Tetrad test

Last updated September 11, 2019

The tetrad test is a series of behavioral paradigms in which rodents treated with cannabinoids such as THC show effects.^[1]^[2] It is widely used for screening drugs that induce cannabinoid receptor-mediated effects in rodents. The four behavioral components of the tetrad are spontaneous activity, catalepsy, hypothermia, and analgesia. Common assays for these behavioral paradigms are as follows:

Cannabinoid receptors, located throughout the body, are part of the endocannabinoid system, which is involved in a variety of physiological processes including appetite, pain-sensation, mood, and memory.

Spontaneous activity (or hypomotility) is determined by an open field test, in which a mouse is placed in a cage with perpendicular grid lines, usually spaced by approx. 1 inch. An experimenter counts the number of line crossings by the mouse in a given amount of time.
Catalepsy is determined by the bar test. The mouse is placed on a bar oriented parallel to and approximately 1 inch off of the ground. If the mouse remains immobile on the bar for typically more than 20 seconds, it is considered cataleptic.
Hypothermia (reduced body temperature) is determined by using a rectal probe to measure the rectal temperature.
Analgesia is usually determined by the hot plate or tail immersion test. In the hot plate test, the mouse is placed on a heated plate, typically between 54 and 58°C. An experimenter measures the time it takes for the animal to raise its feet or jump off of the hot plate. In the tail immersion test, the mouse is immobilized and its tail is placed into a warm water bath, typically also between 54 and 58°C. An experimenter measures the time it takes for the mouse to remove its tail from the water bath.

Developed by Calvin S. Hall, the open field test is an experimental test used to assay general locomotor activity levels, anxiety, and willingness to explore in animals in scientific research. However, the extent to which behavior in the open field measures anxiety is controversial.

Catalepsy is a nervous condition characterized by muscular rigidity and fixity of posture regardless of external stimuli, as well as decreased sensitivity to pain.

Hypothermia is defined as a body core temperature below 35.0 °C (95.0 °F) in humans. Symptoms depend on the temperature. In mild hypothermia there is shivering and mental confusion. In moderate hypothermia shivering stops and confusion increases. In severe hypothermia, there may be paradoxical undressing, in which a person removes their clothing, as well as an increased risk of the heart stopping.

Direct CB1 agonists, such as THC (the psychoactive component of marijuana), or WIN 55,212-2, have effects in all components of the tetrad and induce hypomotility, catalepsy, hypothermia, and analgesia in rodents. Accordingly, all true "tetrad effects" are not observed following treatment with antagonists or inverse agonists of CB1 such as rimonabant. Data have shown, that also CB2 receptors are involved in the tetrad effects induced by cannabinoids, and other, associated with CB1 agonism.^[3]

An agonist is a chemical that binds to a receptor and activates the receptor to produce a biological response. Whereas an agonist causes an action, an antagonist blocks the action of the agonist, and an inverse agonist causes an action opposite to that of the agonist.

WIN 55,212-2 is a chemical described as an aminoalkylindole derivative, which produces effects similar to those of cannabinoids such as tetrahydrocannabinol (THC) but has an entirely different chemical structure.

A receptor antagonist is a type of receptor ligand or drug that blocks or dampens a biological response by binding to and blocking a receptor rather than activating it like an agonist. They are sometimes called blockers; examples include alpha blockers, beta blockers, and calcium channel blockers. In pharmacology, antagonists have affinity but no efficacy for their cognate receptors, and binding will disrupt the interaction and inhibit the function of an agonist or inverse agonist at receptors. Antagonists mediate their effects by binding to the active site or to the allosteric site on a receptor, or they may interact at unique binding sites not normally involved in the biological regulation of the receptor's activity. Antagonist activity may be reversible or irreversible depending on the longevity of the antagonist–receptor complex, which, in turn, depends on the nature of antagonist–receptor binding. The majority of drug antagonists achieve their potency by competing with endogenous ligands or substrates at structurally defined binding sites on receptors.

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Levonantradol (CP 50,556-1) is a synthetic cannabinoid analog of dronabinol (Marinol) developed by Pfizer in the 1980s. It is around 30x more potent than THC, and exhibits antiemetic and analgesic effects via activation of CB₁ and CB₂ cannabinoid receptors. Levonantradol is not currently used in medicine as dronabinol or nabilone are felt to be more useful for most conditions, however it is widely used in research into the potential therapeutic applications of cannabinoids.

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Cannabinoid receptor type 1 (CB₁), also known as cannabinoid receptor 1, is a G protein-coupled cannabinoid receptor that in humans is encoded by the CNR1 gene. The human CB₁ receptor is expressed in the peripheral nervous system and central nervous system. It is activated by: endocannabinoids, a group of retrograde neurotransmitters that include anandamide and 2-arachidonoylglycerol (2-AG); plant phytocannabinoids, such as the compound THC which is an active ingredient of the psychoactive drug cannabis; and, synthetic analogs of THC. CB1 is antagonized by the phytocannabinoid tetrahydrocannabivarin (THCV).

The cannabinoid receptor type 2, abbreviated as CB₂, is a G protein-coupled receptor from the cannabinoid receptor family that in humans is encoded by the CNR2 gene. It is closely related to the cannabinoid receptor type 1, which is largely responsible for the efficacy of endocannabinoid-mediated presynaptic-inhibition, the psychoactive properties of tetrahydrocannabinol, the active agent in cannabis, and other phytocannabinoids (plant cannabinoids). The principal endogenous ligand for the CB₂ receptor is 2-arachidonoylglycerol (2-AG).

N-Arachidonoyl dopamine (NADA) is an endocannabinoid that acts as an agonist of the CB₁ receptor and the transient receptor potential V1 (TRPV1) ion channel. NADA was first described as a putative endocannabinoid (agonist for the CB₁ receptor) in 2000 and was subsequently identified as an endovanilloid (agonist for TRPV1) in 2002. NADA is an endogenous arachidonic acid based lipid found in the brain of rats, with especially high concentrations in the hippocampus, cerebellum, and striatum. It activates the TRPV1 channel with an EC₅₀ of approximately of 50nM which makes it the putative endogenous TRPV1 agonist.

Tetrahydrocannabinol-C4 chemical compound

Tetrahydrocannabinol-C4, also known as THC-C4 and butyl-THC, is a homologue of tetrahydrocannabinol (THC), the active component of cannabis. They are only different by the pentyl side chain being replaced by a butyl side chain. It is unknown whether THC-C4 is an agonist, partial agonist, or antagonist at the cannabinoid receptors. The propyl analog, THCV, is a cannabinoid receptor type 1 and cannabinoid receptor type 2 antagonist, while THC is a CB₁ agonist. THC-C4 has rarely been isolated from cannabis samples, but appears to be less commonly present than THC or THCV. It is metabolised in a similar manner to THC. Similarly to THC, it has 7 double bond isomers and 30 stereoisomers.

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AM-411 is an analgesic drug that is a cannabinoid agonist. It is a derivative of Δ8-THC substituted with an adamantyl group at the 3-position, demonstrating that the binding pocket for the alkyl chain at this position can accommodate significant bulk.

AMG-3 (part of the AM cannabinoid series) is an analgesic drug which is a cannabinoid agonist. It is a derivative of Δ8THC substituted with a dithiolane group on the 3-position side chain. AMG-3 is a potent agonist at both CB₁ and CB₂ receptors with a K_i of 0.32nM at CB₁ and 0.52nM at CB₂, and its particularly high binding affinity has led to it being used as a template for further structural development of novel cannabinoid drugs. It has sedative and analgesic effects, with analgesia lasting for up to 36 hours after administration.

GW-405,833 (L-768,242) is a drug that acts as a potent and selective partial agonist for the cannabinoid receptor subtype CB₂, with an EC₅₀ of 0.65 nM and selectivity of around 1200x for CB₂ over CB₁ receptors. Animal studies have shown it to possess antiinflammatory and anti-hyperalgesic effects at low doses, followed by ataxia and analgesic effects when the dose is increased. Selective CB₂ agonist drugs such as GW-405,833 are hoped to be particularly useful in the treatment of allodynia and neuropathic pain for which current treatment options are often inadequate.

S-14671 is a naphthylpiperazine derivative which acts as a 5-HT_1A receptor agonist (pK_i = 9.3) with high efficacy and exceptional in vivo potency, and also as a 5-HT_2A and 5-HT_2C receptor antagonist (both are pK_i = 7.8). It displays only low and non-significant affinity for 5-HT_1B and 5-HT₃ sites.

AM-2233 is a drug that acts as a highly potent full agonist for the cannabinoid receptors, with a K_i of 1.8 nM at CB₁ and 2.2 nM at CB₂ as the active (R) enantiomer. It was developed as a selective radioligand for the cannabinoid receptors and has been used as its ¹³¹I derivative for mapping the distribution of the CB₁ receptor in the brain. AM-2233 was found to fully substitute for THC in rats, with a potency lower than that of JWH-018 but higher than WIN 55,212-2.

AM-6545 is a drug which acts as a peripherally selective silent antagonist for the CB₁ receptor, and was developed for the treatment of obesity. Other cannabinoid antagonists such as rimonabant have been marketed for this application, but have subsequently been withdrawn from sale because of centrally mediated side effects such as depression and nausea. Because AM-6545 does not cross the blood–brain barrier to any significant extent, it does not produce these kinds of side effects, but has still been shown to effectively reduce appetite and food consumption in animal studies.

References

↑ Little, P.J.; Compton, D.R.; Johnson, M.R.; Melvin, L.S.; Martin, B.R. (1988). Pharmacology and stereoselectivity of structurally novel cannabinoids in mice. J Pharmacol Exp Ther 247: 1046–51.
↑ Fride, E., A. Perchuk, F. S. Hall, G. R. Uhl, and E. S. Onaivi. 2006. Behavioral methods in cannabinoid research. Pp. 269–290 in E. S. Onaivi, ed. Marijuana and Cannabinoid Research: Methods and Protocols. Humana Press, Totowa, NJ.
↑ Liu, Qing-Rong; Canseco-Alba, Ana; Zhang, Hai-Ying; Tagliaferro, Patricia; Chung, Monika; Dennis, Eugene; Sanabria, Branden; Schanz, Norman; Escosteguy-Neto, Joao Carlos (2017-12-12). "Cannabinoid type 2 receptors in dopamine neurons inhibits psychomotor behaviors, alters anxiety, depression and alcohol preference". Scientific Reports. 7. doi:10.1038/s41598-017-17796-y. ISSN 2045-2322. PMC 5727179 . PMID 29234141.

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[1] Little, P.J.; Compton, D.R.; Johnson, M.R.; Melvin, L.S.; Martin, B.R. (1988). Pharmacology and stereoselectivity of structurally novel cannabinoids in mice. J Pharmacol Exp Ther 247: 1046–51.

[2] Fride, E., A. Perchuk, F. S. Hall, G. R. Uhl, and E. S. Onaivi. 2006. Behavioral methods in cannabinoid research. Pp. 269–290 in E. S. Onaivi, ed. Marijuana and Cannabinoid Research: Methods and Protocols. Humana Press, Totowa, NJ.

[3] Liu, Qing-Rong; Canseco-Alba, Ana; Zhang, Hai-Ying; Tagliaferro, Patricia; Chung, Monika; Dennis, Eugene; Sanabria, Branden; Schanz, Norman; Escosteguy-Neto, Joao Carlos (2017-12-12). "Cannabinoid type 2 receptors in dopamine neurons inhibits psychomotor behaviors, alters anxiety, depression and alcohol preference". Scientific Reports. 7. doi:10.1038/s41598-017-17796-y. ISSN 2045-2322. PMC 5727179 . PMID 29234141.