Synthetic cannabinoids: Analysis and metabolites

Cannabimimetics (commonly referred to as synthetic cannabinoids), a group of compounds encompassing a wide range of chemical structures, have been developed by scientists with the hope of achieving selectivity toward one or the other of the cannabinoid receptors CB1 and CB2. The goal was to have compounds that could possess high therapeutic activity without many side effects. However, underground laboratories have used the information generated by the scientific community to develop these compounds for illicit use as marijuana substitutes. This chapter reviews the different classes of these “synthetic cannabinoids” with particular emphasis on the methods used for their identification in the herbal products with which they are mixed and identification of their metabolites in biological specimens.     

Synthetic cannabinoids and potential reproductive consequences

Increases in emergency room visits due to abuse of designer drugs, popularly known by the street names “K2” and “Spice,” are a cause for social, judicial, and clinical concerns. The psychoactive components in these herbal drugs mainly consist of different synthetic cannabinoids, and users of these street drugs are primarily within the age group of 12 to 20 years old. The abusive use of synthetic cannabinoids results in anxiety, nausea, vomiting, tachycardia, elevated blood pressure, tremors, seizures, hallucinations, and paranoid behavior, but the effects of maternal use of synthetic cannabinoids during pregnancy are ambiguous due to limited studies in humans and a relative short history of the drugs. In this review, we discuss the known and potential adverse effects of synthetic cannabinoids on human pregnancy using knowledge gathered from studies in mice and limited studies in humans. In mice, multiple sites and stages of pregnancy are potential targets of synthetic cannabinoids, including preimplantation embryo development, oviductal embryo transport, implantation, placentation, and parturition. It is anticipated that maternal use of synthetic cannabinoids would result in severely compromised female fertility and pregnancy outcome.     

Cannabis reinforcement and dependence: role of the cannabinoid CB1 receptor.

Awareness of cannabis dependence as a clinically relevant issue has grown in recent years. Clinical and laboratory studies demonstrate that chronic marijuana smokers can experience withdrawal symptoms upon cessation of marijuana smoking and have difficulty abstaining from marijuana use. This paper will review data implicating the cannabinoid CB1 receptor in regulating the behavioral effects of Delta(9)-tetrahydrocannobinol (THC), the primary psychoactive component of cannabis, across a range of species. The behavioral effects that will be discussed include those that directly contribute to the maintenance of chronic marijuana smoking, such as reward, subjective effects, and the positive and negative reinforcing effects of marijuana, THC and synthetic cannabinoids. The role of the CB1 receptor in the development of marijuana dependence and expression of withdrawal will also be discussed. Lastly, treatment options that may alleviate withdrawal symptoms and promote marijuana abstinence will be considered.     

Emergence and properties of spice and bath salts: A medicinal chemistry perspective

Over the past five years the number of internet sites advertising “legal highs” has literally exploded, as have user reports of experiences (both pleasurable and frightening) with these substances and the number of emergency room visits by users. Although the majority of these “legal highs” have been described as bath salts and herbal extracts, most contain neither plant derived compounds nor components of personal hygiene products. So-called “bath salts” largely contain synthetic analogs of the natural compound Khat; spice-related materials, claimed to be “legal marijuana,” are mostly synthetic analogs of cannabinoid receptor ligands that were developed as research tools. This review describes the emergence and properties of these two groups of “legal highs” from a medicinal chemist's perspective.     

Moving around the molecule: Relationship between chemical structure and in vivo activity of synthetic cannabinoids

Originally synthesized for research purposes, indole- and pyrrole-derived synthetic cannabinoids are the most common psychoactive compounds contained in abused products marketed as “spice” or “herbal incense.” While CB₁ and CB₂ receptor affinities are available for most of these research chemicals, in vivo pharmacological data are sparse. In mice, cannabinoids produce a characteristic profile of dose-dependent effects: antinociception, hypothermia, catalepsy and suppression of locomotion. In combination with receptor binding data, this tetrad battery has been useful in evaluation of the relationship between the structural features of synthetic cannabinoids and their in vivo cannabimimetic activity. Here, published tetrad studies are reviewed and additional in vivo data on synthetic cannabinoids are presented. Overall, the best predictor of likely cannabimimetic effects in the tetrad tests was good CB₁ receptor affinity. Further, retention of good CB₁ affinity and in vivo activity was observed across a wide array of structural manipulations of substituents of the prototypic aminoalkylindole molecule WIN55,212-2, including substitution of an alkyl for the morpholino group, replacement of an indole core with a pyrrole or phenylpyrrole, substitution of a phenylacetyl or tetramethylcyclopropyl group for JWH-018's naphthoyl, and halogenation of the naphthoyl group. This flexibility of cannabinoid ligand–receptor interactions has been a particular challenge for forensic scientists who have struggled to identify and regulate each new compound as it has appeared on the drug market. One of the most pressing future research needs is determination of the extent to which the pharmacology of these synthetic cannabinoids may differ from those of classical cannabinoids.     

Distinct pharmacology and metabolism of K2 synthetic cannabinoids compared to Δ-THC: Mechanism underlying greater toxicity?

K2 or Spice products are emerging drugs of abuse that contain synthetic cannabinoids (SCBs). Although assumed by many teens and first time drug users to be a “safe” and “legal” alternative to marijuana, many recent reports indicate that SCBs present in K2 produce toxicity not associated with the primary psychoactive component of marijuana, ?⁹-tetrahydrocannabinol (Δ⁹-THC). This mini-review will summarize recent evidence that use of K2 products poses greater health risks relative to marijuana, and suggest that distinct pharmacological properties and metabolism of SCBs relative to Δ⁹-THC may contribute to the observed toxicity. Studies reviewed will indicate that in contrast to partial agonist properties of Δ⁹-THC typically observed in vitro, SCBs in K2 products act as full cannabinoid receptor type 1 (CB1R) and type 2 (CB2R) agonists in both cellular assays and animal studies. Furthermore, unlike Δ⁹-THC metabolism, several SCB metabolites retain high affinity for, and exhibit a range of intrinsic activities at, CB1 and CB2Rs. Finally, several reports indicate that although quasi-legal SCBs initially evaded detection and legal consequences, these presumed “advantages” have been limited by new legislation and development of product and human testing capabilities. Collectively, evidence reported in this mini-review suggests that K2 products are neither safe nor legal alternatives to marijuana. Instead, enhanced toxicity of K2 products relative to marijuana, perhaps resulting from the combined actions of a complex mixture of different SCBs present and their active metabolites that retain high affinity for CB1 and CB2Rs, highlights the inherent danger that may accompany use of these substances.     

Autophagy activation by novel inducers prevents BECN2-mediated drug tolerance to cannabinoids

Cannabinoids and related drugs generate profound behavioral effects (such as analgesic effects) through activating CNR1 (cannabinoid receptor 1 [brain]). However, repeated cannabinoid administration triggers lysosomal degradation of the receptor and rapid development of drug tolerance, limiting the medical use of marijuana in chronic diseases. The pathogenic mechanisms of cannabinoid tolerance are not fully understood, and little is known about its prevention. Here we show that a protein involved in macroautophagy/autophagy (a conserved lysosomal degradation pathway), BECN2 (beclin 2), mediates cannabinoid tolerance by preventing CNR1 recycling and resensitization after prolonged agonist exposure, and deletion of Becn2 rescues CNR1 activity in mouse brain and conveys resistance to analgesic tolerance to chronic cannabinoids. To target BECN2 therapeutically, we established a competitive recruitment model of BECN2 and identified novel synthetic, natural or physiological stimuli of autophagy that sequester BECN2 from its binding with GPRASP1, a receptor protein for CNR1 degradation. Co-administration of these autophagy inducers effectively restores the level and signaling of brain CNR1 and protects mice from developing tolerance to repeated cannabinoid usage. Overall, our findings demonstrate the functional link among autophagy, receptor signaling and animal behavior regulated by psychoactive drugs, and develop a new strategy to prevent tolerance and improve medical efficacy of cannabinoids by modulating the BECN2 interactome and autophagy activity.     

The stimulation of ketogenesis by cannabinoids in cultured astrocytes defines carnitine palmitoyltransferase I as a new ceramide-activated enzyme

The effects of cannabinoids on ketogenesis in primary cultures of rat astrocytes were studied. Delta9-Tetrahydrocannabinol (THC), the major active component of marijuana, produced a malonyl-CoA-independent stimulation of carnitine palmitoyltransferase I (CPT-I) and ketogenesis from [14C]palmitate. The THC-induced stimulation of ketogenesis was mimicked by the synthetic cannabinoid HU-210 and was prevented by pertussis toxin and the CB1 cannabinoid receptor antagonist SR141716. Experiments performed with different cellular modulators indicated that the THC-induced stimulation of ketogenesis was independent of cyclic AMP, Ca2+, protein kinase C, and mitogen-activated protein kinase (MAPK). The possible involvement of ceramide in the activation of ketogenesis by cannabinoids was subsequently studied. THC produced a CB1 receptor-dependent stimulation of sphingomyelin breakdown that was concomitant to an elevation of intracellular ceramide levels. Addition of exogenous sphingomyelinase to the astrocyte culture medium led to a MAPK-independent activation of ketogenesis that was quantitatively similar and not additive to that exerted by THC. Furthermore, ceramide activated CPT-I in astrocyte mitochondria. Results thus indicate that cannabinoids stimulate ketogenesis in astrocytes by a mechanism that may rely on CB1 receptor activation, sphingomyelin hydrolysis, and ceramide-mediated activation of CPT-I.     

Role of lipids and lipid signaling in the development of cannabinoid tolerance

Cannabinoid agonists such as Delta9-tetrahydrocannabinol (THC) produce a wide range of pharmacological effects both in the central nervous system and in the periphery. One of the most striking features of cannabinoids such as THC is the magnitude to tolerance that can be produced upon repetitive administration of this substance to animals. Relatively modest dosing regimens are capable of producing significant tolerance, whereas greater than 100-fold tolerance can be obtained with aggressive treatments. While cannabinoid tolerance has been studied quite extensively to establish its relevance to the health consequences of marijuana use, it has also proven to be a valuable strategy in understanding the mechanism of action of cannabinoids. The discovery of the endocannabinoid system that contains two receptor subtypes, CB1 and CB2, associated signaling pathways, endocannabinoids (anandamide and 2-arachidonoylglycerol) and their synthetic and degradative pathways has provided a means of systematically evaluating the mechanism of cannabinoid tolerance. It is well known that the CB1 cannabinoid receptor is down-regulated in states of cannabinoid tolerance along with uncoupling from its second messenger systems. Endocannabinoid levels are also altered in selected brain regions during the development of tolerance. While it is reasonable to speculate that a likely relationship exists between receptor and endocannabinoid levels, at present, little is known regarding the biological signal that leads to alterations in endocannabinoid levels. It is also unknown to what degree synthetic and degradative pathways for the endocannabinoids are altered in states of tolerance. The discovery that the brain is abundant in fatty acid amides and glycerols raises the question as to what roles these lipids contribute to the endocannabinoid system. Some of these lipids also utilize the endocannabinoid metabolic pathways, produce similar pharmacological effects, and are capable of modulating the actions of anandamide and 2-arachidonoylglycerol. In addition, there are dopamine, glycine, and serotonin conjugates of arachidonic acid that may also contribute to the actions of endocannabinoids. A systematic examination of these lipids in cannabinoid tolerance might shed light on their physiological relevance to the endocannabinoid system.     

Behavioral pharmacology of designer cathinones: A review of the preclinical literature

“Bath salts” is one street name for a family of synthetic cathinones that display pharmacological effects resembling cocaine and commonly abused amphetamines. Despite extensive legislation aimed at the criminalization of bath salts, several designer cathinones are gaining a foothold in the illicit drug scene; for example, in the United Kingdom, mephedrone (4-methylmethcathinone, MEPH) is highly popular among drug abusers whereas, in the United States, MDPV (methylenedioxypyrovalerone) and methylone are highly prevalent. To date, knowledge about the hazards of designer cathinones is based mostly on hospital reports and anecdotal evidence derived from online surveys. Despite the paucity of preclinical studies directed toward designer cathinones, a number of invaluable findings arising from those studies are enabling scientists to develop their neuropharmacological profiles. Despite their commonalities in chemical structures, synthetic cathinones possess distinct neuropharmacological profiles and produce different behavioral effects, including unique effects on locomotor activity, learning, anxiety, thermoregulation, and abuse liability. The present review will discuss the behavioral effects of MEPH, MDPV, and methylone and compare those effects to established psychostimulant drugs. The rise in the use of designer cathinones in the United States and abroad justifies further investigations into these compounds, both for a greater understanding of the danger that “bath salts” pose to the public, and to provide insight into replacement cathinones as they emerge onto the market.     

Cannabinoid-hormone interactions in the regulation of motivational processes

There is a bi-directionality in hormone-cannabinoid interactions: cannabinoids affect prominent endocrine axes (such as the hypothalamic-pituitary-gonadal), and gonadal hormones modulate cannabinoid effects. This review will summarize recent research on these interactions, with a specific focus upon their implications for motivated behavior. Sexual behavior will serve as a “case study.” I will explore the hypothesis that ovarian hormones, in particular estradiol, may serve to release estrous behavior from endocannabinoid inhibition. Hormonal regulation of the endogenous cannabinoid system also affects processes that underlie drug abuse. This review will briefly discuss sex differences in behavioral responses to cannabinoids and explore potential mechanisms by which gonadal hormones alter cannabinoid reward. An examination of this research informs our perspective on how hormones and endocannabinoids may affect drug-seeking behavior as a whole and the development of addiction.     

An Overview on the Biochemistry of the Cannabinoid System

About 40 years ago, cannabinoids were considered as the substances responsible for the psychoactive properties of marijuana and other derivatives of Cannabis sativa, whereas their medicinal use remained unexplored. However, with the discovery of the endocannabinoid system 20 years later, the compounds able to modify this system are being reconsidered for their therapeutic potential. Thus, the term "cannabinoid" includes now much more compounds than those present in C. sativa derivatives, for instance, numerous synthetic cannabinoids obtained by modifications from plant-derived cannabinoids or from the compounds that behave as endogenous ligands for the different cannabinoid receptor types. The term "cannabinoid" should also refer to some prototypes of selective antagonists for these receptors. The explanation for this exponential growth in cannabinoid pharmacology is the discovery and characterization of the endocannabinoid signaling system (receptors, ligands, and inactivation system) which plays a modulatory role mainly in the brain but also in the periphery. The objective of the present review article was to give an overview of the present state-of-the-art of biochemistry of the endocannabinoid system. Other authors in this volume will review their functions in the brain, their alterations in a variety of neurological and psychiatric pathologies, and the proposed therapeutic benefits in these diseases of new cannabinoid-related compounds that improve the pharmacological properties of classic cannabinoids.     

Cannabinoids and the hippocampal glucocorticoid receptor: recent findings and possible significance.

It has long been recognized that cannabinoids, including delta 9-tetrahydrocannabinol (THC), the major psychoactive substance of marijuana, bear structural similarities to steroid hormones. The hippocampal region of the brain is particularly rich in glucocorticoid receptors (GCRs), and the region also displays dense autoradiographic binding by synthetic cannabinoids. The present report summarizes studies conducted on cannabinoid interaction with hippocampal GCRs, both in vivo and in vitro. Young rats treated for 8 months with THC displayed anatomic and cellular changes in the hippocampus similar to those seen in older, untreated rats, or in rats treated with high levels of glucocorticoids. Binding of [3H]dexamethasone in cytosol prepared from adrenalectomized rat hippocampus was reduced in the presence of 100-fold molar excess of unlabeled THC. However, further increases of THC concentration, to 20,000-fold excess, could displace no more than 50% of radiolabeled dexamethasone. Scatchard analysis of the binding produced a parallel competition plot for THC, versus the plot for dexamethasone, which may reflect a noncompetitive or allosteric interaction with hippocampal GCR. Cannabidiol, a nonpsychoactive cannabinoid, displayed less competition than THC in all parameters. Treatment of adrenalectomized rats for 14 days with 10 mg/kg THC produced down-regulation of hippocampal GCR binding in a manner also reported following high glucocorticoid administration. Although an initial oral administration of THC to intact rats stimulated release of plasma corticosterone, daily repetition of treatment for 7 and 14 days failed to elicit further corticosterone secretion. Taken together, the results indicate that THC may possess some agonist-like properties of glucocorticoids at the hippocampal GCR site.(ABSTRACT TRUNCATED AT 250 WORDS)     

The neurobiology of cannabinoid dependence: sex differences and potential interactions between cannabinoid and opioid systems.

Cannabis is the most widely used illicit drug in many western countries. Its psychoactive ingredient, delta9-tetrahydrocannabinol (THC), produces a variety of effects in animals and humans that are probably mediated by specific cannabinoid receptors in the brain and interactions with several neurotransmitter and neuromodulator systems. For instance, recent research has revealed an important mutual functional relationship between cannabinoids and endogenous opioid systems in mediating the pharmacological and behavioral actions produced by these agents, including their reinforcing effects. Perinatal exposure to and interactions between cannabinoids and opioids might also have long-term behavioral consequences lasting into adulthood. In this work, we present preliminary evidence examining the potential effects of maternal exposure to THC on the motivational properties of morphine in male and female adult rats, as measured by an intravenous opiate self-administration paradigm.     

Discovery and characterization of endogenous cannabinoids.

The characterization of cannabinoid receptors and signal transduction mechanisms provided the impetus for the searching for endogenous ligands for this system. The result was a family of fatty acid derivatives that interact with cannabinoid receptors to varying degrees. The two ligands that have received the most attention are anandamide (AN) and 2-arachidonolyl-glycerol (Ara-Gl). They are both present in central as well as peripheral tissues. Mechanisms for the synthesis and metabolism of AN have been described. Presently, the physiological stimuli for production and release of AN are unknown. As a result, elucidation of its physiological role remains elusive. However, it seems reasonable to conclude that both AN and 2-Ara-Gl interact with cannabinoid receptors in both peripheral and central tissue to produce a wide range of effects. Administration of these ligands to laboratory animals produce effects that are quite similar to those elicited by delta9-tetrahydrocannabinol (THC), the psychoactive constituent in marijuana. Nevertheless, there are some pharmacological differences between the plant-derived THC and the endogenous cannabinoids that could be due to either pharmadynamic or pharmacokinetics dissimilarities. Extensive structure-activity relationship studies have provided some vital insights into the actions of the endogenous ligands. First and foremost, systematic structural alterations in AN have additional support that it is acting at the cannabinoid receptors in a fashion similar to that of THC. Development of metabolically stable analogs of AN, as well as those with greater receptor affinity, have helped substantiate AN and THC similarities. Nevertheless, pharmacological differences remain between the endogenous and exogenous ligands. Whether these differences are due to the nature of their interaction with the cannabinoid receptors, activation of unique signaling pathways, interactions with non-cannabinoid receptors, or pharmacokinetic considerations remain to be resolved.     

Prospects for cannabinoids as anti-inflammatory agents

The marijuana plant (Cannabis sativa) and preparations derived from it have been used for medicinal purposes for thousands of years. It is likely that the therapeutic benefits of smoked marijuana are due to some combination of its more than 60 cannabinoids and 200-250 non-cannabinoid constituents. Several marijuana constituents, the carboxylic acid metabolites of tetrahydrocannabinol, and synthetic analogs are free of cannabimimetic central nervous system activity, do not produce behavioral changes in humans, and are effective antiinflammatory and analgesic agents. One cannabinoid acid in particular, ajulemic acid, has been studied extensively in in vitro systems and animal models of inflammation and immune responses. This commentary reviews a portion of the work done by investigators interested in separating the medicinal properties of marijuana from its psychoactive effects. Understanding the mechanisms of the therapeutic effects of nonpsychoactive cannabinoids should lead to development of safe effective treatment for several diseases, and may render moot the debate about "medical marijuana".     

Synergistic interactions between cannabinoid and opioid analgesics

Cannabinoids and opioids both produce analgesia through a G-protein-coupled mechanism that blocks the release of pain-propagating neurotransmitters in the brain and spinal cord. However, high doses of these drugs, which may be required to treat chronic, severe pain, are accompanied by undesirable side effects. Thus, a search for a better analgesic strategy led to the discovery that delta 9-tetrahydrocannabinol (THC), the major psychoactive constituent of marijuana, enhances the potency of opioids such as morphine in animal models. In addition, studies have determined that the analgesic effect of THC is, at least in part, mediated through delta and kappa opioid receptors, indicating an intimate connection between cannabinoid and opioid signaling pathways in the modulation of pain perception. A host of behavioral and molecular experiments have been performed to elucidate the role of opioid receptors in cannabinoid-induced analgesia, and some of these findings are presented below. The aim of such studies is to develop a novel analgesic regimen using low dose combinations of cannabinoids and opioids to effectively treat acute and chronic pain, especially pain that may be resistant to opioids alone.     

Cannabinoid receptors in atherosclerosis

PURPOSE OF REVIEW: Recent findings suggesting that cannabinoid receptors are potential targets for the treatment of atherosclerosis are reviewed. RECENT FINDINGS: Cannabinoids, such as Delta9-tetrahydrocannabinol, the major psychoactive compound of marijuana, their synthetic analogs and endogenous cannabinoid ligands, produce their biological effects by interacting with specific receptors. In the apolipoprotein E knockout mouse model of atherosclerosis, Delta9-tetrahydrocannabinol was shown to inhibit disease progression through pleiotropic effects on inflammatory cells. Blocking of cannabinoid receptor CB2, the main cannabinoid receptor expressed on immune cells, abolished the observed effects. The development of novel cannabinoid receptor ligands that selectively target CB2 receptors or pharmacological modulation of the endocannabinoid system might offer novel therapeutic strategies in the treatment of atherosclerosis. Several reports demonstrating an implication of the endocannabinoid system in different inflammatory conditions support this hypothesis. SUMMARY: The immunomodulatory capacity of cannabinoids is now well established and suggests a broad therapeutic potential of cannabinoids for a variety of conditions, including atherosclerosis. New strategies based on nonpsychotropic cannabinoid receptor ligands or compounds modulating endocannabinoid synthesis or stability might solve the problem of the unwanted side effects associated with cannabinoid administration.     

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.