Cross-talk of cannabinoid and endocannabinoid metabolism is mediated via human cardiac CYP2J2

Highlights

• Cytochrome P450 (CYP) 2J2 metabolizes 6 cannabinoids from cannabis.

• CYP2J2 forms unique hydroxyl metabolites from the cannabinoids.

• The cannabinoids are better substrates than endogenous substrates of CYP2J2.

• Cannabinoids inhibit the endocannabinoid, anandamide, metabolism noncompetitively.

• Δ9-Tetrahydrocannabinol is the strongest inhibitor of anandamide metabolism.

Abstract

Phytocannabinoids have well-known cardiovascular implications. For instance, Δ9-tetrahydrocannabinol (Δ9-THC), the principal component of cannabis, induces tachycardia in humans. In order to understand the impact of phytocannabinoids on human cardiovascular health, there is a need to study the metabolism of phytocannabinoids by cardiac cytochromes p450 (CYPs). CYP2J2, the primary CYP of cardiomyocytes, is responsible for the metabolism of the endocannabinoid, anandamide (AEA), into cardioprotective epoxides (EET-EAs). Herein, we have investigated the kinetics of the direct metabolism of six phytocannabinoids (Δ9-THC, Δ8-tetrahydrocannabinol, cannabinol, cannabidiol, cannabigerol, and cannabichromene) by CYP2J2. CYP2J2 mainly produces 1′/1″-OH metabolites of these phytocannabinoids. These phytocannabinoids are metabolized with greater catalytic efficiency compared to the metabolism of AEA by CYP2J2. We have also determined that the phytocannabinoids are potent inhibitors of CYP2J2-mediated AEA metabolism, with Δ9-THC being the strongest inhibitor. Most of the inhibition of CYP2J2 by the phytocannabinoids follow a noncompetitive inhibition model, and therefore dramatically reduce the formation of EET-EAs by CYP2J2. Taken together, these data demonstrate that phytocannabinoids are directly metabolized by CYP2J2 and inhibit human cardiac CYP2J2, leading to a reduction in the formation of cardioprotective EET-EAs.

Introduction

Cannabis sativa has been used for centuries throughout human history for both its psychoactive effects and medicinal properties. Increasingly, legalization of cannabis for medical and recreational use is gaining worldwide support, in conjunction with trends of increased cannabinoid potency. Therefore, studying the effects of cannabinoids derived from cannabis on human health is of medical and scientific interest.

Cannabinoids are broadly classified into three categories depending on their source: (1) endocannabinoids (eCB) that are endogenously produced derivatives of polyunsaturated fatty acids (PUFAs) in animals; (2) phytocannabinoids (pCBs) that are derived from plants; and (3) synthetic cannabinoids. Psychoactive pCBs include Δ9-tetrahydrocannabinol (Δ9-THC), the primary psychoactive component of the plant, Δ8-tetrahydrocannabinol (Δ8-THC), and cannabinol (CBN). Some of the most abundant non-psychoactive pCBs in cannabis include cannabichromene (CBC), cannabidiol (CBD), and cannabigerol (CBG) (Fig. 1).

Phytocannabinoids have well-known cardiovascular implications that have been difficult to interpret due to variations regarding their effects in different species. For instance, the cardiovascular effects of THC in animals versus humans are contradictory [1,2]. Δ9-THC induces tachycardia in humans, and only reproduces similar results in conscious monkeys; and prolonged exposure resulted in a reduction in elevated heart rate, as is seen in humans with developed tolerance [3]. In other animal models, Δ9-THC induces bradycardia [[4], [5], [6]]. Interpreting animal model data is further complicated using anesthesia. Experiments using anaesthetized [5] versus non-anaesthetized [4] rats did and did not exhibit tolerance to bradycardia symptoms, respectively, despite increased Δ9-THC administration. This lack of consensus in cross-species studies, changing variables in experimental design, and the psychoactivity of pCBs have obfuscated focus on discerning the exact cardiovascular implications of cannabis. Therefore, in order to understand the impact of pCBs on human cardiovascular health, there is a need to study the metabolism of pCBs by human cardiac enzymes. Of interest are the cytochromes P450 (CYPs), the primary enzymes that are involved in drug metabolism in the human body.

CYPs are known for their ability to metabolize diverse xenobiotics, synthesize steroids, and be involved in fatty acid metabolism [7]. CYPs generally require electrons donated by cytochrome P450 reductase (CPR) in order to oxidize their substrates. Previously, it was demonstrated that pCBs inhibit the metabolism of drugs by microsomal CYPs (1A1 [8,9], 1A2 [8], 1B1 [10], 2A6 [11], 2B6 [[10], [11], [12]], 2C8 [12], 2C9 [[13], [14], [15]], 2C11 [16], 2C19 [17], 2D6 [18], 3A4 [12,19], 3A5 [12,19], and 3A11 [20]). Currently, there is absence of any mechanistic study on the metabolism of pCBs by CYP2J2, the most abundant CYP expressed in the cardiomyocytes of the heart [8,9].

CYP2J2 is involved in the metabolism of both ω−3 and ω−6 eCBs leading to the formation of eCB epoxides that are vasodilatory, anti-platelet aggregatory, anti-inflammatory, and overall cardioprotective [21]. Anandamide (AEA) was the first eCB discovered. It is derived from the ω−6 fatty acid, arachidonic acid (AA) (Fig. 1A) [22]. AEA was shown to be metabolized by several CYPs, including CYP2J2, forming different regioisomers of epoxyeicosatrienoyl ethanolamides (EET-EAs) (Fig. 1A) [23,24]. CYP2J2 has also been shown to metabolize several drugs, and many of which are known to be cardiotoxic [[25], [26], [27], [28]].

Despite structural differences between eCBs and pCBs, both of these classes of cannabinoids interact with the endocannabinoid system (ECS) in the body. The ECS system consists of an ensemble of eCBs and eCB-like mediators, their corresponding receptors, and metabolic enzymes involved in ligand formation and degradation [29]. The ECS is involved in homeostatic functions dynamically regulating the functionality of the immune, reproductive, gastrointestinal, and central nervous systems, in addition to that of the brain, liver, and heart [30]. Physiological modulation by the ECS is largely dependent upon the nature and location of the diseased state [31].

The activity of the ECS is primarily elicited via molecular recognition of both pCBs and eCBs by cannabinoid receptors 1 and 2 (CBR1 and CBR2). ECS regulatory roles in the cardiovascular system are complex as the two receptors elicit varied responses [32,33]. Activation of CBR1 translates to cardiovascular complications by decreasing blood flow and inducing vasoconstriction [[34], [35], [36], [37], [38]]; while CBR2 activity suppresses inflammatory responses of endothelial cells and monocytes [36,[39], [40], [41]] and is also suggested to provide a protective role against cardiovascular diseases through induced vasodilation [40]. Thus, conflicting natures of CBR1 and CBR2 activation make the ECS a contributor to the generation, as well as amelioration, of cardiovascular disease.

Previously, we showed that CYP2J2 converts AEA into EET-EAs and similar epoxides from ω−3 fatty acid ethanolamides that are derived from DHA and EPA [42]. During inflammation, the levels of these epoxides are comparable to AEA [42]. Inasmuch as CYP2J2 generates cardioprotective epoxides from eCBs and also metabolizes several drugs, CYP2J2 is a prime enzyme for studying the potential effects of pCBs on eCB metabolism. It has been hypothesized that AEA metabolism by unknown CYPs is inhibited by pCBs [43]. Herein, we determine the direct metabolism of selected pCBs by CYP2J2 and evaluate their effects on AEA metabolism. We have determined that the 6 pCBs tested (Δ9-THC, Δ8-THC, CBC, CBD, CBG, and CBN) are all metabolized by CYP2J2 to form new hydroxyl products of the pCBs, with various di-oxygenated products as well. The catalytic efficiencies of the pCB turnovers are similar to or greater than that of AEA. Using CYP2J2-nanodiscs, we measured the kinetics of AEA metabolism by CYP2J2. Further, we have determined that the pCBs are potent inhibitors of AEA metabolism. We determined that CBG is a competitive inhibitor of AEA, whereas Δ9-THC, Δ8-THC, CBD, CBN, and CBC are noncompetitive inhibitors. Of these, Δ9-THC is the strongest inhibitor of AEA metabolism and reduces CYP2J2-mediated AEA metabolism to 20% of the uninhibited activity. Our study demonstrates that the pCBs, especially Δ9-THC, inhibits CYP2J2 and prevent the metabolism of endogenous substrates such as AEA.