rabbits to examine the potential role of diminished GABA transmission [112]. Other studies have indicated that Δ⁸-THC inhibits adenylate cyclase and subsequently cAMP in a dose-dependent manner, though this mechanism is not thought to be the origin of behavioral alterations [104].

When Δ⁸-THC is administered before a decision-making-based test, it can lead to discriminative behavior, indicating alterations in a state-dependent manner. The study explored the induction and reversal of memory and learning impairment as a means to differentiate between substances, achieved by altering the ‘correct’ choice [113]. Experiments conducted by Jarbe et al. at various doses collectively suggest that Δ⁸-THC enhanced the abilities of rats to pass the T-maze. Individuals subjected to higher doses of Δ⁸-THC required fewer trials to acquire the maze-learned behavior [114,115]. This result was in agreement with another study that identified the discriminative stimulation effects of Δ⁸-THC [116]. However, in contrast, Doty et al. found that rats subjected to phencyclidine (PCP) were responsive to Δ⁸-THC. Although this did not directly assess the potential discriminative effect of Δ⁸-THC itself, these authors did find that Δ⁸-THC altered the dose–response curve of PCP’s discriminative stimulus. The mechanism of this interaction remains unclear and likely involves multiple factors. Interestingly, this is one of the few effects where Δ⁸-THC did not exhibit a clear dose dependency [117].

Evaluating the effects of Δ⁸-THC on pigeons suggested a dose-dependent effect of Δ⁸-THC on pigeons trained with Δ⁹-THC, thus mirroring similar findings reported in rats [118]. Utilizing the fixed behavioral test-resistance to the extinction of avoidance responses in rats, pure Δ⁸-THC led to the quickest cessation of avoidance [86]. Examining different behavioral symptoms, Δ⁸-THC exhibits activity in humans similar to Δ⁹-THC. When assessed through both oral and intravenous administration, it demonstrates a relative potency ratio of 2:3. Doses ranging from 1 to 6 mg of Δ⁸-THC intravenously induce a broad spectrum of effects reminiscent of cannabis [33].

In a short-term study, dose-dependent pain relief and body temperature decrease were observed in rats after Δ⁸-THC administration. These effects were noticeable within the range of doses of Δ⁸-THC and accompanied by complete tolerance [119]. In the vaporized form, employing the tail withdrawal assay in male rats, a mixture containing three parts CBD to one-part Δ⁸-THC produced an immediate analgesic effect when compared to vapor from the control vehicle [120]. In the same study, immediately after exposure, the mixture induced a reduction in body temperature, leading to a hypothermic effect compared to exposure to the control vehicle [120].

Upon oral ingestion, Δ⁸-THC exhibited an analgesic effect that was dose-dependent when measured by the hot-plate method. Interestingly, this analgesic effect was found to be comparable in potency to Δ⁹-THC, but both were notably less potent than morphine [121]. Conversely, when administered intrathecally rather than orally, Δ⁸-THC was reported to be less potent than Δ⁹-THC employing the tail-flick test [122]. In a combination study, to understand the mechanism of analgesic action, while morphine pretreatment did amplify the analgesic effect, administering naloxone to mice beforehand did not impede it. This suggests that the pain-relieving effects brought about by Δ⁸-THC do not arise from a direct interaction with the opiate receptor [122].

Another study demonstrated the interactions in the use of Δ⁸-THC and opioids within intracellular systems. The potential shared points of interaction include Gi/o protein-coupled receptors. These receptors lead to a decrease in both calcium entry into neurons and the calcium content. This subsequently results in a reduction of cAMP levels and induces hyperpolarization of neurons. These effects are facilitated through the involvement of both ATP- and apamin-sensitive potassium channels [123]. El-Alfy et al. were able to corroborate and establish statistical significance for an existing, though less potent, dose-dependent antinociceptive effect of Δ⁸-THC relative to Δ⁹-THC when assessing for antidepressant-like properties [124]. In a more comprehensive investigation, Welch and his team administered CB1 and CB2 selective antagonist (SR141716A) alongside Δ⁸-THC using the tail-flick test to uncover any potential analgesic role through the cannabinoid receptors. The authors noted varying degrees of antagonism when switching between routes of administration for both SR141716A and Δ⁸-THC. This implies a potentially intricate role of cannabinoid receptors involving various subtypes and distinct localization throughout the central nervous system [125].

A statistically significant dose-dependent reduction in audiogenic response score, indicating diminished seizure activity, was observed in female rats at all three doses of Δ⁸-THC administered with an effective dose for complete seizure inhibition of 6.5 mg/kg, exceeding the upper limit of the trial protocol [126]. Another study explored diverse methods, including kindling to provoke seizures, a higher intraperitoneal dose of 10 mg/kg of Δ⁸-THC was required to significantly reduce seizure manifestations, with significant outcomes defined as >25% protection of the population [127]. In a different experimental animal model, seizures were induced in Senegalese baboons through phototherapy and kindling. Observations revealed dose-dependent prevention and reduction of kindled seizures from electrical stimulation, but no such effect was observed with light-induced seizures after intraperitoneal injections of Δ⁸-THC [128].

The same research group once again employed kindling and a similar seizure staging model, this time assessing another animal model: cats. In contrast to their previous findings, Δ⁸-THC demonstrated no significant effect on suppressing or reducing seizure behavior during any stage of kindling in cats at doses ranging from 0.5 to 4 mg/kg [129]. An alternative study utilized cobalt-epileptic rats observing the activity at the epileptic focus, suggesting that the potential anticonvulsant activity of Δ⁸-THC may not be consistent across different types of seizures and could potentially exacerbate convulsant activity in seizures arising from other causes [130]. Interestingly, both Δ⁸-THC and Δ⁹-THC were determined to have similar relative potency in anticonvulsant activity for PTZ-induced seizures in mice, contrary to previous studies in rats. Additionally, the same study showed that Δ⁸-THC exhibited an additional protective effect against seizures when combined with phenobarbital, but not with diphenylhydantoin [131].

In a short-term study, dose-dependent effects on the reduction of heart rate were observed in rats while complete tolerance to the decrease in heart rate was established within only 13 days [119]. In a conflicting study, Gong and colleagues conducted a three-part experiment examining bronchodilation and tolerance to Δ⁸-THC. They observed dose-dependent responses. These responses included statistically significant inducible bronchodilation and tachycardia [132]. Smiley KA observed arrhythmia and reduced contractility on perfused rat hearts [133]. A subsequent in vitro study in rats investigated changes in contractility by measuring nitric oxide production recording a significant reduction [134]. Another study suggested alterations in the activity of monoamine oxidase enzymes in the hearts of live rats as a potential mechanism for bronchodilation and tachycardia. An early study that compared intravenous and intra-arterial Δ⁸-THC in rats mentioned a potential role as a peripheral vasoconstrictor with intra-arterial administration, while the intravenous administration led to immediate increases in blood pressure, followed by subsequent drop and a decrease in heart rate [135].

Δ⁸-THC, when administered orally, led to dose-dependent reductions in intestinal motility in mice. This effect was assessed by monitoring the transit of a charcoal meal through the digestive tract. Specifically, as the dosage of Δ⁸-THC increased, there was a corresponding decrease in the pace at which the charcoal meal moved through the intestines [121]. In a study targeted the food intake, a very low dose of Δ⁸-THC (0.001 mg/kg) caused increased food consumption and a tendency to improve cognitive function, without cannabimimetic side effects suggesting that the low dose might serve as a potential therapeutic agent in the treatment of weight disorders [107]. Inversely, rats experienced weight loss, decreased intake, bradycardia, and decreased body temperature after administration of Δ⁸-THC in another study; however, partial or complete tolerance developed to many of these physiologic changes [132]. Emesis prevention in radiation-treated shrews showed a statistically significant dose-dependent response at single doses of 5 mg/kg and 10 mg/kg of Δ⁸-THC. The prevention of emesis could be reversed with a CB1, but not with a CB2 antagonist. It is noteworthy that Δ⁸-THC demonstrated greater efficacy in preventing emesis compared to Δ⁹-THC, despite having a lower binding affinity for CB1 and CB2 receptors presenting a unique scenario [101]. In another study on emesis prevention using oral Δ⁸-THC administration every 6 h for 24 h in eight children undergoing multiple cycles of antineoplastic regimens for hemolytic malignancies over 8 months, the study found that administering Δ⁸-THC over 24 h effectively prevented emesis on the day of treatment and for the subsequent two days [136]. On the other hand, a recent retrospective case report suggests that frequent oral administration of Δ⁸-THC may be associated with cannabinoid hyperemesis syndrome indicating a paradoxical, dose-dependent, and time-dependent effect. The factors that could be involved in such results include a potential biphasic or antagonist effect at higher doses, in addition to the receptor downregulation and subsequent alterations in the endocannabinoid system [73].

Several scientific in vitro and in vivo studies have explored the potential anti-cancer effects of Δ⁸-THC. These investigations aim to understand how this compound may impact different kinds of cancer cells and their proliferation. To provide a clear overview of these findings, a selection of studies has been compiled and organized in the following Table 2 for easy reference and analysis.

The diverse array of studies outlined in Table 2 collectively underscores the multifaceted antitumor properties of Δ⁸-THC. The findings consistently reveal its ability to impede critical processes such as DNA synthesis, cancer cell growth, and cellular respiration across a spectrum of cell types, including murine leukemia cells, human cancer cells, and lymphoma cells. Notably, Δ⁸-THC demonstrates promise in inhibiting multidrug resistance in certain cancer cells and induces a range of cellular responses, from apoptosis and autophagy to modulation of molecular markers associated with cell cycle progression. Furthermore, in vivo experiments on mice carrying lung adenocarcinoma suggest that Δ⁸-THC may not only retard tumor growth but also enhance the life span of treated animals. These cumulative results support the potential therapeutic relevance of Δ⁸-THC in combating cancer through diverse mechanisms, warranting further exploration and clinical investigation.

Numerous scientific investigations have focused on understanding the immunomodulatory properties of Δ⁸-THC. These studies aim to unravel how this compound can influence the immune system. To facilitate a comprehensive grasp of these findings, a subset of these studies has been thoughtfully consolidated and presented in Table 3 for easy reference and analysis.

In summary, the diverse array of studies examining the effects of Δ⁸-THC across different cells, organs, and systems underscores its multifaceted immunomodulatory impact. Δ⁸-THC demonstrates a range of outcomes, from inhibiting lymphocyte proliferation and inducing cell death in macrophages to influencing immune responses in vivo. Notably, it exhibits both immunosuppressive effects, as observed in the reduction of hemolytic plaque-forming cells and the suppression of autoimmune encephalomyelitis, and modulatory effects on behavior, evidenced by the regulation of lever-pressing behavior through intricate signaling pathways in the brain. Additionally, Δ⁸-THC appears to antagonize oxidative stress, suggesting potential protective effects on B lymphocytes and fibroblasts. The presented findings collectively highlight the complex and context-dependent nature of Δ⁸-THC’s interactions within the biological systems studied, emphasizing the need for further research to elucidate its therapeutic immunomodulatory potential and potential adverse effects.

The mechanism of Δ⁸-THC in corneal injury treatment was found to be mediated by cannabinoid receptors, as a study observed reduced pain and inflammation after corneal injury in mice with topical administration of Δ⁸-THC [156]. Interestingly, additional lipid-based vehicles for topical preparation of different Δ⁸-THC-containing topical formulations enhanced corneal penetration and minimized systemic absorption [157]. Similar results were obtained after the application of a submicron emulsion as an ocular vehicle for Δ⁸-THC as a treatment for intraocular pressure in rabbits [158]. An early study involving eight rabbits observed a decrease in the intraocular pressure following intravenous administration of Δ⁸-THC, but this effect was not dose-dependent [159]. In another study, topical Δ⁸-THC did eventually lead to a reduction in intraocular pressure in rabbits [160]. Further studies by the same team indicated that the effect was likely not centrally mediated or a result of the drug crossing the blood–brain barrier [161].

In the vaporized form, a concentration of 10 mg of Δ⁸-THC initially induced hyperlocomotion in male rats within the first 30 min of the session, followed by a hypo locomotor effect later in the session [120]. In mice, both Δ⁸-THC and Δ⁹-THC demonstrated a notable decrease in spontaneous activity, leading to reduced awareness. They also caused a loss of muscle coordination, diminished sensorimotor responses, and elicited a hunched posture and gait reminiscent of the effects induced by narcotic analgesics. Additionally, when administered intravenously, both Δ⁸-THC and Δ⁹-THC exhibited a quicker onset of activity compared to intracerebral administration [87].

In an investigation into its anti-androgenic properties, Δ⁸-THC demonstrated remarkable effectiveness as an inhibitor of mitochondrial O₂ consumption within human sperm cells. This finding underscores the significant impact that Δ⁸-THC can exert on the metabolic processes crucial for sperm function and reproductive physiology [162]. In another study, rats that were exposed chronically to Δ⁸-THC displayed significantly reduced body weight and lower levels of follicle-stimulating hormone and luteinizing hormone in comparison to the control group. However, it is worth noting that these hormone levels did recover once the drug was discontinued. The precise clinical implications of these effects on fertility remain uncertain and warrant further investigation [163].

The antidepressant effects of Δ⁸-THC were evaluated, using the automated mouse-forced swim and tail suspension tests. Interestingly, doses of 1.25, 2.5, and 5 mg/kg administered intraperitoneally demonstrated a U-shaped dose-response in terms of their antidepressant action [124].

Following the receipt of 104 reports detailing adverse events in individuals who had consumed Δ⁸-THC products between December 1, 2020, and February 28, 2022, the Food and Drug Administration (FDA) released a report underscoring the potential hazards linked with Δ⁸-THC products and emphasized the necessity for regulatory oversight and public awareness. The FDA report highlights five major significant concerns regarding Δ⁸-THC products [38]. Firstly, these products have not undergone FDA assessment or approval for safe usage, potentially posing risks to public health. There are worries about inconsistent formulations, incorrect labeling, and varying Δ⁸-THC levels. Some products may be misleadingly labeled as “hemp products”, potentially leading consumers to underestimate their psychoactive effects. Secondly, the report expresses apprehension about products claiming therapeutic benefits without FDA approval. This could endanger consumers, as the safety and efficacy of such products have not been confirmed. The data presented indicates possible adverse events, particularly among pediatric patients, emphasizing the need for caution. Thirdly, Δ⁸-THC shares similar psychoactive effects with Δ⁹-THC, indicating a comparable level of impairment. Fourthly, concerns are raised about the manufacturing process, which may involve potentially harmful chemicals and lead to contaminants in the final product. Finally, manufacturers’ packaging Δ⁸-THC products in ways appealing to children is a notable concern, as this could lead to unintentional exposure [38].

On 14 September 2020, the Centers for Disease Control and Prevention (CDC) announced an alert, warning healthcare workers and the public about the recent rise in adverse health events related to Δ⁸-THC consumption. It highlights that emergency room visits mentioning Δ⁸-THC as the primary complaint were mostly concentrated in southern states without legal adult-use marijuana access. In these cases, Δ⁸-THC products are often marketed as hemp products, potentially leading to accidental intoxication due to consumer confusion. The CDC advises caution, noting that Δ⁸-THC products can be intoxicating, may have misleading labels, and should be kept away from children [164].

In the same context, chemists expressed serious concerns over impurities in Δ⁸-THC, as many Δ⁸-THC products available may not be entirely pure, containing other cannabinoids in addition to uncertain by-products with unclear health effects. The lack of oversight and testing raises safety worries. Regulators are struggling to manage Δ⁸-THC’s sales and chemists stress the importance of enhanced regulation and transparency in production. They also caution against potential risks stemming from unregulated synthesis methods [165]. In light of this, the findings of a recent study combining techniques such as NMR, HPLC, and MS revealed that the tested products contain not only 10 different Δ⁸-THC related structures but also several impurities, present in concentrations significantly higher than what is stated on the certificates of analysis for these products [66].

A recent investigation into self-reported effects linked to Δ⁸-THC revealed similarities to those reported during acute cannabis intoxication. The most commonly reported adverse events were related to psychiatric disorders, followed by respiratory, thoracic, and mediastinal issues, as well as nervous system problems. The most frequently cited symptoms in these reports included feelings of anxiety, coughing, and paranoia [166]. This study supported a prior investigation that detailed cases of two individuals, aged 19 and 20, experiencing acute psychiatric distress after consuming a Δ⁸-THC product. These cases demonstrate a conceivable link in time between the consumption of products containing Δ⁸-THC and the emergence of manic or psychotic symptoms, implying a potential relationship between dosage and response [167]. In pediatric cases, the clinical manifestations resulting from exposure to Δ⁸-THC encompass a spectrum of effects, which extend beyond but are not confined to alterations in mental status. These include presentations such as episodes reminiscent of seizures and irregularities in vital signs [168].

In an early assessment of tolerance to Δ⁸-THC, after chronic administration and mice sacrifice, the livers demonstrated significant increases in tyrosine aminotransferase activity with acute administration; however, little-to-no increased activity was noted after 8 and 12 weeks of chronic administration of Δ⁸-THC [169]. Additionally, the Birmingham School of Medicine has reported recently a notable surge in the frequency of emergency department admissions, both in their own hospital and in several others, attributed to the utilization of Δ⁸-THC [170]. In a study on the antiemetic effects of oral Δ⁸-THC in eight children undergoing hemolytic malignancies over an 8-month period, the authors observed minimal incidence of anxiolytic or psychotropic effects, taking into consideration the challenges in both identifying and self-reporting these symptoms in young children [136].

It is worth noting that tolerance to the biological effects of Δ⁸-THC has been a recurring observation in several scientific publications. These studies have consistently pointed out that over time, the response to Δ⁸-THC can undergo significant alterations. This phenomenon has been extensively documented, shedding light on the complex nature of how the body interacts with this compound. Mice became completely tolerant to the hypothermic effects and partially tolerant to extended phenobarbital-induced sleeping times and catalepsy within 38 days of daily intravenous Δ⁸-THC administration [171,172]. Moreover, Δ⁸-THC -tolerant mice were cross-tolerant to the body temperature-lowering effects of chlorpromazine, but not to morphine or pentobarbital [173,174]. When considering morphine interactions, a study did find that morphine-tolerant mice treated with Δ⁸-THC induced heightened catalepsy rather than cross-tolerance [175]. Furthermore, the development of cross-tolerance to the prolongation of pentobarbital-induced sleep by Δ⁸-tetrahydrocannabinol and 11-hydroxy-Δ⁸-tetrahydrocannabinol was reported in mice [172]. In a study exploring the reducing power of Δ⁸-THC against seizure manifestations, chronic administration of intraperitoneal 15 mg/kg Δ⁸-THC rapidly led to tolerance in preventing seizure activity. Only two intraperitoneal injections were required to induce tolerance [127]. At elevated levels of Δ⁸-THC, the ability to counteract epileptic activity seems to diminish in Senegalese baboons following neural discharge at the targeted stimulation site [128]. In a short-term study regarding the cardiovascular system, a relationship between the administered dose (2 mg/kg) of Δ⁸-THC and a decrease in heart rate was evident. Notably, a complete tolerance to the heart rate reduction occurred in just 13 days [118]. The immunomodulatory effects of 60 mg/kg of Δ⁸-THC against direct hemolytic plaque-forming cells in the mice spleen showed a state of reduced responsiveness (hyporesponsiveness) developed when a pretreatment regimen was employed [150].

Some research articles have focused on the potential of Δ⁸-THC withdrawal. In general, the decrease in exploratory behavior with short-term and long-term administration of Δ⁸-THC supports the CNS depressant effects. Significant changes in rat’s defecation (potentially indicative of mood changes) and grooming behaviors (potentially indicative of withdrawal symptoms) were noted [176]. In morphine-dependent mice, increased jumping and forepaw tremors upon administration of naloxone were considered consistent with withdrawal. The administration of Δ⁸-THC was able to inhibit these behavioral modifications. Also, high doses of Δ⁸-THC induced hyperactivity in morphine-dependent mice rather than ataxia/catalepsy [175].

The upregulation of the CB1 receptors coupled with the mitigation of withdrawal symptoms with CB1 stimulation indicates that dysregulation of the endocannabinoid system may have a role in opiate-induced withdrawal [175]. A contradictory study showed that a single administration of 10 mg/kg of Δ⁸-THC was insufficient to suppress naloxone-induced morphine withdrawal [177]. Abrupt cessation in rats after chronic administration of Δ⁸-THC for 30 days increased spontaneous yawning in a similar way to that of opioid withdrawal [178]. On the other hand, the acute administration portion of the study identified that Δ⁸-THC reduced yawning induced by dopaminergic and cholinergic agonists [178]. Further investigations into the potential dopaminergic modulation of Δ⁸-THC may clarify any potential for inducing overuse-associated behaviors.

Modulation of the arachidonic acid pathway and the stimulation of the prostaglandin EP3 receptors may also be involved in dependence and withdrawal [178]. Furthermore, with this induced prostaglandin synthesis, the rats’ brain reduces natural production and becomes dependent on THC for prostaglandin maintenance [178]. The injection of 10 mg/kg diclofenac, an inhibitor of prostaglandin synthesis, intraperitoneally 30 min before the synthetic CB1 antagonist, SR 141716A, induced withdrawal symptoms in mice that were naïve to cannabinoids. These signs closely resembled those of Δ⁸-THC withdrawal symptoms. Conversely, the administration of 10 mg/kg Δ⁸-THC i.p., 15 min before SR 141716A, prevented the manifestation of these withdrawal signs. These findings imply that abrupt cessation of Δ⁸-THC may cause a decrease in prostaglandin levels that contributes to the withdrawal syndrome [179]. Anggadiredj and colleagues’ study suggested that the potential for Δ⁸-THC to provoke withdrawal symptoms is not yet well understood and warrants further research [179].

As of now, there is no approved medication widely and consistently proven effective for cannabinoids dependence and withdrawal [180,181]. Explored medications include those alleviating symptoms of cannabinoids withdrawal (such as dysphoric mood and irritability), those influencing endogenous cannabinoid receptor function, and those successful in treating other substance abuse or psychiatric conditions [182]. Among these, some drugs such as buspirone demonstrated efficacy against cannabinoid dependence in controlled clinical trials [183]. Also, nabiximols, in a randomized clinical trial, were utilized as a successful agonist replacement therapy against cannabinoid withdrawal [184]. In two case reports, it was demonstrated that dronabinol effectively diminished withdrawal symptoms and alleviated the subjective effects associated with cannabinoids [185]. Recent pre-clinical studies also highlight the potential of fatty acid amide hydrolase inhibitors like URB597, endocannabinoid-metabolizing enzymes, and nicotinic alpha7 receptor antagonists such as methyllycaconitine [186].

Resources discussing the clinical trials on Δ⁸-THC are very limited; however, most of its potential benefits are dependent on marketing claims. The first clinical study was conducted in 1973 on six participants for the comparison between Δ⁸-THC and Δ⁹-THC following orally and intravenously administered doses [33]. The study participants were orally administered drug-containing chocolate cookies containing 20 mg and 40 mg of Δ⁸-THC and 20 mg Δ⁹-THC. The trials were conducted over three weeks intervals. Both Δ⁸-THC and Δ⁹-THC clinical effects were evaluated by a narrative log covering every 30 min for up to 5 h with the records of pulse rate, blood pressure, and conjunctival color. The study results showed that all three treatments produced similar somatic, psychic, and perceptual effects. The lower Δ⁸-THC dose (20mg) produced the least clinical effects with slower onset and shorter duration of action [33].

For intravenous administration, both Δ⁸-THC and Δ⁹-THC solutions in 95% Ethanol (1 mg per 0.20 mL) were injected into a normal saline solution after the start of dripping. In a single experiment, three volunteers received Δ⁸-THC, while four subjects received Δ⁹-THC. Δ⁸-THC was administered to the three subjects in a total of six separate doses. The participants experienced the same qualitative symptoms of Δ⁹-THC. Moreover, the intensity and duration of clinical effects produced by the treatment with Δ⁸-THC were dose-dependent. Generally, the study showed that Δ⁸-THC produced slightly weaker effects than Δ⁹-THC. The authors concluded that both Δ⁸-THC and Δ⁹-THC produce the same clinical effects when administered intravenously, however, Δ⁸-THC exhibited approximately 2/3 the potency of Δ⁹-THC, when administered orally [33].

In 1995, Mechoulam’s group conducted a small clinical trial on Δ⁸-THC in children, aged 3–13 years with different hematologic cancers. Eight pediatric cancer patients who were expected to vomit upon receiving an antineoplastic treatment received an oral dose of Δ⁸-THC oil solution at a dose of 18 mg/m² two hours before each chemotherapy session. The selected subjects were monitored for up to 8 months with a total number of 480 treatments. Complete prevention of vomiting was achieved with almost no side effects upon the use of Δ⁸-THC treatments, regardless of the antineoplastic protocol followed [136].

In conclusion, more clinical research is required to determine the potential benefits of Δ⁸-THC; however, legal concerns make these types of studies challenging.

Not applicable.