Hexahydrocannabinol: pharmacokinetics, systemic toxicity, and acute behavioral effects in Wistar rats

Abstract

Background

Hexahydrocannabinol (HHC) is a new psychoactive substance known for its mind-altering effects and temporary legal status. It is widely used in parts of the Europe and United Kingdom as a legal alternative to ∆9-tetrahydrocannabinol, yet little research has explored its effects and safety. This study examined how HHC is processed in the body, its toxicity, and its impact on behavior in male Wistar rats.

Methods

A 1:1 mixture of (9R)-HHC and (9S)-HHC was administered via intragastric gavage at doses of 1, 5, and 10 mg/kg. Behavioral effects were assessed using the open field test and the prepulse inhibition of acoustic startle response.

Results

Two hours after the highest dose (10 mg/kg), peak concentrations of HHC were detected in blood and brain tissue. The Organization for Economic Co-operation and Development 423 toxicity test classified HHC as a Category 4 substance, estimating a lethal dose of 1000 mg/kg. Compared to controls (administered by sunflower oil), 10 mg/kg HHC reduced movement, increased anxiety, and impaired sensory processing.

Conclusions

Overall, HHC crosses the blood–brain barrier, exhibits mild toxicity, and induces behavioral effects similar to tetrahydrocannabinol. Its dose-dependent anxiogenic properties and impact on information processing highlight the importance of the appropriate dosing in any potential therapeutic use.

Significance Statement.

This study investigated how hexahydrocannabinol (HHC) behaves in the body, its potential toxicity, and its effects on the behavior of male Wistar rats. Hexahydrocannabinol is a new psychoactive compound that has attracted attention for its mind-altering effects and its temporary legal status. It is widely used as a legal and available alternative to tetrahydrocannabinol, including by underage users. Despite this, there has been little research into its safety and effects on living organisms. Based on our findings, hexahydrocannabinol readily crosses the blood–brain barrier and has low toxicity. It also produces effects similar to tetrahydrocannabinol, including restricted movement, increased anxiety, and impaired sensory processing.

INTRODUCTION

Hexahydrocannabinol (HHC) is a hydrogenated derivative of tetrahydrocannabinol (THC) which shares similar properties, such as binding to cannabinoid CB₁ receptors (CB₁R) that mediate its psychoactive effects.¹ It is found in low concentrations in the Cannabis sativa plant,² but is predominantly synthesized from precursors such as ∆⁸-tetrahydrocannabinol (∆⁸-THC), ∆⁹-tetrahydrocannabinol (∆⁹-THC), or cannabidiol (CBD).^3,4 Although first described in the 1940s,^5,6 Hexahydrocannabinol did not appear on the drug market until 2021.⁷ Its current popularity is evidenced by police seizures and the detection of HHC metabolites in the urine of recreational drug users.⁸ Along with its analogs, hexahydrocannabinol acetate, hexahydrocannabinol propionate, and tetrahydrocannabidiol, HHC is classified as a novel psychoactive compound within the category of semi-synthetic cannabinoids that replace controlled THC products.^9,10

Hexahydrocannabinol exists in different isomeric forms, each with distinct effects on its chemical properties and pharmacological activities.¹¹ These isomers differ in stereochemistry at the C9 position. The R-epimer (9R)-HHC, with the equatorial methyl substituent, is primarily responsible for the psychoactive effects, whereas the S-epimer (9S)-HHC possessing axial methyl substituent has a significantly lower binding affinity to CB₁R.^1,12,13 Studies in various animal models, including rhesus monkeys and laboratory rodents, have confirmed these findings, showing that (9R)-HHC induces effects similar to Δ⁹-THC intoxication, such as drowsiness, sedation, and reduced responsiveness to external stimuli.^14,15 Gas chromatography–mass spectrometry analyses of seized products have shown varying ratios of 9R- to 9S-epimers, ranging from 1:1 to 2:1.¹⁵

Recreational users typically consume HHC by smoking or by oral ingestion, for example, in baked goods, gummy bears, liquids, or capsules. The typical dose varies depending on the route of administration and the user experience but is generally between 5 and 60 mg.¹⁶ Anecdotal reports describe the effects of HHC as mind-numbing, relaxing, and sleep-inducing. It is considered less intense than Δ⁹-THC, but more potent than Δ⁸-THC.¹⁷ As with other cannabinoids, the psychoactive effect is not always the primary reason for use; HHC is also taken to reduce pain and/or anxiety. Although the overall experience is mostly positive, 20% of respondents report adverse effects such as nausea, dizziness, and anxiety.¹⁸

Despite the popularity of HHC among recreational users, including adolescents, relatively few studies have examined its in vivo effects. Therefore, this study used a standard battery of measurements to describe the pharmacokinetic profile, systemic toxicity, and behavioral effects of novel synthetic compounds in laboratory rats.^19–21 To maximize the translational validity of the animal model, a 1:1 mixture of 9R- and 9S-HHC epimers was administered intragastrically in doses relevant to human use. Brain and serum levels of the isomers were studied over 24 h, with peak concentrations expected within 60-120 min, as with closely related cannabinoids.^22–24 Systemic toxicity was assessed using oral administration in the toxicity assay 423, as defined by the Organization for Economic Co-operation and Development (OECD). Additionally, as behavioral changes such as catalepsy, reduced motor activity, and impaired information filtering are typically induced by THC-like cannabinoids,^25,26 the open field test (OFT) and prepulse inhibition of acoustic startle response (PPI ASR) were used to assess these effects in HHC.

MATERIALS AND METHODS

Animals

Wistar rats (VELAZ, Prague, Czech Republic) were housed in pairs in home cages placed in a room with a controlled temperature (22 ± 2°C), lighting (12/12 h light/dark) and humidity (30%-70%); water and food were provided ad libitum. Except for toxicity assessments, only male rats were used to minimize variability given the documented influence of hormonal fluctuations across the estrous cycle in female rats on behavioral responses to psychoactive compounds.^27,28 All rats were habituated to laboratory conditions during the first 10 days after delivery, regularly handled and weighed. The rats weighed 200-275 g at the beginning of the testing. A total of 80 rats were used for behavioral tasks (n = 10 per group): OFT (N = 40) and PPI ASR (N = 40). Eight additional male rats were used for pharmacological sampling (n = 8 per time point) (for sampling at 30 min); the rest of the experimental group (n = 40) consisted of rats that had been used previously in behavioral tasks. Finally, nine additional female rats were used for acute toxicity assessment, following the OECD protocol for estimating acute toxicity (see below).

All procedures followed the principles of laboratory animal care of the National Committee for the Care and Use of Laboratory Animals (Czech Republic) and Guidelines of the European Union (86/609/EU). The National Committee approved the Care and Use of Laboratory Animals (Czech Republic) protocol under MZDR 48237/2017-3/OVZ.

Drugs and Chemicals

Hexahydrocannabinol was synthesized and purified by the University of Chemistry and Technology in Prague at the Forensic Laboratory of Biologically Active Compounds. The resulting mixture was certified to be more than 95% pure, and the 9R- to 9S-epimers ratio was determined to be 1:1 (analyzed by NMR and HPLC-UV). Chemicals used in an LC/MS analysis were of LC/MS grade and together with other necessary chemicals purchased from Merck (United States). Other chemicals for sample preparation procedures were of p. a. grade supplied by Merck (United States).

Dosage

The doses of HHC for behavioral studies and pharmacokinetics (PKs) were selected according to the potency of related compounds, such as THC and CBD.²⁴ They were 1, 5, and 10 mg/kg for behavioral experiments and 10 mg/kg for PKs measurements. Doses for acute toxicity reflected OECD 423 assay (see below) (300 and 2000 mg/kg). In all cases, the compound was dissolved in sunflower oil and administered by an oral gavage at a volume of 0.5 mL/kg; an equivalent volume of sunflower oil was used as a vehicle in controls.

Pharmacokinetics

Rats were intragastrically administered by HHC 10 mg/kg and decapitated after 0.5, 1, 2, 4, 8, and 24 h (n = 8 per time point). Serum was obtained by allowing the blood to clot in a refrigerator for 1 hr. The clotted blood was centrifuged for 10 min at 3500 rpm, 10°C, and the serum was then collected. The brains were removed from the skull and thoroughly rinsed in physiological saline following the methodology used in our previous paper.²⁴ Both serum and brains were stored at –80°C until analysis. Drug concentrations in the serum and dissected brain tissue were calculated as ng/mL or ng/g, respectively.

Determination of 9R and 9S Epimers of HHC in Serum and Brain Samples Using LC–MS/MS

LC/MS analysis: A 1290 Infinity liquid chromatography system (Agilent Technologies, USA) was used, and the chromatography separation was achieved with a Kinetex PFP 100 Å (3 × 150 mm, 2.6 μm particle size) column equipped with a guard column (both Phenomenex, USA). Mobile phases consisted of 0.1% (v/v) acetic acid with 10 mM ammonium acetate in water (solvent A) and pure methanol (solvent B). The time profile of the gradient was as follows: the mobile phase composition started at 50% B (v/v), increased from 50% to 80% B in 5 min and was kept constant for 6 min. Then, the amount of B was increased to 100% B at 0.5 min, held for 1.5 min at 100% B, and decreased to 50% B in 0.5 min. Then, the column was re-equilibrated for 1.5 min. The total run time was 15 min. The flow rate was 0.400 mL/min and the column temperature was set constant at 30 °C.

The analytes were detected with a 6460 QqQ mass spectrometer (Agilent Technologies, USA) in positive electrospray (ESI+) ionization mode. The optimized ion source settings were as follows: gas temperature 350 °C, gas flow 12 L/min, nebulizer 50 psi, sheath gas temperature 320 °C, sheath gas flow 12 L/min, and capillary voltage 4500 V. The protonated form of HHC has a nominal mass of 317.3 m/z. For quantification, multiple reaction monitoring was used. The following transitions were monitored: 317.3 to 123.0 for quantification, and 317.3 to 81.2 for confirmation, using collision energies of 37 and 25 eV, respectively. Commercial Δ⁸- and Δ⁹-THC were used as internal standards (ISs) as their structures and chromatographic behavior closely correspond with HHC epimers. To track the ISs Δ⁸- and Δ⁹-THC, the acquisition was set for transitions of 315.2 to 193.1 and 315.2 to 123.0, using collision energies of 18 and 34 eV, respectively. HHC was quantified using an external matrix-matched calibration. The limit of quantification was 1 ng/mL for serum and 10 ng/g for brain tissue.

Serum Pre-Treatment

The procedure was conducted as follows: (1) dilution of 200 μL of serum with 190 μL of acetonitrile and spiked with 10 μL of IS mixture (Δ⁸- and Δ⁹-THC, both at concentration 500 ng/mL); (2) vortex mixing for 2 min; (3) addition of 800 μL of hexane to the mixture and vortex mixing for 15 min; (4) centrifugation for 10 min at 25°C (10 000 rpm); (5) evaporation of 640 μL of supernatant to dryness under a gentle stream of nitrogen gas; (6) reconstitution with 160 μL of 0.1% (v/v) acetic acid with 10 mM ammonium acetate in methanol:water (1:1, v/v) and vortex mixing for 10 min; (7) centrifugation for 10 min at 25 °C (10 000 rpm); (8) transfer of 140 μL of the resulting solution into a glass LC vial with insert.

Brain Pre-Treatment

The procedure was conducted as follows: (1) addition of 250 μL of deionized water to 100 mg of brain tissue; (2) homogenization with 200 mg of zirconium oxide beads in a BulletBlender for 1 min (speed 6); (3) addition of 90 μL of acetonitrile and spiked with 10 μL of IS mixture (Δ⁸- and Δ⁹-THC, both at concentration 1000 ng/mL) and homogenization (BulletBlender, 1 min, speed 6); (4) vortex mixing for 5 min; (5) addition of 800 μL of hexane to the mixture and homogenization (BulletBlender, 1 min, speed 6); (6) centrifugation for 10 min at 25°C (10 000 rpm); (7) evaporation of 640 μL of supernatant to dryness under a gentle stream of nitrogen gas; (8) reconstitution with 1 mL of acetonitrile:water (4:1, v/v) and vortex mixing for 10 min; (9) transfer of 900 μL of the resulting solution onto Captiva EMR Lipid cartridge (Agilent Technologies, USA); (10) sorption and flow under gravity; (11) column drying under vacuum for 5 min; (12) column washing with 500 μL of acetonitrile:water (4:1, v/v) and drying under vacuum for 5 min; (13) evaporation of the resulting solution to dryness under a gentle stream of nitrogen gas; (14) reconstitution with 400 μL of 0.1% (v/v) acetic acid with 10 mM ammonium acetate in methanol:water (1:1, v/v) and vortex mixing; (15) sonification of the obtained solution for 15 min and transfer into a glass LC vial with insert.

Systemic Toxicity

Toxicity Assessment

Acute oral toxicity was assessed using the acute toxic class method (OECD Guideline No. 423, 2001).²⁹ The initial dose for the unknown substances (300 mg/kg, based on the protocol) was administered to three female rats in the first step. Following a single dose administration, body temperature was measured rectally three times, with a 1-h interval between each measurement. The condition of the rats was individually monitored at least twice per hour during the first 24 h and then observed daily for 14 days. Animals showing severe distress or a moribund condition were humanely euthanized. Organs (brain, heart, lungs, liver, kidneys, and spleen) of these deceased rats preserved in a 4% buffered formalin solution, underwent pathological-anatomical analysis. The tissues were processed, embedded in paraffin blocks, sectioned into 4-μm-thick slices, and stained with the hematoxylin–eosin (HE) technique. Two pathologists independently performed the microscopic analysis.

Histopathological Investigation

The brain, heart, lung, liver, kidney, and spleen were collected from two experimental and one control specimen and preserved in a 4% buffered formalin solution for subsequent pathological-anatomical examination. The entire brain was sampled in frontal sections, capturing all the main anatomical structures. Tissue specimens underwent processing and embedding in paraffin blocks. Sections of 4 μm were prepared and stained with HE. Two pathologists (A.S., Q.H.B.) conducted microscopic analyses in a double-blinded manner.

Behavioral Effects

All experiments were conducted during the daytime (between 07:00 and 15:00 h) under the standard laboratory conditions described previously and were initiated 120 min after the HHC administration. One hour before the testing, the rats were habituated to the testing room in their home cages. The design was chosen to ensure comparability to our previous studies on closely related substances.^19–21

Open Field Test

The OFT was initiated by placing the rat in the central part of an empty black arena (80 × 80 cm) surrounded by 40 cm high walls. The arena was placed in a soundproof room with even, dim lighting (60 lx). A rat was allowed to move freely within the arena for 30 min, and its behavior was recorded and quantified using the software EthoVision XT v. 14.0. (Noldus, Netherlands). The arena was virtually divided into a 5 × 5 grid of identical squares for data processing; 16 were placed around the arena walls (peripheral zones) and nine were situated centrally (central zones). The following parameters were measured: trajectory length divided into 5-min intervals (centimeter; corrected for deviations of <3 cm), time spent in the central zone (s; T_centre = ∑_{time central zones}), and probability of occurrence in the peripheral zone (thigmotaxis = Σf_{peripheral zones}/Σf_{all zones}, where f = frequency of occurrence in a zone). The described procedure is consistent with our previous studies.

Prepulse Inhibition of the Acoustic Startle Response

Prepulse inhibition of acoustic startle response was measured in the startle chambers (SR-LAB, San Diego Instruments, CA, USA), each consisting of an evenly lit and soundproof enclosure, a high-frequency loudspeaker (which produced acoustic stimuli and background noise at 75 dB) and a Plexiglas stabilimeter with an inner diameter of 8.7 cm. Startle response amplitudes were detected using a piezoelectric accelerometer and digitized.

Two days before the measurement, the rat was habituated to the startle boxes for 5 min during which five presentations of a pulse stimulus alone (125 dB, 40 ms) were delivered over a white background noise (75 dB). The PPI ASR measurement began with a habituation period of 5 min of background white noise (75 dB), followed by 72 trials with an inter-trial interval (ITI) of 15-30 s (mean ITI: 22.5 s). Six pulse-alone trials (125 dB, 40 ms) were performed to establish baseline ASR. Subsequently, 60 trials were conducted in a pseudo-random order: (A) pulse alone: 40 ms 125 dB; (B) prepulse alone: 20 ms 83 dB or 91 dB; (C) prepulse-pulse: 20 ms 83 dB or 91 dB prepulse, a variable (30, 60, or 120 ms) inter-stimulus interval (mean 70 ms), then 40 ms 125 dB pulse; (D) 60 ms no stimulus. Finally, six pulse-alone trials were performed. Habituation was calculated as the percentage reduction in ASR from the initial six baseline trials to the last six trials. Mean ASR was obtained from the pulse-alone trials. Animals with a mean ASR response lower than 10 arbitrary units were excluded from analyses as non-responders. Prepulse inhibition was calculated using the formula: (100 − (mean response for the prepulse–pulse trial/startle response for the single pulse trials) × 100).

Statistical Analysis

Pharmacokinetics

Both (9R)-HHC and (9S)-HHC serum concentration-time data were analyzed using a Nonlinear mixed-effects modeling approach with the Stochastic Approximation Expectation–Maximization algorithm in Monolix Suite software 2021R2 (Lixoft SAS, Antony, France). Several structural models were evaluated to fit the concentration–time data: one- or two-compartment models with first-order or Michaelis–Menten elimination kinetics, and first-order absorption kinetics with or without lag time (the delay before a drug enters systemic circulation). The model parameters were assumed to be log-normally distributed. The best structural model was selected based on the lowest objective function value and minimal relative standard errors of the estimated PK parameters.

Both final population models were subsequently implemented in Simulx software 2021R2 (Lixoft SAS, Antony, France) to simulate the theoretical distribution of (9R)-HHC and (9S)-HHC concentration–time profiles in the population. This simulation involved 100 replicates of the original dataset of rats following oral administration of a 10 mg/kg mixture of HHC epimers using the Monte Carlo method.

Elimination half-life (t_1/2) was calculated based on linear regression of semi-logarithmic concentration–time data in the elimination phase using the following formula:

where the slope is defined by a linear regression line.

The brain/serum concentration ratio was calculated in each rat and the mean blood/serum concentration ratio of (9R)-HHC and (9S)-HHC was compared in each sampling time using a paired t-test. A paired t-test was also used for the comparison of both serum and brain concentration at each sampling time point.

Behavioral Effects

Behavioral data were analyzed using linear mixed models (LMMs) and one-way analysis of variance (ANOVA), following the methodology of previous studies.^19,20 The experiment was conducted as a parallel design with 10 rats per group. Significant main effects were explored using appropriate post hoc tests. When comparing HHC groups to the control group, a two-sided Dunnett’s test was applied. In the OFT, treatment conditions (control; HHC at 1, 5, and 10 mg/kg) served as the independent variable. Dependent variables included the length of trajectory, time spent in the center, and the probability of thigmotaxis, with time (5-min time blocks) as a within-subject factor. For the PPI ASR test, treatment conditions (control; HHC at 1, 5, and 10 mg/kg) were again the independent variable, with habituation, the acoustic startle response (ASR), and PPI as the dependent variables.

An alpha level of P < .05 (two-tailed) was used to determine statistical significance. All analyses and visualizations were performed using R software (version 4.0.5) and R Studio (version 1.4.1717). Data are presented as mean ± standard deviation (SD) or median with interquartile ranges (Q₁/Q₃), and individual values depending on the distribution of the data and the type of used analysis.

RESULTS

Pharmacokinetics

A one-compartment model with first-order elimination kinetics and first-order absorption kinetics with lag time best fits the concentration–time data. Both (9R)-HHC and (9S)-HHC PK models were parameterized in terms of lag time (lag), absorption rate constant (Ka), apparent volume of distribution (Vd/F), and apparent clearance (CL/F). The estimates of PK parameters in the population models are summarized in Table 1.

Table 1.

Estimates of the R-HHC and S-HHC population pharmacokinetic models.

	R-HHC population PK model		S-HHC population PK model
PK parameter	Estimate	RSE (%)	Estimate	RSE (%)
Tlag (h)	0.49	6.65	0.49	10.0
Ka (h−1)	0.47	24.5	0.41	18.8
Vd/F (mL)	1514.9	43.5	1925.9	33.4
CL/F (mL/h)	2457.4	25.2	3732.1	23.8

Abbreviations: R-HHC, R-hexahydrocannabinol; S-HHC, S-hexahydrocannabinol; RSE, relative standard error. Tlag is lag time (the finite time taken for a drug to appear in systemic circulation). Ka is absorption rate constant. Vd/F is apparent volume of distribution. CL/F is apparent clearance

Monte Carlo simulation of theoretical distribution of (9R)-HHC and (9S)-HHC serum concentration-time profiles in rats after oral administration of 10 mg/kg of HHC epimer mixture is presented in Figure 1.

Figure 1.

Monte Carlo simulation of the theoretical distribution of R-hexahydrocannabinol (R-HHC) and S-hexahydrocannabinol (S-HHC) serum concentration - time profiles in rats after oral administration of 10 mg/kg of HHC racemic mixture. The solid line represents the median, and the shaded bands represent the 90% prediction interval percentiles of the simulated concentration distribution.

The elimination half-life of (9R)-HHC and (9S)-HHC was 1.14 and 1.21 h, respectively.

Mean ± standard deviation serum concentration–time, brain concentration–time, and brain/serum concentration ratio–time profiles of (9R)-HHC and (9S)-HHC after oral administration of 10 mg/kg HHC epimer mixture are shown in Figure 2.

Figure 2.

Mean ± SD blood/serum concentration ratio–time profiles of R-hexahydrocannabinol (R-HHC) and S-hexahydrocannabinol (S-HHC) in rats after oral administration of 10 mg/kg of HHC racemic mixture. ^*P = .0444; ^**P = .0019 (paired t-test).

Systemic Toxicity

Toxicity Assessment

All three females completely recovered within 24 h after being administered a single HHC dose of 300 mg/kg. Measurements taken at 30, 60, and 90 min following compound administration indicated hypothermia (body temperature below 35°C) and reduced locomotion in all observed animals. The animals were further observed for 2 weeks before being euthanized. The second administration of 300 mg/kg produced the same outcome. The three animals which were orally administered 2000 mg/kg of HHC, experienced hypolocomotion and hypothermia before being lethal for two of the three animals within 8 h. The recovered animal was euthanized after 2 weeks of observation. Based on the results, HHC was classified as a category 4 compound according to the Globally Harmonized System (GHS) of Classification and Labeling of Chemicals, indicating low to moderate acute toxicity. This category corresponds to an estimated LD₅₀ between 300 and 2000 mg/kg. Following the OECD 423 protocol, the exact LD₅₀ value is estimated based on the number of animal deaths observed at a fixed dose. Since two out of three animals died following administration of 2000 mg/kg, the LD₅₀ for HHC can be estimated at ~1000 mg/kg.

Histopathological Investigation

Numerous areas of congestion within the interalveolar septa, with a predilection for perivascular regions, were observed in the lungs. Despite thorough examination, the brain, liver, heart, spleen, and kidneys observations did not exhibit significant disparities compared to the control. Given the nonspecific macroscopic and microscopic findings, we hypothesized that the animals likely succumbed to malignant arrhythmia. It is essential to highlight that this condition belongs to a group of diseases lacking a distinctive histomorphological correlation.

Behavioral Effects

Open Field Test

A LMM incorporating fixed effects for time (5-min intervals), treatment, and individual random effects explained significantly more variance in trajectory length compared to models with only time (5-min intervals) or treatment (ANOVA; P=.013). Both the 5 and 10 mg/kg groups showed significantly reduced activity than the control group (5 mg/kg: t(40) = −2.240, P = .030; 10 mg/kg: t(40) = −2.609, P = .013), while the 1 mg/kg group did not (Figure 3A).

Figure 3.

Results of the Open field test (OFT) and Prepulse inhibition of acoustic startle response (PPI ASR) are presented in the panel. (A) OFT: trajectory length in 5-min intervals, mean ± SEM for each dosage at 5-minute intervals are presented, Linear Mixed Model (LMM); (B) OFT: thigmotaxis, individual values are shown; (C) OFT: an example of characteristic trajectories for each treatment; (D) OFT: time in the centre, individual values are shown; (E) PPI ASR: Acoustic startle response (ASR), individual values are shown; (F) PPI ASR: Prepulse Inhibition (PPI), individual values are shown. (B) – (F) ANOVA followed by Dunnett's post hoc; significant differences from the control group values are marked by ^*P < .05, ^**P < .01, ^***P < .001.

Treatment also affected thigmotaxis (F(3,36) = 4.17, P = .013). No significant differences between the 1 mg/kg, 5 mg/kg, and control groups were detected; however, the 10 mg/kg group showed a considerably increased probability of thigmotaxis (P = .004) (Figure 3B). Furthermore, a borderline significant effect of treatment on the time spent in the center of the arena was found (one-way ANOVA; F(3,36) = 2.66, P = .063). The Dunnett test revealed no significant differences for the 1 and 5 mg/kg groups compared to the control, but the 10 mg/kg treated rats spent considerably less time in the center (P = .048) (Figure 3D).

Prepulse Inhibition of Acoustic Startle Response

One-way ANOVA revealed no significant effect of treatment on habituation. The ASR was significantly affected by treatment (one-way ANOVA; F(3,36) = 10.29, P < .001) since the Dunnett test indicated that the highest dose of 10 mg/kg significantly reduced motor responses (P < .001) compared to the control group, whereas the 1 and 5 mg/kg doses showed no significant differences (Figure 3E). In comparison to the control group, prepulse inhibition (PPI) was significantly impaired in the 10 mg/kg group (one-way ANOVA; F(3,36) = 10.25, P < .001), while no impairment was observed in the 1 and 5 mg/kg groups (Figure 3F).

DISCUSSION

The present study has clarified several aspects of the effect of HHC in rats treated intragastrically with a single bolus of a 1:1 mixture of 9R- and 9S-HHC isomers: (1) the drug is well absorbed and readily crosses the blood–brain barrier; (2) peak concentrations were observed 120 min after gavage, followed by a slow elimination phase, with the longest retention in brain tissue; (3) the drug exhibits relatively low toxicity, comparable to that of THC; (4) HHC overdose led to hypothermia, inactivity, bradypnea, ataxia, and muscle relaxation with no significant histopathological changes; (5) HHC displays psychoactive properties, characterized by (a) moderate, dose-dependent inhibition of locomotor activity, (b) anxiogenesis (anxiety-inducing effects), (c) disruption of cognitive processing and information handling.

Pharmacokinetics

Pharmacokinetic models showed that the PK characteristics of absorption and distribution (Tlag, Ka, Vd/F) are similar in both isomers, whereas elimination, characterized by CL/F, exhibits a higher degree of difference—specifically, the clearance of the (9S)-HHC is about 50% higher than that of the (9R)-HHC (Table 1). This observation has a physiologically plausible explanation. While the absorption and distribution of a substance depend mainly on its physicochemical properties such as water/fat solubility, acid–base ionization, etc., which do not differ much between isomers, elimination, which in this case is mediated by metabolism, requires stereoselective interaction with enzymes, and therefore, it is common for each of the isomers to use a different metabolic pathway.^30,31

There was almost no time lag between the maximum concentration in brain tissue and serum in rats following the oral administration of a 10 mg/kg HHC isomeric mixture. The difference in brain/serum concentration ratio of HHC isomers observed at the elimination phase of the PK profile (4 and 8 h after administration) further supports our previous explanation. Since (9S)-HHC exhibits a higher elimination rate but has a similar distribution to (9R)-HHC, its serum concentration declines more rapidly than in brain tissue. Consequently, its brain/serum concentration ratio is significantly lower compared to (9R)-HHC.

Systemic Toxicity

The toxicity of cannabinoids is influenced by factors such as the route of administration, dose, sex, and individual susceptibility.³² When administered orally to female rats, HHC was classified as a GHS category 4 compound (with estimated LD₅₀ 1000 mg/kg), comparable to the reported oral LD₅₀ of THC (~800-1270 mg/kg).³³ Following an HHC overdose, symptoms such as hypothermia, inactivity, bradypnea, ataxia, and muscle relaxation were observed, consistent with effects documented in mammals (including rats, dogs, and monkeys) exposed to toxic levels of THC.³³ Acute symptoms of cannabinoid toxicity may manifest shortly after ingestion and include hepatic and renal toxicity, as well as tachycardia.³⁴ Our findings suggest that a single high dose of HHC did not result in significant histopathological changes. Given the lack of distinct histomorphological features and the clinical presentation of the overdosed animals, it is hypothesized that the mortality was likely due to malignant arrhythmia. This is consistent with side effects observed in users of synthetic cannabinoids.³⁵ However, further investigation is needed to confirm this hypothesis.

Behavioral Effects

The behavioral effects of HHC in humans include THC-like psychoactive effects such as euphoria, relaxation, sedation, impaired motor function, altered perception, increased anxiety, and analgesia.⁷ Consistent with these effects, we observed decreased overall locomotor activity and impaired habituation to the apparatus following the administration of moderate and higher doses (5 and 10 mg/kg) of HHC. These patterns align with findings from studies on HHC in mice¹⁵ and investigations of THC effects in rodent models.^24,36,37 This contrasts with several THC studies showing dose-dependent bidirectional effects, such as hyperlocomotion at low doses and hypolocomotion or sedation at higher doses.^25,26 In addition, the higher dose of HHC (10 mg/kg) increased anxiety, as indicated by reduced time spent in the center of the arena and increased thigmotaxis (i.e., the tendency of rodents to move along the walls, reflecting anxiety-like behavior). A similar, though less pronounced, trend was observed in the 5 mg/kg dose group, while no significant effect was noted at the lowest dose of 1 mg/kg.

In humans, certain aspects of the psychoactive effects induced by cannabinoids are mediated through their impact on sensorimotor gating.³⁸ This results in the detection and enhancement of stimuli that should have been filtered out, which can induce psychotic symptoms.^39,40 Consistent with this, cannabinoid-induced impairment of information processing has been observed in rodent studies, as evidenced by affected PPI.⁴¹ In our study, a similar pattern was observed at the highest dose of HHC (10 mg/kg), suggesting an information processing deficit comparable to that induced by both phyto-⁴² and synthetic cannabinoids,⁴³ but see.⁴⁴

It is important to note that unlike habituation, which was not significantly affected by any of the factors, the level of ASR varied considerably with dose. Rats administered 10 mg/kg exhibited a reduced motor response to the startle stimulus, indicating a high degree of sedation. Although the ASR value was not below the critical threshold (determined based on the sensitivity of the apparatus), the interpretation of the results may be confounded by a floor effect.⁴⁵ However, for HHC, the relationship between locomotor activity in the OFT, ASR, and information processing deficits is not straightforward, since the moderate dose (5 mg/kg) reduces locomotor activity but does not affect motor response or disrupts PPI.

Taken together, the observed dose-dependent anxiogenic effects of HHC and its impact on information filtering underscore the importance of proper dosing in potential therapeutic applications—particularly given that HHC-induced anxiety and psychotic symptoms have also been reported in humans.^18,46

Author Contributions

Klára Šíchová (Conceptualization, Data curation, Investigation, Methodology [equal], Writing—original draft [lead], Writing—review & editing [equal]), Barbara Mallarino (Data curation, Formal analysis, Investigation [equal], Writing—original draft [equal], Writing—review & editing [supporting]), Lucie Janecková (Conceptualization [supporting], Data curation [supporting], Formal analysis [equal], Investigation [equal], Methodology [equal], Validation [supporting], Writing—original draft [supporting], Writing—review & editing [supporting]), Petr Palivec (Conceptualization [supporting], Formal analysis [equal], Investigation [equal], Methodology [equal], Validation [supporting], Writing—original draft [supporting], Writing—review & editing [supporting]), Magdaléna Vagnerová (Conceptualization [equal], Data curation [supporting], Formal analysis [equal], Investigation [equal], Methodology [equal], Validation [equal], Writing—original draft [supporting], Writing—review & editing [supporting]), Cestmír Vejmola (Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Visualization [equal], Writing—original draft [supporting], Writing—review & editing [equal]), Marek Nikolic (Formal analysis, Software [equal], Writing—original draft [supporting], Writing—review & editing [supporting]), Lucie Olejníková-Ladislavová (Conceptualization [supporting], Data curation [supporting], Investigation [equal], Methodology [equal], Validation [equal], Writing—original draft [supporting], Writing—review & editing [supporting]), Kristýna Mazochová (Conceptualization [equal], Investigation [equal], Methodology [equal], Writing—original draft [supporting], Writing—review & editing [equal]), Pavel Ryšánek (Conceptualization [supporting], Formal analysis [equal], Investigation [equal], Methodology [equal], Writing—original draft [supporting], Writing—review & editing [supporting]), Martin Šíma (Conceptualization, Investigation, Methodology, Visualization [equal], Writing—original draft [supporting], Writing—review & editing [supporting]), Adam Šafanda (Formal analysis [equal], Methodology [supporting], Writing—original draft [supporting], Writing—review & editing [supporting]), Bui Quang Hiep (Formal analysis [equal], Methodology [supporting], Writing—original draft [supporting], Writing—review & editing [supporting]), Isis Rita Anzel Koutrouli (Data curation, Investigation, Methodology [equal], Writing—original draft [supporting], Writing—review & editing [supporting]), Martin Kuchar (Conceptualization [equal], Funding acquisition [lead], Methodology [supporting], Project administration [equal], Resources [equal], Supervision [equal], Validation [equal], Writing—original draft [supporting], Writing—review & editing [equal]), Tomas Pálenícek (Conceptualization [equal], Funding acquisition [lead], Methodology [supporting], Project administration [supporting], Supervision [equal], Validation [equal], Writing—original draft [supporting], Writing—review & editing [equal]).

Funding

This work was financially supported by the Ministry of the Interior of the Czech Republic (VK01010212 - New psychoactive substances: forensic-toxicology research center).

Conflicts of Interest

T.P. declares to have shares in “Psyon s.r.o.,” and has founded “PSYRES—Psychedelic Research Foundation.” T.P. has shares in “Společnost pro podporu neurovědního výzkumu s.r.o.” and reports consulting fees from GH Research and CB21-Pharma outside the submitted work. T.P. is involved in Compass Pathways, MAPS, GH-Research, Ketabon clinical trials with psilocybin, MDMA, 5-MeO-DMT, ketamine, and MDMA outside the submitted work.

Data Availability

The data that support the findings of this study are available from the corresponding author upon reasonable request. Some data may not be made available because of privacy or ethical restrictions.