Pharmacokinetics of Phytocannabinoid Acids and Anticonvulsant Effect of Cannabidiolic Acid in a Mouse Model of Dravet Syndrome

This is an open access article published under a Creative Commons Non-Commercial No Derivative Works (CC-BY-NC-ND) Attribution License, which permits copying and redistribution of the article, and creation of adaptations, all for non-commercial purposes. Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX pubs.acs.org/jnp Pharmacokinetics of Phytocannabinoid Acids and Anticonvulsant Effect of Cannabidiolic Acid in a Mouse Model of Dravet Syndrome † ‡ † † § † ⊥ Lyndsey L. Anderson, , Ivan K. Low, Samuel D. Banister, , Iain S. McGregor, , † ‡ and Jonathon C. Arnold*, , † Lambert Initiative for Cannabinoid Therapeutics, Brain and Mind Centre, The University of Sydney, Sydney, New South Wales 2050, Australia ‡ Discipline of Pharmacology, Faculty of Medicine and Health, The University of Sydney, Sydney, New South Wales 2006, Australia § School of Chemistry, Faculty of Science, The University of Sydney, Sydney, New South Wales 2006, Australia ⊥ School of Psychology, Faculty of Science, The University of Sydney, Sydney, New South Wales 2006, Australia ABSTRACT: Cannabis sativa produces a complex mixture of many bioactive molecules including terpenophenolic compounds known as phytocannabinoids. Phytocannabinoids come in neutral forms (e.g., Δ9- tetrahydrocannabinol, THC; cannabidiol, CBD; etc.) or as acid precursors, which are dominant in the plant (e.g., Δ9-tetrahydrocannabinolic acid, THCA; cannabidiolic acid, CBDA; etc.). There is increasing interest in unlocking the therapeutic applications of the phytocannabinoid acids; however, the present understanding of the basic pharmacology of phytocannabinoid acids is limited. Herein the brain and plasma pharmacokinetic profiles of CBDA, THCA, cannabichromenic acid (CBCA), cannabidivarinic acid (CBDVA), cannabigerolic acid (CBGA), and cannabigerovarinic acid (CBGVA) were examined following intraperitoneal administration in mice. Next it was examined whether CBDA was anticonvulsant in a mouse model of Dravet RX/+ syndrome (Scn1a mice). All the phytocannabinoid acids investigated were rapidly absorbed with plasma tmax values of between 15 and 45 min and had relatively short half-lives (<4 h). The brain−plasma ratios for the acids were very low at ≤0.04. However, when CBDA was administered in an alternate Tween 80-based vehicle, it exhibited a brain−plasma ratio of 1.9. The anticonvulsant potential of CBDA was examined using this vehicle, and it was found that CBDA significantly increased the temperature threshold at which the Scn1aRX/+ mice had a generalized tonic-clonic seizure. Cannabis sativa L. (Cannabaceae) contains more than 500 CBDA has anxiolytic, anti-inflammatory, and antihyperalgesic 4,5 Downloaded via 217.138.40.154 on November 21, 2019 at 11:13:17 (UTC). potentially bioactive compounds including approximately 140 effects in animal models. Moreover, both CBDA and THCA terpenophenolic molecules called phytocannabinoids. Phyto- have antiemetic effects in shrews and rats, and THCA appears cannabinoids come in neutral forms such as Δ9-tetrahydro- to be the key component mediating the anti-inflammatory 9 cannabinol, THC, and cannabidiol, CBD, or acid forms as Δ - actions of C. sativa extracts in cellular models of inflammatory See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. − tetrahydrocannabinolic acid, THCA, and cannabidiolic acid, bowel disease.6 9 THCA exerts its anti-inflammatory and 1,2 CBDA. The phytocannabinoid acids are formed enzymati- neuroprotective effects by reducing tumor necrosis factor alpha cally and are the most abundant in raw plant material. Neutral (TNF-α) levels modulated by peroxisome proliferator- cannabinoids are formed via decarboxylation of these acid γ 10,11 2 activated receptor gamma (PPAR ). It has been speculated precursor molecules as a result of heat or light exposure. The that CBDA and THCA have anticonvulsant properties, as legalization of medicinal cannabis in many countries around these phytocannabinoids are highly abundant in artisanal C. the world has provoked great interest in the phytocannabinoid sativa oils being used in the community to treat intractable acids, as there is increasing use of cannabis extract formulations childhood epilepsies.3,9 Indeed, it was reported in a patent that that are not heated, smoked, or vaporized. Since these formulations of cannabis are produced via cold extraction or CBDA reduced seizure severity in a rat model of epilepsy; “ ” however, no study has yet documented anticonvulsant effects juicing of raw cannabis material, they contain much higher fi 12 concentrations of the acid forms of the phytocannabinoids.2,3 of this molecule in the peer-reviewed scienti c literature. While research focusing on the therapeutic potential of The capture and dissemination of pharmacokinetic data for phytocannabinoid acids is escalating, there is still a very limited the phytocannabinoid acids is an important contribution in the understanding of the basic pharmacodynamic and pharmacokinetic nature of these abundant components. CBDA and Received: June 30, 2019 THCA may have widespread therapeutic applications, as © XXXX American Chemical Society and American Society of Pharmacognosy A DOI: 10.1021/acs.jnatprod.9b00600 J. Nat. Prod. XXXX, XXX, XXX−XXX Journal of Natural Products Article acids are unknown; thus, the present study characterized the pharmacokinetic profiles of CBDA, THCA, cannabichromenic acid (CBCA), cannabigerolic acid (CBGA), cannabigerovarinic acid (CBGVA), and cannabidivarinic acid (CBDVA) (Figure 1) in mouse plasma and brain. ■ RESULTS AND DISCUSSION Rapid absorption of CBDVA was observed following intraperitoneal (i.p.) administration with a plasma tmax of 15 min and a t1/2 of 49 min (Figure 2A, Table 1). Absorption into the brain was slightly delayed, as the tmax was 30 min; however, the ± Cmax was quite low (0.8 0.1 ng/mg brain; Figure 2B) and elimination was rapid (t1/2 19 min). At 60 min, CBDVA was detected in brain tissue but was below the limit of quantification (LOQ), so a value of 1/2 LOQ (0.25 ng/mg brain) was used. Elimination was complete by 90 min. The brain−plasma ratio (0.02) suggests CBDVA exhibits poor brain penetration. The neutral form of CBDVA, cannabidivarin Figure 1. Chemical structures of phytocannabinoid acids used in this (CBDV), was not detected in the brain or plasma following study. injection of CBDVA, suggesting there is no significant decarboxylation of CBDVA to CBDV in vivo following i.p. search for novel therapeutic applications, revealing optimal injection. timing of dosing for experiments involving in vivo disease Intraperitoneal administration of CBCA in an oil vehicle models. The basic pharmacokinetics of the phytocannabinoid resulted in rapid absorption as the plasma tmax was 30 min with Figure 2. Pharmacokinetic analysis of CBDVA and CBCA in mouse plasma and brain samples. Concentration−time curves for CBDVA in (A) plasma and (B) brain and CBCA in (C) plasma and (D) brain following 5 mg/kg i.p. injections. Phytocannabinoids were administered in a vegetable oil vehicle. Phytocannabinoid concentrations are depicted as both mass concentrations (left y-axis) and molar concentrations (right y- axis). Dashed lines represent limit of quantification (LOQ). Data are expressed as means ± SEM, with n =3−5 per time point. B DOI: 10.1021/acs.jnatprod.9b00600 J. Nat. Prod. XXXX, XXX, XXX−XXX Journal of Natural Products Article a short half-life (t1/2 = 24 min; Figure 2C, Table 1). However, 0.4 CBCA was not detectable in brain tissue (Figure 2D). The ± decarboxylation product of CBCA, cannabichromene (CBC), was not detectable in the brain or plasma following injection of CBCA. ± μ 7.6 2.3 A high maximal plasma concentration (Cmax 34.4 2.1 g/ ± mL; Figure 3A, Table 1) of CBGVA was achieved 15 min post i.p. injection in an oil vehicle. CBGVA exhibited a long half-life (t1/2 = 204 min). Distribution into brain tissue was delayed, not reaching tmax until 90 min (Figure 3B). The half-life of 0.4 63.5 CBGVA in brain, though, was short (t1/2 =29min). ± Concentrations and overall drug exposure (AUC) of CBGVA in brain were significantly lower than plasma values, resulting in a brain−plasma ratio of 0.04. Cannabigerivarin 2.2 2.0 (CBGV) was not detected in the brain following the i.p. ± injection of CBGVA but was present in the plasma (∼0.2% of CBGVA concentrations; the CBGVA administered was 97% pure). THCA was rapidly absorbed (tmax 15 min) and had a relatively short half-life (t1/2 = 67 min) in plasma (Figure 3C, Table 1) in an oil vehicle. Notably, there was a general absence of THCA in brain tissue (Figure 3D). While THCA was 0.4 BLQ 29.6 detectable in brain tissue at some time points, it was below the ± LOQ (a value of 1/2 LOQ 0.25 ng/mg brain was used). Despite not being able to accurately calculate pharmacokinetic parameters in brain tissue, it can be concluded that THCA has 0.5 2.8 poor brain penetration. On comparing the LOQ (0.5 ng/mg ± ± μ brain) of THCA in the brain to the Cmax (2.8 0.4 g/mL) in the plasma, at best the brain−plasma ratio is 0.15. THC was not detected in the brain following i.p. injection of THCA but was found in the plasma (∼1% of plasma THCA 2.1 3.8 ± concentrations; the THCA administered was 95% pure with 5% THC content). Similarly, CBDA was rapidly absorbed following i.p. administration with a plasma tmax of 30 min and Cmax of 29.6 ± 2.2 μg/mL (Figure 4A, Table 1). The half-life of CBDA was quite long (t1/2 = 92 min). Absorption into the brain was slightly delayed, as the tmax was 45 min (Figure 4B); however, 0.5 BLQ 34.4 the elimination was faster (t1/2 = 41 min). Total exposure, ± represented by AUC values, of CBDA in brain tissue was a fraction of total plasma exposure. The brain−plasma ratio (0.04) suggests poor brain penetration by CBDA in an oil vehicle. CBD was not detected in the brain following i.p.

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