Pharmacokinetics of Orally Applied Cannabinoids and Medical Marijuana Extracts in Mouse Nervous Tissue and Plasma: Relevance for Pain Treatment

Cannabis sativa plants contain a multitude of bioactive substances, which show broad variability between different plant strains. Of the more than a hundred naturally occurring phytocannabinoids, Δ9-Tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD) have been the most extensively studied, but whether and how the lesser investigated compounds in plant extracts affect bioavailability or biological effects of Δ9-THC or CBD is not known. We therefore performed a first pilot study to assess THC concentrations in plasma, spinal cord and brain after oral administration of THC compared to medical marijuana extracts rich in THC or depleted of THC. Δ9-THC levels were higher in mice receiving the THC-rich extract. Surprisingly, only orally applied CBD but not THC alleviated mechanical hypersensitivity in the mouse spared nerve injury model, favoring CBD as an analgesic compound for which fewer unwanted psychoactive effects are to be expected.


Introduction
Cannabinoids are bioactive substances found in the Cannabis sativa plants, which show broad variability between different cannabis strains [1,2]. Of the more than a hundred naturally occurring phytocannabinoids that have been identified so far, ∆9-Tetrahydrocannabinol (∆9-THC) and cannabidiol (CBD) are the best studied and have been shown to possess distinct physiological and therapeutic properties [3]. Cannabinoids bind to the G protein coupled (GPCR) CB1 and CB2 receptors, which are widely expressed throughout the nervous system as well as peripheral tissues [4,5], and to different orphan GPCRs, such as GPR55, GPR18, GPR3, GPR6 and GPR12, and peroxisome proliferator activated receptor gamma (PPARγ) [6]. In addition, CBD acts as modest affinity agonist at human 5-HT1a receptor [7,8] and transient receptor potential (TRP) channels TRPV1, TRPV2, TRPV3, TRPV4, TRPA1 and TRPM8 [9].
While THC and its metabolite 11-hydroxy-∆9-THC (OH-THC) are the main psychoactive components of Cannabis sativa, CBD is considered non-psychoactive and can modulate THC-intoxicating and metabolic effects [10,11]. Due to their lipophilic chemical structures, THC and CBD can easily enter all organs including the brain, where they can be detected for more than 24 h after application [12][13][14], despite rapid first-pass hepatic metabolism [15,16].
Already in 1974, cannabis extracts were considered to provide greater pharmacological effects compared to pure cannabinoids [17], which might be due to additional bioactive substances included in extracts, such as minor phytocannabinoids, terpenes or flavonoids [18,19]. This complementary action of the full-spectrum cannabis extracts over the pure cannabinoids is evident mainly from in vitro models using adenocarcinoma cells [20], epithelial cells and colon tissue [21].
For therapeutic interventions, different cannabis preparations exert positive effects for cancer and arthritis pain, headache, as well as mental disorders, such as depression, anxiety, post-traumatic stress and sleeping disorders [22,23]. Interestingly, the combination of THC and CBD increases the anti-allodynic potency by a factor of 200 [24], whereas the combination of cannabinol (CBN) and CBD provides analgesic relief for chronic muscle pain [25]. In addition, the application of a CBD-enriched cannabis extract completely abolishes mechanical allodynia in a mouse model of neuropathic pain [26].
Despite their wide use as painkillers, data on the pharmacokinetics of cannabinoids upon oral administration are sparse, and the effects of the less abundant cannabinoids as well as flavonoids and terpenes on bioavailability, especially in the nervous tissue, are currently not available. We therefore assessed the differences in the pharmacokinetics of cannabinoids and metabolites after oral application of medical marijuana extracts as well as pure THC and CBD compounds in plasma, brain and spinal cord tissues in naive mice and evaluated their analgesic properties in a neuropathic pain mouse model.
The quantification of bioactive substances contained in medical marijuana extracts was performed by working with extract stock solutions prepared with methanol in triplicates. The THC+ extract stock solution was diluted to 400, 40 and 4 µg/mL, and the THC-extract was diluted to 400 and 40 µg/mL, with 50% (v/v) methanol. Calibration or extract samples were mixed with the internal standard (AM stock solution: 428.4 µg/mL in 60% acetonitrile, AM working solution: 1000 ng/mL in 50% methanol) in a 1:1 ratio. After centrifugation at 18,300× g for 90 s, the supernatant was transferred into amber glass vials. Then, 50% (v/v) methanol was used as a blank solvent.

Animals
Adult male C57BL/6J mice (8-10 weeks; Janvier Labs, Le Genest-Saint-Isle, France) were housed under specific pathogen-free (SPF) conditions. Animals were maintained in individual cages at constant room temperature (24 • C) on 12 h light/dark cycle (lights on from 07:00 to 19:00) and had ad libitum access to autoclaved pelleted food and water for an acclimatization phase of 7 days. All procedures involving animals were carried out in accordance with the Ethics Guidelines of Animal Care (Medical University of Innsbruck), as well as the Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes and approved by the Austrian Bundesministerium für Wissenschaft, Forschung und Wirtschaft (permit number GZ: 2020-0.432.251).

Administration of Cannabinoid Formulations
For bioavailability experiments, mice were food deprived overnight preceding administration of single-dose cannabinoid formulations, to control for stomach content. For behavioral experiments, mice had ad libitum access to food for the entire duration of the experiment. Cannabinoid preparations were suspended in sesame oil and stored at −20 • C. Prior to administration, the formulations were sonicated for 15 min at 30 • C. Administration of 5 µL/g cannabinoid formulation was performed by oral gavage using disposable animal feeding needles (20 G, Sigma-Aldrich, Saint Louis, MO, USA). Doses were selected based on literature reports [24,27,28].

Spared Nerve Injury (SNI) Model
SNI surgery was performed as previously described [29]. In short, mice were anesthetized by intraperitoneal (i.p.) injections of 10 mg/kg Xylazine (AniMedica GmbH-a LIVISTO company, Senden, Germany) and 100 mg/kg Ketamine (AniMedica GmbH-a LIVISTO company, Senden, Germany). The skin on the lateral surface of the thigh was incised and the sciatic nerve exposed by separating the biceps femoris muscle through incision of the connective tissue without wounding the muscle. The common peroneal and tibial nerves were ligated with 4-0 Vicryl (Sh-1 plus; Raritan, NJ, USA) and a portion of 3 mm length was excised around the ligation site. Care was taken to avoid any mechanical damage to the sural nerve. After dissection, the muscle and skin were sutured using 4-0 Vicryl. Mice were left to recover at 37 • C until they regained consciousness.

Tissue Collection and Sample Preparation
Time-points for cannabinoid administration and tissue harvesting were planned in a counter balanced design to avoid batch effects. For each formulation, three mice were sacrificed at 0.5, 1, 2, 4, and 6 h after administration. Following deep anesthesia by i.p. injection of Ketamine/Xylazine (Ketamine 100 mg/kg, Xylazine 10 mg/kg), whole blood was drawn via cardiac puncture in 0.5 M EDTA rinsed syringes. Plasma was isolated by centrifugation and collection of the supernatant (9000× g for 10 min at 4 • C). After cardiac puncture, mice were perfused with 1× PBS (Gibco) in order to clear brain and spinal cord tissue of remaining blood, and whole brain and spinal cord tissue samples were collected, frozen on dry ice and stored at −80 • C until further analysis. Whole brain and spinal cord samples were weighed. Whole brain samples were minced and mixed with 70% ethanol (v/v) extraction solvent to obtain a final concentration of approximately 50% (m/v). Whole spinal cord samples were extracted with a four-fold volume extraction solvent under pulsed shaking (600 rpm) for 30 min. In total, 50 µL of plasma, spinal cord or brain homogenate were mixed with 150 µL of the internal standard working solution, ∆9-Tetrahydrocannabinol-D 3 (working solution 5 ng/mL in acetonitrile, (THC-D3; Lipomed, Arlesheim, Switzerland)). Following centrifugation (18,300× g, 1 min), the supernatant was transferred into a 96-well plate.

Analysis of Quantitative Mass Spectrometry Data
Concentrations calculated by mass spectrometry were rounded to four significant digits and accuracies were reported with two decimal places by the chromatographic data processing software Sciex OS/Analytics 2.0.0 or Analyst 1.7.1. Based on the peak area ratios (analyte/internal standard) of the calibration standards, a calibration curve fit was established for each analyte. A quadratic regression model with weighting factor 1/x was used. The bioanalytical method used was not fully validated. However, the performance of each analytical batch was evaluated by the quality of the calibration curve fit as well as the back-calculated concentrations of the calibration standards. Acceptable correlation coefficients (r ≥ 0.99) as well as accuracies meeting specifications of the bioanalytical method validation guideline ICH M10 were obtained (https://www.ema. europa.eu/en/ich-m10-bioanalytical-method-validation-scientific-guideline (Accessed on 16 February 2023); ±15% or ±20% for the lowest concentration level).
Further statistical calculations were performed, and figures were generated using Excel 2016 (Microsoft, Redmond, WA, USA) and Prism 9.0 (GraphPad Software, San Diego, CA, USA). Plasma, brain and spinal cord cannabinoid concentrations at each time point were averaged and the pharmacokinetic parameters were calculated by non-compartmental pharmacokinetic analysis. Pharmacokinetic profiles indicated the actual sampling time and concentration data normalized by tissue wet weight. The linear trapezoidal method was used to calculate the AUC 0-2h (area under concentration-time curve). Brain-plasma and spinal cord-plasma ratios were calculated based on AUCs. The level of statistical significance was predefined at p < 0.05.

Von Frey Test for Mechanical Thresholds
The von Frey test was performed as previously described [29]. In brief, mice were placed in a Plexiglas chamber on an elevated iron-mesh floor and allowed to habituate before testing. To determine paw withdrawal thresholds, a series of custom made calibrated von Frey filaments with uniform tip diameter (diameter: 1.1 mm; forces: 2.8, 4, 5.7, 8, 11.4, 16, 22.6, 32 and 45.3 mN) was presented perpendicularly to the lateral side of the plantar paw surface (sural nerve innervation territory). Withdrawal thresholds were calculated according to the up-and-down method [30,31]. Von Frey results were analysed using repeated measure ANOVA. The level of statistical significance was predefined at p < 0.05.

Data Analysis
For statistical data analyses, GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA) was used. Statistical tests used are specified in the respective methods section as well as in the text or in their respective Figures or Tables. Graphs were plotted in GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA) and figures prepared using CorelDRAW 2021 (Alludo, Ottawa, ON, Canada).

Quantification of Bioactive Cannabinoids and Flavonoids in Medical Marijuana Extracts
Anticipated differences in the composition of bioactive cannabinoids in the THC+ and THC-extracts were confirmed by mass spectrometry analysis. The THC+ extract contained all measured cannabinoids except for CBDA and no flavonoids (Table 2) (Table 3).

Pharmacokinetic Profile of ∆9-THC Bioavailability in Nervous Tissue and Plasma after Application of Medical Marijuana Extracts and Pure Compound
To assess the pharmacokinetic profile of THC in tissues that are involved in the modulation of pain signaling, we determined THC concentrations in brain, spinal cord and plasma following oral gavage of two different doses (i.e., low vs. high) of THC+ (2 vs. 40 mg/kg bodyweight) and THC-extract (40 vs. 1560 mg/kg bodyweight) as well as pure THC compound (1 vs. 20 mg/kg bodyweight) at different time-points (i.e., 30 min, 1 h, 2 h, 4 h, and 6 h after gavage). Upon administration of the high doses of the THC+ extract and pure THC, ∆9-THC was detectable in all tissues 1 h after administration, reaching its maximum concentration at 2 h ( Figure 1A). While ∆9-THC levels in plasma showed a decline after 2 h, they remained elevated in brain and spinal cord tissue until the last measurement. As expected, administration of the high dose of the THC-extract did not induce relevant levels of ∆9-THC in any of the investigated samples. When determining the area under the curve for the initial 2 h post administration (AUC 0-2h ), we found 2-fold higher cumulative concentrations of ∆9-THC after application of the THC+ extract as compared to pure THC compound in both plasma and brain tissue, suggesting an improved bioavailability through more efficient absorption of ∆9-THC from the extract  C, concentration; 2 mean concentration of sample triplicates; the reported concentrations refer to the lowest extract concentration providing an analyte concentration within the calibration range; 3 LLOQ, lower limit of quantification; <LLOQ: no analyte concentration within the calibration range could be found in any of the analysed extract concentrations; 4 N/A, not available.

Pharmacokinetic Profile of ∆9-THC Metabolites in Nervous Tissue and Plasma after Application of Medical Marijuana Extracts and Pure Compound
We next investigated if the concentration of ∆9-THC metabolites differed between the different cannabinoid extracts or compounds in the tissues investigated. For this, we measured the concentrations of the two main secondary metabolites of ∆9-THC, 11-Nor-9carboxy-∆9-THC (COOH-THC) and 11-hydroxy-∆9-THC (OH-THC). As the administration of the low dose for all formulations provided negligible concentrations for ∆9-THC, the metabolites were assessed only after administration of the high dose.
OH-THC was mainly found in brain tissue after application of both THC+ extract and pure THC compound, with a peak concentration at 2 h after application (Figure 2A) and a~1.5-fold increased absorption after application of the THC+ extract compared with the pure THC compound ( Figure 2B). In contrast, COOH-THC was found mainly in one plasma sample of the THC+ extract group (Figure 2C,D). While this sample was displaced far from the remaining samples for this time point, it survived the outlier analysis (Grubb's outlier test, Z = 1.1433 vs. Z critical = 1.1543, p > 0.05). These results suggest tissue-specific degradation mechanisms of ∆9-THC.

Pharmacokinetic Profile of CBD Bioavailability in Nervous Tissue and Plasma after Application of Medical Marijuana Extracts and Pure Compound
When evaluating the CBD bioavailability after oral application of the THC+ and THC-extract, which contained only 0.16% or non-detectable concentrations of CBD, respectively (Tables 2 and 3), as expected, no CBD was measurable. However, application of two different doses of pure CBD compound (1 vs. 20 mg/kg bodyweight) resulted in a dose-dependent absorption of CBD reaching its maximum concentration at 2 h after application ( Figure 3A,B). Interestingly, when directly comparing the absorption of pure THC and pure CBD compounds in the investigated tissues, CBD showed a 2.8-and 1.7-fold higher absorption rate compared with THC absorbance in the brain and spinal cord, respectively. This suggests a better uptake of the cannabinoid CBD in nervous tissue.

Analgesic Potency of Medical Marijuana Extracts and Pure Cannabinoid Compounds
Finally, we tested if oral application of the different cannabis formulations or pure compounds would lead to different analgesic effects. For this, mice that underwent spared nerve injury (SNI) surgery were tested for mechanical hypersensitivity by mechanical threshold determination using von Frey filaments. All mice showed strong mechanical hypersensitivity seven days after surgery, and while a single oral application of vehicle (i.e., sesame oil), THC+ and THC-extract as well as pure THC compound did not affect mechanical withdrawal thresholds, application of CBD alleviated mechanical hypersensitivity 4 h after administration (2-way repeated measures ANOVA followed by Dunnett's multiple comparisons test, 4 h-vehicle vs. pure CBD, p = 0.022; Figure 4).

Pharmacokinetic Profile of CBD Bioavailability in Nervous Tissue and Plasma after Application of Medical Marijuana Extracts and Pure Compound
When evaluating the CBD bioavailability after oral application of the THC+ and THC-extract, which contained only 0.16% or non-detectable concentrations of CBD, respectively (Tables 2 and 3), as expected, no CBD was measurable. However, application of two different doses of pure CBD compound (1 vs. 20 mg/kg bodyweight) resulted in a dose-dependent absorption of CBD reaching its maximum concentration at 2 h after application ( Figure 3A,B). Interestingly, when directly comparing the absorption of pure THC and pure CBD compounds in the investigated tissues, CBD showed a 2.8-and 1.7fold higher absorption rate compared with THC absorbance in the brain and spinal cord, respectively. This suggests a better uptake of the cannabinoid CBD in nervous tissue.

Analgesic Potency of Medical Marijuana Extracts and Pure Cannabinoid Compounds
Finally, we tested if oral application of the different cannabis formulations or pure compounds would lead to different analgesic effects. For this, mice that underwent spared nerve injury (SNI) surgery were tested for mechanical hypersensitivity by mechanical threshold determination using von Frey filaments. All mice showed strong mechanical hypersensitivity seven days after surgery, and while a single oral application of vehicle (i.e., sesame oil), THC+ and THC-extract as well as pure THC compound did not affect mechanical withdrawal thresholds, application of CBD alleviated mechanical hypersensitivity 4 h after administration (2-way repeated measures ANOVA followed by Dunnett's multiple comparisons test, 4 h-vehicle vs. pure CBD, p = 0.022; Figure 4).

Discussion
The current study aimed to provide first insights into whether oral administration of medical marijuana extracts could offer benefits for treatments of patients suffering from pain disorders. We report on the composition and presence of less well investigated phytocannabinoids in Cannabis sativa extracts and provide first results supporting a faster rise

Discussion
The current study aimed to provide first insights into whether oral administration of medical marijuana extracts could offer benefits for treatments of patients suffering from pain disorders. We report on the composition and presence of less well investigated phytocannabinoids in Cannabis sativa extracts and provide first results supporting a faster rise in THC levels already at one hour of administration in plasma and brain, but not in the spinal cord from a THC+ extract. These findings were also reflected by the trajectories of the psychoactive metabolite OH-THC, and this supports oral administration as a possible route for cannabinoid intake for patients suffering from pain disorders. Surprisingly, CBD but not THC alleviated signatures of mechanical hypersensitivity 4 h after administration.
Cannabis products have been tested for various routes of administration and delivery forms in order to enhance their therapeutic effect by increasing the bioavailability of principle compounds CBD and THC. Oral administration of medical marijuana is the prevalent route of administration recommended due to easy administration, prolonged drug action, and reduced toxicity [13,32]. Similar to previous studies in humans and rats, we found that THC reached its peak concentration in mouse plasma, serum or brain approximatively two hours after oral ingestion [11,33,34]. In addition, our studies provide the first data to fill the knowledge gap on tissue distribution at multiple time points following oral administration. The pharmacokinetics of THC when administered as an extract was markedly different from pure THC delivered at an equivalent dose. Specifically, THC peak plasma concentrations were reached substantially faster when administered as a THC+ extract and this may be associated with phytocannabinoids or even the presence of CBD, which is known to affect THC bioavailability [35]. Alterations of gut motility, secretion and resorption may further contribute to this observation [36,37]. Given that pure THC was administered at doses equivalent to that in the THC+ extract, other bioactive substances present in the extract, such as CBC, CBD, CBDV, CBG, CBN and THCV may account for the faster bioavailability of THC by altering its metabolism or distribution [38]. Another possibility may be that CBD and CBN act as inhibitors of enzymes of the cytochrome P450 (CYP) complex resulting in inhibition of THC metabolism in the liver [39,40]. The central nervous system (CNS) is affected by major disorders targeted by cannabinoid therapeutic interventions, such as neurodegeneration, pain and mood disorders and physiochemical properties determine drug penetration into the CNS [41,42]. The presence of other cannabinoids in THC+ extract might support THC bioavailability not only in plasma but also sustained elevated levels in the CNS, which may result in prolonged biological effects. This is also mirrored by the kinetics of THC metabolites OH-THC and COOH-THC. In plasma, both COOH-THC and OH-THC were present for all three THC formulations, whereas OH-THC was the predominant form in the brain. Considering that OH-THC induces physiological effects on its own [33], it may contribute to the therapeutic benefits of cannabinoids. However, there is no difference in OH-THC concentration following co-administration of THC and CBD [11], suggesting that CBD does not affect THC metabolism. However, in our study, the ratio between the bioactive substances of the extract is markedly different and the combination of cannabinoids might interfere with THC metabolism. CBD acts as a neuroprotectant and anti-inflammatory agent with positive safety profiles [43,44]. Our results are in accordance with an earlier study showing that CBD levels continue to rise four hours after administration and a brain-plasma ratio of 3.54 is reported when CBD is orally given to mice [45].
Surprisingly, our behavior data did not match our pharmacokinetic profiles. Neither orally administered THC+ extract nor pure THC alleviated mechanical hypersensitivity induced by nerve injury. These results challenge our understanding on drug dosing, since oral THC at 1 mg/kg or 17.8 mg/kg and intraperitoneal injection at 1.0-4.0 mg/kg or 30 mg/kg had significant analgesic effects [46][47][48], which, however, could be due to different pain models and vehicles used. A significant decrease in mechanical hypersensitivity four hours after administration was only observed for CBD and this was consistent with CBD concentrations measured in the brain and spinal cord, as well as in previous reports [24,48,49].
Our pilot evaluation suggests that unknown Cannabis sativa components appear to affect the bioavailability of psychoactive THC. The data support the necessity of further research aiming at a more precise administration of cannabinoid-based edibles and more precise recommendations for Cannabis sativa over the counter drugs [50]. In addition, variations in extract composition resulting from differences in plant strains and treatments of the harvested material and uncontrolled degradation of cannabinoids by oxygen, light or heating may be problematic. Our current data favor CBD as an analgesic since less psychoactive unwanted effects may be expected [51]. Further investigations exploring larger cohort sizes, different therapeutic windows, oral extracts versus pure compounds and clearly defined medical conditions are important to tailor analgesic cannabinoid therapies precisely to the patients' needs.