Lester M. Bornheim

Dual effects of anandamide on NMDA receptor-mediated responses and neurotransmission.

Abstract: Anandamide is an endogenous ligand of cannabinoid receptors that induces pharmacological responses in animals similar to those of cannabinoids such as Δ9‐tetrahydrocannabinol (THC). Typical pharmacological effects of cannabinoids include disruption of pain, memory formation, and motor coordination, systems that all depend on NMDA receptor mediated neurotransmission. We investigated whether anandamide can influence NMDA receptor activity by examining NMDA‐induced calcium flux (ΔCa2+NMDA) in rat brain slices. The presence of anandamide reduced ΔCa2+NMDA and the inhibition was disrupted by cannabinoid receptor antagonist, pertussis toxin treatment, and agatoxin (a calcium channel inhibitor). Whereas these treatments prevented anandamide inhibiting ΔCa2+NMDA, they also revealed another, underlying mechanism by which anandamide influences ΔCa2+NMDA. In the presence of cannabinoid receptor antagonist, anandamide potentiated ΔCa2+NMDA in cortical, cerebellar, and hippocampal slices. Anandamide (but not THC) also augmented NMDA‐stimulated currents in Xenopus oocytes expressing cloned NMDA receptors, suggesting a capacity to directly modulate NMDA receptor activity. In a similar manner, anandamide enhanced neurotransmission across NMDA receptor‐dependent synapses in hippocampus in a manner that was not mimicked by THC and was unaffected by cannabinoid receptor antagonist. These data demonstrate that anandamide can modulate NMDA receptor activity in addition to its role as a cannabinoid receptor ligand.

Anandamide hydroxylation by brain lipoxygenase:metabolite structures and potencies at the cannabinoid receptor

Anandamide (arachidonyl ethanolamide) is a compound that was identified from porcine brain lipids by its ability to bind to the brain cannabinoid receptor. This study assessed anandamide as a substrate for a brain lipoxygenase and characterised the brain metabolite 12-hydroxyanandamide. Anandamide was also compared with arachidonic acid as a lipoxygenase substrate by examining enzyme kinetics in the presence of either of the two compounds. In addition, a non-mammalian enzyme was used to generate 11- and 15-hydroxy-anandamide in order to compare the cannabinomimetic properties of a range of anandamide derivatives. A ligand displacement assay indicated a large variation in the affinity of anandamide metabolites for the brain cannabinoid receptor. The brain metabolite, 12-hydroxyanandamide had an affinity twice that of anandamide, although the 11- and 15- hydroxy-metabolites were considerably poorer ligands of this receptor. Consistent with the receptor binding data, 12-hydroxyanandamide (unlike 15-hydroxyanandamide) inhibited forskolin-stimulated cAMP synthesis, indicating it to be a functional agonist at the brain cannabinoid receptor. Pharmacological studies of the capacity of anandamide and its metabolites to inhibit the murine vas deferens twitch response indicated the 12-hydroxy-metabolite to be less active than the parent compound, but a better cannabinomimetic than 15-hydroxyanandamide.

Microsomal cytochrome P450-mediated liver and brain anandamide metabolism

Anandamide (AN) is an arachidonic acid congener, found in the brain, that binds to the cannabinoid receptor and elicits cannabinoid-like pharmacological activity. Cytochromes P450 (P450s) are known to oxidize arachidonic acid to a wide variety of metabolites, yielding many physiologically potent compounds. To determine if AN could be similarly oxidized by P450s, its metabolism by mouse liver and brain microsomes was examined. Mouse hepatic microsomal incubation of AN with NADPH resulted in the generation of at least 20 metabolites, determined after HPLC separation by increased UV-absorbance at 205 nm. Pretreatment of mice with various P450 inducers resulted in increased hepatic microsomal formation of several AN metabolites, with dexamethasone being the most effective inducer. Phenobarbital pretreatment resulted in a metabolic profile similar to that observed after dexamethasone pretreatment, whereas 3-methylcholanthrene pretreatment selectively increased the formation of several other metabolites. Clofibrate pretreatment had no effect on hepatic AN metabolism. Polyclonal antibodies prepared against mouse hepatic P450 3A inhibited the formation of several AN metabolites by hepatic microsomes from untreated mice as well as the formation of those metabolites found to be increased after dexamethasone pretreatment. AN metabolism by brain microsomes resulted in the formation of two NADPH- and protein-dependent metabolites. Hepatic P450 3A antibody partially inhibited the formation of only one of these metabolites. Thus, P450 3A is a major contributor to AN metabolism in the liver but not in the brain. The physiological consequences of P450-mediated AN metabolism remain to be determined.

Inhibition of cyclosporine and tetrahydrocannabinol metabolism by cannabidiol in mouse and human microsomes

1. The in vitro and in vivo effects of cannabidiol on mouse and human liver microsomal metabolism of the immunosuppressive drug cyclosporine and the psychoactive compound tetrahydrocannabinol have been examined. 2. Preincubation of mouse or human liver microsomes with cannabidiol decreased the formation of all detectable cyclosporine metabolites by 73-89%. 3. In vivo cannabidiol treatment of mouse similarly decreased the formation of all detectable cyclosporine metabolites by 60-86%. 4. Preincubation of human liver microsomes with cannabidiol selectively decreased the formation of tetrahydrocannabinol metabolites catalyzed by cytochrome P4503A by 60% but had no effect on P4502C9-catalyzed metabolites. 5. Cannabidiol has the potential to clinically affect cyclosporine metabolism which may result in increased cyclosporine blood levels and an increase in its toxic side effects, and likewise may also affect tetrahydrocannabinol and its metabolite levels in man.

Cannabinoid-induced alterations in brain disposition of drugs of abuse.

Marijuana contains a complex mixture of compounds including tetrahydrocannabinol (THC), the major psychoactive constituent, and cannabidiol (CBD), a nonpsychoactive constituent. We have shown previously that CBD pretreatment of mice increases brain levels of THC and have now further characterized this effect and determined whether the brain pharmacokinetics of other drugs are also affected. CBD pretreatment of mice (30-60 min) increased brain levels of THC nearly 3-fold, whereas CBD co-administration did not. Because marijuana is often consumed with other drugs, the influence of cannabinoids on the brain levels of several other drugs of abuse was also determined. CBD pretreatment of mice increased brain levels (2- to 4-fold) of subsequently administered cocaine as well as phencyclidine (PCP). Although CBD pretreatment increased blood and brain levels of cocaine comparably, blood levels of PCP were only modestly elevated (up to 50%). Behavioral tests indicated that the CBD-mediated increases in the brain levels of THC, cocaine, and PCP correlated with increased pharmacological responses. Pretreatment with THC instead of CBD could similarly increase brain levels of cocaine, PCP, and CBD, although with a lower potency than CBD. On the other hand, pretreatment of mice with CBD had no effect on the brain levels of several other drugs of abuse including morphine, methadone, or methylenedioxyphenyl-methamphetamine. These findings demonstrate that cannabinoids can increase the brain concentrations and pharmacological actions of several other drugs of abuse, thereby providing a biochemical basis for the common practice of using marijuana concurrently with such drugs.

Characterization of cannabidiol-mediated cytochrome P450 inactivation

Cannibidiol (CBD) has been shown to impair hepatic drug metabolism in several animal species and to markedly inhibit mouse hepatic microsomal delta 1-tetrahydrocannabinol (THC) metabolism by inactivating specific cytochrome P450s (P450) belonging to the 2C and 3A subfamilies. Elucidation of the mechanism of CBD-mediated P450 inhibition would be clinically very important for predicting its effect on metabolism of THC and the many other clinically important drugs known to be metabolized by P450s 2C and 3A. CBD-mediated inactivation of mouse hepatic microsomal P450s did not decrease hepatic microsomal heme content. However, [14C]CBD was found covalently bound to microsomal protein in an approximately equimolar ratio to P450 loss. Immunoprecipitation of microsomal protein with antibodies raised against either P450 2C or 3A revealed that approximately equal amounts of [14C]-CBD were bound to each of these P450s after CBD-mediated inactivation. Furthermore, this specific P450 binding was equivalent to P450 loss and accounted for nearly all of the microsomal [14C]CBD irreversible binding. Although > 80% of the enzyme activities attributed to P450s 2C and 3A were inactivated by CBD at the anticonvulsant dose of 120 mg/kg, P450 2C was approximately 3-fold more sensitive than P450 3A at the lower CBD doses tested. CBD analogs were synthesized in order to elucidate the chemical pathways for CBD-mediated P450 inactivation in vivo. Oxidations at allylic carbon positions or saturation of either the exocyclic double bond or both double bonds of the terpene moiety did not markedly affect the inhibitory properties of the analogs. Methylation of both phenolic groups of the resorcinol moiety completely blocked the P450-inhibitory properties of this analog, revealing the involvement of a free hydroxyl group in the inactivation process. Rotation of the resorcinol moiety in abnormal-CBD did not impair the inhibitory properties of the analog, suggesting that the position of the hydroxyl group relative to the terpene ring is unimportant. Further studies are required to fully understand the chemical basis of CBD-mediated P450 inactivation.

Induction and genetic regulation of mouse hepatic cytochrome P450 by cannabidiol

Cannabidiol (CBD) has been shown to be a selective inactivator of cytochromes P450 (P450s) 2C and 3A in the mouse and, like many P450 inactivators, it can also induce P450s after repeated administration. The inductive effects of CBD on mouse hepatic P450s 2B, 3A, and 2C were determined using cDNA probes, polyclonal antibodies, and specific functional markers. P450 2B10 mRNA was increased markedly after repeated CBD administration and correlated well with increased P450 2B immunoquantified content and functional activity. On the other hand, although the 2-fold increase in P450 3A mRNA detected after repeated CBD administration was consistent with the increased immunoquantified P450 3A protein content, the lack of an observable increase in P450 3A-specific functional activity suggested subsequent inactivation of the induced P450 3A. Repeated CBD treatment increased P450 2C mRNA content 2-fold, but did not increase either the P450 2C immunoquantified content or its functional activity. The effect of CBD treatment on the ability of tetrahydrocannabinol (THC) to induce P450 2B was also determined. A THC dose that did not induce P450 2B significantly was administered alone or in combination with a CBD dose that markedly inactivated P450s 2C- and 3A but submaximally increased P450 2B functional activity. The combination of THC and CBD did not increase P450 2B-catalyzed activity significantly over that observed after CBD treatment alone. Thus, prior CBD-mediated P450 inactivation does not appear to increase the ability of THC to induce P450 2B. To further characterize the relationship between P450 inactivation and induction, several structurally diverse CBD analogs with varying P450 inactivating potentials were tested for their ability to induce P450 2B. At least one CBD analog that is an effective P450 inactivator failed to induce P450 2B, while at least one CBD analog that is incapable of inactivating P450 was found to be a very good P450 2B inducer. It therefore appears that inherent structural features of the CBD molecule rather than its ability to inactivate P450 determine P450 2B inducibility. The complex effects of CBD treatment on P450 inactivation and induction have the potential to influence the pharmacological action of many clinically important drugs known to be metabolized by these various P450s. The mechanism of CBD-mediated P450 induction remains to be elucidated but does not appear to be related to CBD-mediated P450 inactivation.

The influence of side chain modifications of the heme moiety on prosthetic acceptance and function of rat hepatic cytochrome P-450 and tryptophan pyrrolase

The relative potential of various structural isomers (III, XIII) and various 2,4-side chain modified analogs of heme (iron-protoporphyrin IX) to incorporate into rat liver hemoproteins, cytochrome P-450(s), and tryptophan pyrrolase was examined. Such assessments for hepatic cytochrome P-450 relied on generation of reconstitutible apocytochrome(s) P-450 by suicidal alkylation of the existing prosthetic heme moiety by allylisopropylacetamide (AIA) in vivo. Subsequent replacement of the prosthetic heme was brought about by incubating the apocytochrome(s) P-450-enriched preparations with a particular heme isomer or analog. Structure-function relationships of the reconstituted isozymes were assessed in microsomal preparations by monitoring cytochrome P-450 content (structure) and its mixed function oxidase activity (function). In parallel, the relative ability of these heme isomers and analogs to functionally constitute hepatic tryptophan pyrrolase was also assessed by monitoring the relative increase in holoenzyme activity when preparations deliberately enriched in constitutible apoenzyme were incubated with each of these compounds. The findings reveal that 2,4-side chain modifications on the heme IX skeleton markedly influence the function of the constituted hemoproteins possibly by affecting their structural assembly through steric, electronic, and/or hydrophobic interactions with the corresponding apoproteins. Furthermore, these studies not only reveal that the structural specifications of the active prosthetic site of rat liver cytochrome P-450(s) differ from those of tryptophan pyrrolase, but also that the structural specifications of these mammalian hemoproteins for their prosthetic heme differ considerably from those reported for their bacterial counterparts.

Effects of unsaturated side-chain analogs of tetrahydrocannabinol on cytochromes P450

The ability of unsaturated side-chain analogs of Delta(8)-tetrahydrocannabinol (THC) to selectively inactivate mouse hepatic cytochromes P450 3A11 and 2C29 was examined. THC side-chain analogs were preincubated with mouse hepatic microsomes and NADPH for various times before dilution and determination of Delta(9)-THC metabolism specific for P450s 3A11 and 2C29. THC-enyl analogs had little or no effect on P450 3A11 but inactivated P450 2C29 in a time-dependent manner, with approximately 50% inactivation observed after a 30-min preincubation. THC-ynyl analogs were less selective in their P450 inactivation but appeared to be more effective than their corresponding enyl analogs. THC-ynyl analogs inactivated P450s 3A11 and 2C29 in a time-dependent manner and could inactive 40-80% of their activities after a 30-min preincubation. The THC-ynyl analogs were nearly as effective as cannabidiol, a well-characterized inactivator of these mouse P450s. Despite their ability to inactivate P450 in vitro, neither the THC-enyl nor the THC-ynyl analogs were very effective after in vivo administration. Unsaturated side-chain THC analogs may be useful in the development of specific P450 inactivators.