Khat (Catha edulis F.) and cannabinoids: Parallel and contrasting behavioral effects in preclinical and clinical studies

Pharmacology, Biochemistry and Behavior 138 (2015) 164–173

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Pharmacology, Biochemistry and Behavior journal homepage: www.elsevier.com/locate/pharmbiochembeh

Review

Khat (Catha edulis F.) and cannabinoids: Parallel and contrasting behavioral effects in preclinical and clinical studies Berhanu Geresu Department of Pharmacology and Clinical Pharmacy, School of Pharmacy, College of Health Sciences, Addis Ababa University, Addis Ababa, Ethiopia

a r t i c l e

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Article history: Received 13 March 2015 Received in revised form 17 September 2015 Accepted 27 September 2015 Keywords: Khat Catha edulis Endocannabinoids Cathinone Behavioral effects Cannabis

a b s t r a c t After a brief outline of Catha edulis F. (khat) and the cannabinoid systems, the interactions between the pharmacological effects of khat and cannabinoids will be reviewed. Khat chewing is a widespread habit that has a deeprooted sociocultural tradition in Africa and the Middle East. Experimental studies conducted to investigate khat's central and peripheral effects have revealed an amphetamine-like mechanism of action mediated through the dopaminergic system. The endocannabinoid system comprises the receptors, the endogenous agonists and the related biochemical machinery responsible for synthesizing these substances and terminating their actions. Endocannabinoids are synthesized “on demand” from membrane phospholipids and then rapidly cleared by cellular uptake and enzymatic degradation. Khat and cannabinoids produce a body of parallel and contrasting behavioral effects. Concurrent consumption of khat and cannabinoids may increase the risk of getting or precipitating psychosis, has rewarding and motivational effect, increases the threshold of pain perception and impairs learning and memory. On the other hand, the action of cannabis to enhance food intake is likely to reduce khat's appetite suppressant effects. © 2015 Elsevier Inc. All rights reserved.

Contents 1. 2.

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Introduction . . . . . . . . . . . . . . . . . . . . . Khat, Catha edulis foresk . . . . . . . . . . . . . . . 2.1. Botany and epidemiology . . . . . . . . . . . . 2.2. Cultivation . . . . . . . . . . . . . . . . . . 2.3. Chemistry and constituents . . . . . . . . . . . 2.4. Pharmacokinetics of khat . . . . . . . . . . . . Pharmacology of cannabinoids . . . . . . . . . . . . . 3.1. Cannabinoid receptors . . . . . . . . . . . . . 3.2. Cannabinoid receptor signaling . . . . . . . . . 3.3. Endogenous cannabinoid receptor ligands . . . . 3.4. Biosynthesis and inactivation of endocannabinoids 3.4.1. Biosynthesis . . . . . . . . . . . . . 3.4.2. Inactivation . . . . . . . . . . . . . . Behavioral effects of khat and cannabinoids . . . . . . . 4.1. Anxiety-like behavior . . . . . . . . . . . . . 4.2. Psychosis . . . . . . . . . . . . . . . . . . . 4.3. Mood disorders . . . . . . . . . . . . . . . . 4.4. Aggressive behavior . . . . . . . . . . . . . . 4.5. Feeding behavior . . . . . . . . . . . . . . . 4.6. Reward and motivation . . . . . . . . . . . . 4.6.1. Self-administration . . . . . . . . . . 4.6.2. Discriminative stimulus properties . . . 4.6.3. Conditioned place preference . . . . . .

E-mail address: [email protected].

http://dx.doi.org/10.1016/j.pbb.2015.09.019 0091-3057/© 2015 Elsevier Inc. All rights reserved.

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4.7. Analgesia . . . . . . 4.8. Motor activity . . . . 4.9. Cognition and memory 5. Concluding remarks . . . . References . . . . . . . . . . .

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1. Introduction

2.2. Cultivation

Khat (Catha edulis F.) has been consumed for centuries by people living around the horn of Africa, East Africa and the Middle East (Connor et al., 2002; Carlini, 2003). Cathinone is the main psychoactive alkaloid of fresh khat leaves and has the same indirect sympathomimetic mechanism of action as amphetamine (AL-Hebshi and Skaug, 2005). The effects observed following khat consumption are generally of central stimulation and include euphoria, excitation, anorexia, increased respiration, hyperthermia, analgesia and increased sensory stimulation (Feyissa and Kelly, 2008). Khat has also been shown to increase locomotion and alter performance in several behavioral paradigms in rodents (Oyungu et al., 2007; Bedada and Engidawork, 2010). Cannabinoids constitute a diverse class of compounds that include Δ9-Tetrahydrocannabinol (Δ9-THC), the principal psychoactive constituent of marijuana, and synthetic compounds such as HU-210. Administration of cannabinoids in animal models produces various behavioral changes, many of which are mediated by the centrally expressed cannabinoid type 1 (CB1) receptor (Ledent et al., 1999). The CB1 receptor can be found in several areas of the brain such as the frontal cortex, basal ganglia, hippocampus, amygdala and brainstem and it has been implicated in the regulation of learning and memory as well as depression, anxiety and pain (Carvalho et al., 2010). In rodents, both Δ9-THC, WIN 55,212-2, and CP 55,940 alter specific types of behavior, including nociception, motor activity, memory, and feeding (Chaperon and Thiébot, 1999). It has been reported that cannabinoids can produce anxiolytic or anxiogenic like behavioral response in rats under the elevated plus maze experiment (Onaivi et al., 1990). Several studies have shown the existence of an interaction between the endocannabinoid system (ECS) and some drugs of abuse, such as opioids (Scavone et al., 2013), nicotine (Viverosa et al., 2006; Werling et al., 2009) and cocaine (Xi et al., 2012). In particular, CB1 receptor blockade reduces the behavioral effects of many drugs of abuse, including marijuana, morphine, ethanol, and nicotine (Balerio et al., 2006; Biala and Kruk, 2008). As far as our knowledge goes, this review is the first in its kind in the area to critically analyze the interaction of cannabinoids and khat, although the two agents have the capacity to produce pleasant sensation.

Khat is cultivated by farmers across a wide geographical area: the southern shores of the Red Sea, the Southern parts of the Arabian Peninsula, the mountains of the Yemen Arab Republic, and Eastern and Southern African regions, including the Harar Plateau of Ethiopia, the Meru districts of Kenya, the Jima district of Ethiopia, Tanzania, Uganda, Zaire and Zimbabwe (McKee, 1987).

2. Khat, Catha edulis foresk 2.1. Botany and epidemiology Khat is a name generally used for Catha edulis, a dicotyledonous evergreen shrub of the family Celastraceae. The khat tree has a slender bole and white bark. In Yemen, the trees range from 1 to 10 m in height, while in Ethiopian highlands they can reach heights of 18 m (Al-Motarreb et al., 2002; AL-Hebshi and Skaug, 2005). The use of khat has traditionally been confined to the regions where khat is grown. In recent years, however, the economic importance and consumption of khat leaves have increased dramatically which had allowed a much wider distribution in USA, UK and other European countries (Feyissa and Kelly, 2008). The habit of khat chewing is largely confined to inhabitants of the countries of Eastern Africa and South-Western Arabia. In Yemen approximately 80% of adult men in the major cities and 90% of adult men in the villages of regions in which khat is produced are regular chewers (Mela and McBride, 2009).

2.3. Chemistry and constituents The constituents of khat vary with the geographical location of the plant. Fresh khat leaves contains alkaloids of the phenylalkylamine type (basic fraction) known as khatamines, more than 14 alkaloids of the sesquiterpene polyester type (weakly basic fairly lipophilic fraction) known as cathedulins, large amount of polyphenols (including tannins and flavonoid glycosides) and volatile oil, triterpenes, sterols, amino acids, ascorbic acid and sugar alcohols (Dhaifalah and Šantavý, 2004). Cathinone is the main psychoactive alkaloid of fresh khat leaves and has the same indirect sympathomimetic mechanism of action as amphetamine. Therefore, cathinone may be called, like khat, the “natural amphetamine”. (+)-Norpseudoephedrine and (−)-norephedrine (cathines) are much less active than cathinone. Both of khat's major active ingredients–cathine and cathinone–are phenylalkylamines, meaning they are in the same class of chemicals as amphetamines. Cathinone and cathine have a very similar molecular structure to amphetamine (AL-Hebshi and Skaug, 2005). 2.4. Pharmacokinetics of khat During chewing, the alkaloids from khat leaves are effectively liberated with about 80% of cathinone and cathine, and over 90% of norephedrine. The alkaloids from blood samples were assayed using gas chromatography–mass spectrometry. The absorption of the constituents of khat is said to have two phases, the first being at the buccal mucosa, plays a major role in the absorption of alkaloids. The second phase is following swallowing of the juice, at the stomach and/or small intestine (Toennes et al., 2003). A study done on five volunteer healthy adults by Halket et al. (1995) revealed that the euphoric effects of khat start after about 1 h of chewing. Blood levels of cathinone start to rise within 1 h and peak plasma levels are obtained 1.5–3.5 h after the onset of chewing. The study also showed that maximum plasma levels ranged from 41 to 141 ng/ml (mean 83 ng/ml) after a 1 h chewing dose of 60 g fresh khat leaves per subject (cathinone: 0.8–1 mg/kg body weight). Cathinone was barely detectable at 7.5 h and not detectable any more after 24 h. A double blind placebo controlled study by Widler et al. (1994) on six drug-naive volunteers receiving a single dose of khat corresponding to 0.8 mg/kg body weight showed that maximal plasma concentrations of cathinone (127 ± 53 ng/ml) were attained after 127 ± 30 min. The terminal elimination half-life was 260 ± 102 min. Maximal plasma concentrations of norephedrine (110 ± 51 ng/ml) and norpseudoephedrine (89 ± 49 ng/ml were observed after 200 ± 134 and 183 ± 73 min, respectively. The main metabolite of cathinone was identified as (−)norephedrine. Metabolism is rapid and occurs already during first passage through the liver. Only 2% of administered cathinone was

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found unchanged in the urine. In humans, norephedrine and norpseudoephedrine are slowly absorbed and excreted almost unchanged in urine (Brenneisen et al., 1986). 3. Pharmacology of cannabinoids 3.1. Cannabinoid receptors To date two cannabinoid receptors have been identified, the CB1 and the CB2 receptor which belong to the large family of the G-proteincoupled receptors (GPCR) (Grotenhermen, 2006). CB1 receptors are expressed predominantly in the central nervous system (CNS) with particularly high levels in cerebellum, hippocampus and basal ganglia. In fact, of all known neurotransmitter and hormone receptors, the CB1 receptor is by far the most abundant in the mammalian brain. CB1 receptors are also expressed, albeit at much lower levels, in the peripheral nervous system as well as on the cells of the immune system, in the heart, vascular tissues, and the testis (Liu et al., 2000; Bonz et al., 2003). In contrast to the CB1 receptor, which is highly conserved in mice, rats, and humans, the CB2 receptor is much more divergent. 3.2. Cannabinoid receptor signaling CB1 and CB2 receptors couple primarily to the Gi/o subtypes of G protein and their signaling is remarkably complex. Although coupling to adenylate cyclase through Gi/o usually results in inhibition of cyclase activity through the release of Giα isoforms (Rhee et al., 1998). Cannabinoids can also inhibit different types of calcium channels and activate certain potassium channels via G protein βγ subunits. Cannabinoids can activate members of all three families of multifunctional mitogenactivated protein kinases, including p44/42 MAP kinase and JUN-terminal kinase and activate the phosphatidylinositol-3-kinase pathway. Furthermore, cannabinoids can regulate the activity of phosphatases, as exemplified the CB1-mediated regulation of calcineurin (protein phosphatase 2b) or the activation of mitogen-activated protein kinase phosphatase 1, which plays an important role in the anti-inflammatory action of endocannabinoids (Pacher et al., 2006). Endocannabinoid signaling appears to occur via a retrograde mechanism, where stimulation of the postsynaptic neuron triggers the biosynthesis of endocannabinoids, which are released and transported by poorly understood mechanisms to activate CB1 receptors expressed primarily on the presynaptic terminal to inhibit the release of neurotransmitters (Howlett, 2005). Inhibition of the presynaptic release of GABA from hippocampal neurons, for example, can result in depolarization-induced suppression of inhibition (DSI) or of glutamate from cerebellar climbing fibers that originate in the inferior olive or from parallel fibers of cerebellar granule cells results in depolarization-induced suppression of excitation (DSE). It is noteworthy that while DSE should provide a negative feedback mechanism for damping down high synaptic activity, DSI is expected to exacerbate intense synaptic activity (Pertwee and Ross, 2002). 3.3. Endogenous cannabinoid receptor ligands In the early 1990s, anandamide (AEA) and 2-arachidonoylglycerol (2-AG) were discovered and characterized as the first endogenous ligands for CB receptors. Subsequently, other possible endocannabinoids have been proposed, such as noladin ether, virodhamine and arachidonoyldopamine, but their natural occurrence and their roles are still unclear (Solinas et al., 2006). 3.4. Biosynthesis and inactivation of endocannabinoids 3.4.1. Biosynthesis It is generally accepted that AEA is generated by calcium dependent enzymatic transfer of arachidonic acid from the sn-1 position of

membrane phospholipids to the primary amine of phosphatidylethanolamine (PE) to form N-arachidonoyl phosphatidylethanolamine (NArPE), followed by hydrolysis to give AEA (Solinas et al., 2006; Liu et al., 2008). 2-AG is synthesized from arachidonoyl-containing diacylglycerol (DAG) species by sn-1-specific diacylglycerol lipase-α and -β (DAGLα and DAGLβ). Characterization of DAGL (−/−) mice confirmed a primary role for DAGLα in 2-AG formation in the brain and DAGLβ in peripheral tissues such as the liver. DAG precursors are themselves synthesized from membrane phospholipids with most evidence suggesting that the major 2-AG biosynthetic pathway is hydrolysis of sn-2 arachidonoyl phosphatidylinositol 4,5-bisphosphate (PIP2) species by PLCβ (Hashimotodani et al., 2005). 3.4.2. Inactivation After cellular uptake, AEA and 2-AG are subject to metabolism by the fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. In addition, AEA and 2-AG have been shown to undergo oxidation by cyclooxygenase-2 (COX-2) and the 12- and 15lipoxygenases (12-LOX and 15-LOX). Catalytic modification of AEA and 2-AG not only serves as a mechanism for the augmentation of cellular uptake and cessation of extracellular signaling but also regulates the intracellular signaling events of these two endocannabinoids (Dinh et al., 2002; Di Marzo, 2006). 4. Behavioral effects of khat and cannabinoids Khat and cannabis, which share some biological actions, are used frequently in combination, particularly among adolescents and young adults. Different elegant preclinical (Table 1) and clinical (Table 2) studies showed that the two drugs have parallel and contrasting behavioral effects, reviewed below. 4.1. Anxiety-like behavior The overall effect of khat on anxiety behavior is difficult to predict, it depends on the test paradigm, dose and animal species. The open field simultaneously provides measures of locomotion, exploration and anxiety. In the test, increased number of centre square frequencies (horizontal locomotion) and duration are indicative of low anxiety because the animals have exploratory behavior. On the other hand, increased line crossings and rearing frequencies (vertical locomotion) are reflective of increased locomotion, exploration and/or a lower level of anxiety since the animals have a thigmotaxic rather than exploratory behavior (Podhorna and Brown, 2002; Prut and Belzung, 2003). In a study done to enumerate the effect of single and daily khat extract on anxiety behavior in mice, khat extract had mixed effects on

Table 1 Summary of the parallel and contrasting effects of khat and cannabinoids in pre-clinical studies. Anxiety like behavior Khat Enhanced central locomotion and reduced anxiety Kimani and Nyongesa (2008) Increased thigmotactic movement and enhanced Kimani and Nyongesa (2008) anxiety Cannabinoid agonists Low doses usually induce an anxiolytic-like effect Berrendero and Maldonado (2002) High doses reduce anxiety Marco et al. (2004) Analgesia Khat Khat extract showed analgesic effect at higher Connor et al. (2002) dose Cannabinoid agonists WIN-55,212-2, HU-210 and JWH-133 reduced Guerrero et al. (2008); pain in rat models of neuralgia Potenzieri et al. (2008)

B. Geresu / Pharmacology, Biochemistry and Behavior 138 (2015) 164–173 Table 2 Summary of the parallel and contrasting effects of khat and cannabinoids in clinical studies. Psychosis Khat Khat consumption precipitates psychosis. Cannabinoid agonists Cannabis use is higher in schizophrenic patients. Depression Khat Khat chewing is associated with depression. Cannabinoid agonists Cannabis abuse may increase the risk of developing depressive symptoms. Aggression Khat Causes violent behavior Cannabinoid agonists Increase aggressive behavior Reduce aggression Feeding behavior Khat Suppress appetite Cannabinoid agonists Stimulate eating

Cox and Rampes, 2003; Odenwald et al. (2007) Bersani et al. (2002)

Hassan et al. (2002); Nielen et al. (2004) Degenhart (2003)

Berardelli et al. (1980); Cox and Rampes (2003); Odenwald et al. (2007) Myerscough and Taylor (1985) Cherek and Dougherty (1995)

Kalix (1983) Mattes et al. (1994)

the levels of anxiety depending on the parameter looked at. Based on line crossing the results are indicative of reduction of anxiety in mice in a dose-dependent manner. On the other hand, the result of the effect of khat extract on rearing and centre square frequencies is indicative of enhancement of anxiety levels, with high doses increasing it while low doses reducing the anxiety (Kimani and Nyongesa, 2008). Cannabinoids are able to display both anxiogenic- and anxiolyticlike effects depending upon doses, animal models, specific test conditions, and strains (Onaivi et al., 1990; Rey et al., 2012). Overall, low doses of cannabinoid agonists usually induce an anxiolytic-like effect (Berrendero and Maldonado, 2002; Marco et al., 2004), whereas higher doses cause the opposite response. More recently, Marinho et al. (2015) showed that the highest dose of rimonabant, a CB1 antagonist, abolished ethanol- and cocaine-induced hyperlocomotion and behavioral sensitization without modifying spontaneous and central locomotor activity in the open field apparatus in mice. As in animal models, in humans contradictory findings have been reported regarding effect of cannabinoid agonists in anxiety disorders. A double-blind study on eight volunteer subjects revealed a large increase in the level of anxiety in healthy volunteers after ingestion of Δ9-THC which was blocked by cannabidiol (Zuardi et al., 1982). However, another double-blind placebo-controlled study showed that chronic treatment with nabilone, a synthetic cannabinoid, decreased anxiety (Fabre and McLendon, 1981). Consideration should be given to subjective behavior, number of volunteer subjects and the actual experiment environment which may alter the effect of cannabinoids on anxiety level. Some results suggest that the endocannabinoid system is involved in the control of emotional behavior via CB1 receptors. Neuroanatomical studies showed that this receptor is expressed at high levels in brain regions involved in the control of fear and anxiety, such as the basolateral amygdala, the anterior cingulated cortex, the prefrontal cortex, and the paraventicular nucleus (PVN) of the hypothalamus (Tsou et al., 1998). Previous studies implicated the role of the hypothalamic–pituitary–adrenal (HPA) axis, the neuroendocrine system in responses to cannabinoid induced emotional stress (Wenger et al., 1997). However, Di et al. (2003) pointed out a mechanism of rapid glucocorticoid feedback inhibition of the HPA involving a release of endocannabinoid and the activation of CB1 receptors in the PVN.

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4.2. Psychosis There have been growing reports of khat-induced psychosis in khat producing areas and in immigrants residing in Europe and United States of America. The characteristics of psychosis following the use of khat were mainly of two types: manic psychosis and paranoid or schizophrenia spectrum disorder. It is postulated that khat consumption precipitates psychosis by either increasing the risk in already vulnerable individuals or affecting the course of a psychotic disorder and the maintenance of symptoms (Odenwald et al., 2007). In schizophrenic psychosis the patients typically present with paranoid delusions, fear, a hostile perception of the environment, auditory hallucinations, ideas of reference, thought alienation and a tendency to isolate themselves, or alternatively displaying aggressive behavior towards others. In manic psychosis the patient presented with hyperactivity, shouting, pressure of speech, grandiose delusions with flight of ideas and tangential thought processes and a labile mood varying from euphoria to anger (Cox and Rampes, 2003). Locomotor sensitization paradigm has been widely studied as animal models of psychosis, after intermittent oral administration of (−) cathinone or C. edulis extract in rats. Results from previous studies suggest that the psychotic effects of C. edulis or cathinone are mediated, at least in part, by dopamine in the meso–striato–cortico limbic pathway. This pathway is believed to play a central role in the induction, maintenance and expression of sensitization following repeated administration of psychostimulants (Banjaw and Schmidt, 2005). Several elegant clinical findings suggest that schizophrenia may be associated with functional anomalies in the endocannabinoid signaling system. A retrospective study showed that the prevalence of regular or problematic cannabis use is higher in schizophrenic patients than in the general population (Bersani et al., 2002). Some methodological limitations should also be considered including the following problems in defining the real onset of psychosis and substance abuse; information gathered from and based on self-reports of subjects whose reliability is variable; lack of follow-up, and difficulty in using a population of cannabis-only consumers and normal control subjects to be studied prospectively in comparison with schizophrenic patients. Similarities between some effects of cannabinoid intoxication and some symptoms of schizophrenia, especially regarding cognitive disturbances, hallucinations, perceptual distortion, and paranoia, have been shown in a longitudinal cohort report (Fergusson et al., 2003). Finally heavy abuse of cannabis can be considered as a factor eliciting relapse in patients with schizophrenia and possibly a premorbid precipitant (Linszen et al., 1997). An in vivo microdialysis study showed that activation of dopamine D2 -like receptors by quinpirole increased AEA release in rat dorsal striatum. Thus, the high levels of AEA found in schizophrenics' cerebrospinal fluid (CSF) might result from an overstimulation of D2like receptors, due to the activation of dopamine neurotransmission in these patients. Alternatively, increased CSF AEA levels may reflect a primary “hypercannabinergic” state, which may occur in schizophrenic patients. In rats, autoradiography and in situ hybridization studies showed that CB1 receptors are highly expressed in basal ganglia, limbic structures (hippocampus, olfactory bulbs, and septum), and cerebellum (Mailleux and Vanderhaeghen, 1992). In mice, a study demonstrated that systemic administration of the synthetic cannabinoid agonists CP55940 and WIN55212-2 induced Fos expression within A10 DA neurons. This effect, probably mediated by CB1 receptors because it was prevented by rimonabant, also appeared to be dependent upon an activation of noradrenergic neurotransmission (Patel and Hillard, 2003). These results are consistent with earlier works showing that cannabinoids increased the firing rate of dopamine neurons in the ventral tegmental area (VTA) and the substantia nigra (SN) and enhanced dopamine release in the medial prefrontal cortex (mPFC) and the nucleus accumbens (NAcc) (Chen et al., 1990).

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4.3. Mood disorders Khat chewing can induce a substantial degree of mood disturbances, particularly depression in healthy subjects. Depression associated with khat chewing has been reported by several authors. In most of the reports it is seen as a consequence of cessation of khat chewing (reactive depressive mood). A case report showed that, the severity of depression varied from agitation and sleep disturbances to severe depression with suicidality (Nielen et al., 2004). Hassan et al. (2002) longitudinally studied the effect of khat chewing in human mood using the HAD (Hospital Anxiety and Depression) Scale. They reported that khat chewing results in a functional mood disorder consisting of predominantly reactive depressive mood (seen an hour after acute khat administration), and it might exacerbate symptoms in patients with pre-existing mood disorder. It is thought to be mediated by the sympathomimetic action of cathinone (Hassan et al., 2002). Other mood disorders such as khat-induced behavioral syndrome described as hypomania have also been reported by several authors (Nencini et al., 1984). In humans, the existence of a causal association between cannabis use and depression remains controversial. An epidemiological study of a possible causal role of marijuana use in the development of major depressive episode showed that, the risk of first major depressive episode was moderately associated with the number of occasions of marijuana use and with more advanced stages of marijuana use (Chen et al., 2002). Some longitudinal studies in adults reported that cannabis abuse may increase the risk of developing depressive symptoms (Degenhart, 2003). Animal studies also provided contradictory results. In the mouse forced-swimming or tail-suspension tests, two procedures predictive of an antidepressant activity (Stéru et al., 1987), rimonabant and AM251, a CB1 receptor antagonist/inverse agonist, induced an antidepressant like reduction of the time spent immobile, at doses that did not affect locomotor activity (Shearman et al., 2003). Consistent with the CB1 receptors' role in such effects, the AM251-induced reduction of immobility did not occur in CB1 knockout mice subjected to the forced-swimming test (Shearman et al., 2003). Together, these results suggest that an endogenous cannabinoid tone may contribute to the maintenance of mood, probably through a modulation of monoaminergic pathways.

male mice exhibited aggressive reactions toward a nonaggressive intruder following a 7-day chronic treatment with AEA at a very small dose (0.01 mg/kg/day). A larger dose (1 mg/kg/day) produced no noticeable effect, whereas in similar conditions, AEA (10 mg/kg/day) reduced agonistic behavior and enhanced defensive conducts in otherwise spontaneously aggressive mice, an effect perhaps linked to motor deficits induced by such a high dose. In human subjects, Myerscough and Taylor (1985) showed that a low dose (0.1 mg/kg) of oral Δ9-THC tended to increase aggressive responses. In contrast, subjects receiving a larger dose (0.4 mg/kg) behaved in a relatively nonaggressive manner throughout the reaction time task experimental session. On the other hand, using the point-subtraction aggression paradigm, Cherek and Dougherty (1995) found that smoked marijuana reduced the enhanced rate of aggressive responding induced by a shift from low to high level of provocation. A link between central 5-HT levels and aggressive behavior has been established in humans and animals (Chiavegatto and Nelson, 2003). High aggression in humans is correlated with low CSF concentrations of the 5-HT metabolite 5-hydroxyindoleacetic acid (5-HIAA), suggesting a diminution of 5-HT turnover. In aggressive laboratory animals, brain 5-HT turnover is also reduced, and pharmacological manipulations of the serotoninergic system substantiate a negative correlation between 5-HT neurotransmission and aggressive behavior. Accordingly, mice lacking the 5-HT transporter (5-HTT knockout) were found to be less aggressive than wild-type controls (Holmes et al., 2002). Interestingly, the CB receptor agonist CP55940 inhibited the production of nitric oxide (NO), an effect probably mediated through CB1 receptors, because it was reversed by rimonabant. NO modulates many behavioral and neuroendocrine responses, and its synthetic enzyme NO synthase has been found in high densities within emotion-regulating brain regions (Nelson et al., 1997). The excessive aggressive and impulsive traits of neuronal NO synthase knockout mice (nNOS −/−) have been attributed to reductions in 5-HT turnover (Chiavegatto and Nelson, 2003). This suggests that possible modifications of the endogenous cannabinoid system would contribute, via NO-related mechanisms, to local changes in 5-HT levels leading to aggressive behavior. 4.5. Feeding behavior

4.4. Aggressive behavior The use of khat is associated with an enhanced aggressive behavior both in human subjects and in animal studies. A review by Cox and Rampes (2003) revealed that, there are traditional claims that prolonged abuse of khat may influence the behavioral characteristics of individuals and lead to heinous violence. In a community based study in Somalia, there was evidence of the presence of disruptive and violent behavior among chronic khat users (Odenwald et al., 2007). In animal studies, Berardelli et al. (1980) observed a spontaneous burst of aggressive behavior in rats after intraperitoneal administration of cathinone, similar to that seen with amphetamines. Banjaw and Schmidt (2006) have reproduced this phenomenon using isolation induced aggression paradigm, in which repeated oral administration of C. edulis or (−) cathinone enhanced aggressive behavior of isolated rats. Similar to amphetamine, neurochemical correlates revealed depletion of serotonin and its corresponding metabolites in both anterior and posterior striatum, which suggest that aggression in this paradigm is enhanced presumably by decreasing the level of serotonin and its metabolites (Banjaw and Schmidt, 2006). In normal animals, it seems that acute administration of cannabis or Δ9-THC might reduce aggressive behavior, probably due to a suppressant effect on locomotor activity and a depression of general motivation (Frischknecht, 1984). Miczek (1978) showed that when a resident animal (mouse, rat, or squirrel monkey) is confronted by an intruder Δ9THC (0.25 to 2 mg/kg) decreased species specific attack behavior. A study by Sulcova et al. (1998) demonstrated that singly housed timid

Anorexia, a characteristic effect of amphetamine-like substances, is a consequence of khat chewing. This feature of khat has been used for centuries to alleviate the sensation of hunger (Kalix, 1983). Therapeutically, the khat alkaloid cathine (norpseudoephedrine) and norephedrine have been widely used as appetite suppressants in the modern world. Marked loss of appetite after khat chewing may be attributed to a combination of a central amphetamine-like effect and delay in gastric emptying (Hassan et al., 2002). Both isomers of cathinone and cathine markedly inhibited the food intake of rats at intracerebroventricular doses of 300 and 500 μg per animal respectively. Systemic acute as well as chronic administration of the two alkaloids in rats showed similar effects, however they were reported to be less potent than (+)-amphetamine. It was reported that within a week there was development of tolerance to this effect of cathinone, and the weight reducing effect disappeared within 3–4 weeks. Two models have been proposed to explain the reduction in food intake of psychostimulants like cathinone. According to some who advocate the Pavlovian homeostatic model, the suppression of intake is due to loss of appetite, which results in a failure to seek food or to eat it. On the other hand, enhanced locomotion and/ or stereotyped response interfere with locating, approaching, and orienting to food (Wolgin and Munoz, 2006). There is increasing evidence of endocannabinoids' role in the regulation of appetite. Exogenous cannabinoids (smoked marijuana, Δ9-THC, dronabinol) have been shown to stimulate eating in humans dose and route of administration dependently as shown by a four with-in subject

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design studies (Mattes et al., 1994). In rodents, exogenous and endogenous cannabinoids also promote overeating, although some discrepancies exist in this respect (Giuliani et al., 2000). The effects of cannabinoid agonists on feeding behavior are easier to observe when spontaneous eating is low, i.e., in animals fed ad libitum or when they are provided highly palatable food. For example, in free-feeding Lewis rats, given the choice between chow and highly palatable food, Δ9THC al low dose induced a short-lasting (30 to 60 min), binge-like pattern of palatable food eating. However, higher doses of Δ9-THC were inactive or even induced hypophagia. Acute systemic injections of AEA or WIN55212-2, enhanced food intake in presatiated rats (but not in 24-h food-deprived animals) and, curiously, were inactive on central administration (Koch and Matthews, 2001). A possible reciprocal link may exist between endocannabinoid mechanisms and leptin, an appetite-suppressing peptide hormone. Leptin, released from adipocytes, is believed to signal the nutritional status to brain areas controlling appetite. It directly stimulates the action of anorexigenic agents and suppresses the effects of orexigenic peptides, thereby decreasing appetite. The administration of leptin, in the range of doses active to reduce food intake and body weight in non-obese animals fed ad libitum, lowered the hypothalamic levels of 2-AG and AEA in both normal rats and obese mice (Halaas et al., 1997), suggesting that endocannabinoids may be under the negative control of this hormone. Emerging findings suggest the existence of heterodimers between CB1 cannabinoid receptor and hypocretin receptor-1, which have common physiological functions in the regulation of appetite (Flores et al., 2013). 4.6. Reward and motivation 4.6.1. Self-administration Khat chewing has been described as ‘pleasurable’ and the behavior of repetitive chewing of khat leaves has been labeled as from of ‘psychic dependence’, characterized by compulsive khat consumption. Self-administration studies using (−) cathinone have illustrated the role of this alkaloid as a dependence producing compound among the khat alkaloids enhancing the behavior of animals that gives them access to the substance (Kalix, 1983). Johanson and Schuster (1981) have reported that intravenous infusions of cathinone will maintain responding in rhesus monkeys, which had been previously trained to lever press for cocaine injections. When monkeys were given the choice of self-administering cathinone and cocaine, they chose both equally often (Johanson and Schuster, 1981). Similar results were obtained in rhesus monkeys, which were first trained to self-administer cocaine intravenously by lever presses, after which progressive ratio tests were conducted (Yanagita, 1986). Progressive ratio tests, which utilize the breaking point generated by increasing the fixed-ratio requirement, are important measures of motivation to take the drug and to compare the reinforcement magnitude of several psychostimulants (Willner, 1997). The final ratios obtained for (−) cathinone were similar with amphetamine, and roughly half of those of cocaine in monkeys (Yanagita, 1986). In addition to the aforementioned studies on primates, Gosnell et al. (1996) have demonstrated intravenous self-administration of cathinone in rats under a continuous reinforcement schedule. They also reported that pre-treatment with D1-type receptor antagonist SCH 23390, increased the number of infusions, which suggests a role for D1 type dopamine receptors in mediating its reinforcing effects. Significant intravenous self-administration of WIN55,212-2 was reported in food restricted Long Evans rats by Fattore et al. (2001). Selfadministration of WIN55, 212-2 was dose-dependent (with peak responding at 12.5 μg/kg/injection) and behavior extinguished (although very slowly) when vehicle was substituted for WIN55,212-2. Vehicle-like responding was also found when the cannabinoid CB1 receptor antagonist rimonabant was administered before the session. Soria et al. (2005) studied the effect of CB1 receptors on cocaine self-administration in mice. They reported that both the breaking points

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achieved and, consequently, the cocaine intake in progressive ratio schedule were lower in CB1 knockout mice in comparison with wildtype animals. Accordingly, the cocaine dose–response curve was flattened in the knockout group. This study clearly demonstrated the role of the cannabinoid system in drug self-administration. Tanda et al. (2000) were the first to report robust and persistent selfadministration of THC in nonhuman primates. Self-administration of THC was maintained at rates as high as those of cocaine in the same experimental conditions, was dose dependent, extinguished rapidly when vehicle was substituted for THC or when rimonabant was administered before the session, and immediately returned to normal levels when THC was made available again. Whereas the monkeys in this study had a history of cocaine self-administration behavior that had been extinguished for several weeks before the study began. Like THC, the endogenous cannabinoid AEA and its synthetic analog methanandamide maintain high rates of self-administration behavior when they are intravenously administered. Self-administration of AEA and methanandamide was dose dependent (with both compounds being about 10 times less potent than THC), and was sensitive to vehicle extinction and to pharmacological blockade of CB1 receptors by rimonabant (Justinova et al., 2005). 4.6.2. Discriminative stimulus properties The discriminative stimulus properties of drugs in animals are considered to be predictive of their subjective effects in humans. Animals trained to detect cathinone react as if they had received cathinone when injected with amphetamine and cocaine but not when injected with opioids, benzodiazepines or fenfluramine (Goudie et al., 1986). Similarly, in rats trained to discriminate the interoceptive cues produced by (−) cathinone, the administration of (+) cathinone and (+) cathine produced (−) cathinone like responding. In an experiment to discriminate intraperitoneal administration of cathinone using a foodmotivated, two-lever discrimination procedure Schechter et al. (1992) reported that, direct microinjection of cathinone into the NAcc is sufficient to produce discriminative stimuli. The drug discrimination procedure is used to not only test the similarity and dissimilarity of the mechanism of action of psychoactive drugs, but it can also be used to investigate the production of tolerance after chronic treatment of trained rats. Indeed, it was reported that tolerance tends to develop to cathinone in their ability to control discriminative behavior, indicated by deficits in discriminative performance and shift of the dose response curve to the right (Schechter et al., 1992). Rats or monkeys readily learn to discriminate even relatively low doses of THC from vehicle, although the development of stable discrimination performance usually requires 30 sessions or more. While discriminative stimulus effects of drugs are not a direct measure of reward, it is clear that discriminative effects of drugs play an important role in the initiation and maintenance of drug-taking behavior (Solinas et al., 2006). Importantly, the range of effects measured by drug discrimination are wider than those of direct measures of reward and reinforcement and can include aversive, anxiogenic or anxiolytic effects of cannabinoids. From a practical point of view, discriminative stimulus effects of cannabinoid CB1 receptor agonists are very strong and they provide a behavioral baseline that remains stable over long periods of time (Colpaert, 1999). The discriminative effects of cannabinoids are also pharmacologically selective so that, generally, only cannabinoid CB1 receptor agonists produce discriminative effects similar to those of THC and only CB1 receptor antagonists block them. Several studies have investigated whether endogenous cannabinoid ligands produce THC-like discriminative effects when systemically administered and found that AEA either does not produce THC-like discriminative effects or it does so only at very high doses that dramatically depress rates of responding. Since, under the same or similar conditions, metabolically stable synthetic analogs of AEA such as methanandamide, O-1812 and AM-1346, produce complete generalization to a THC training stimulus, it is likely

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that anandamide's fast metabolic inactivation is responsible for the weak effects observed (Jarbe et al., 2001). 4.6.3. Conditioned place preference Conditioned place preference is a method of assessing the rewarding and motivational effects of drugs of abuse. This behavioral task, which involves the pairing of drug cues with a distinctive environment, has been shown to produce a dose response location preference with intraperitoneal cathinone, similar to cocaine and amphetamine in rats. Furthermore, intracerebroventricular injection of cathinone to rats showed that the lowest dose (0.2 mg/kg) produced neither a conditioned place preference nor a significant increase in activity, whereas the highest (1.6 mg/kg) dose produced both. The report also mentioned that conditioned place preference by cathinone is mediated by dopaminergic neurons and that these same dopaminergic mechanisms subserve increased activation following psychostimulant administration (Schechter and Meehan, 1993). It is generally believed that cathinoneinduced conditioned place preference is mediated by dopaminergic neurons. This contention is supported by evidence that pre-treatment with a dopamine release inhibitor attenuates place preference induced by cathinone (Kalix, 1990). Also, both positive (rewarding) and negative (aversive) effects of drugs can be evaluated within the same experiment, which can be particularly interesting with cannabinoid agonists, where quite contrasting findings ranging from positive place preferences to no effect to place aversions can be found. Lepore et al. (1995) were among the first to evaluate the effects of THC on place conditioning. In their experiments with Long-Evans rats, THC-induced conditioned place preferences were observed but they depended not only on the dose of THC but also on the regimen of THC administration. With place-conditioning procedures, conditioning sessions with drug and vehicle alternate. Thus, when conditioning sessions were performed every day and THC was given every second day, conditioned place preferences developed at THC doses of 2 and 4 mg/kg with no effect at 1 mg/kg, whereas, when conditioning sessions were performed every second day and THC was given every fourth day, preferences only developed at the 1 mg/kg dose of THC and higher THC doses produced conditioned place aversions. Subsequently, a few studies have shown cannabinoid-induced conditioned place preferences in rats and all of them used a standard every day conditioning procedure. One study using the potent synthetic CB1 receptor agonist CP 55, 940 and Wistar rats found conditioned place preferences at a dose of 20 μg/kg but there were no effects at lower or higher doses (Braida et al., 2001); another study using Sprague-Dawley rats and THC found conditioned place preferences at a dose of 0.1 mg/kg but no effect at lower or higher doses (Le Foll et al., 2006); a third study using Wistar rats and WIN55,212-2 found conditioned place preferences at a dose of 1 mg/kg in rats housed in enriched conditions but not in rats housed in standard conditions (Bortolato et al., 2006). 4.7. Analgesia Khat leaves and its constituents have been shown to have analgesic properties in animal experiments. This property is shared by amphetamine. Khat extract was shown to exert analgesic effects in mice, albeit at high doses relative to ibuprofen and amphetamine. It produced analgesic effects in the tail flick test and hot plate test at a lower dose and in acetic acid-induced abdominal constriction assays at a higher dose (Connor et al., 2002). Cathinone has also been shown to cause a long lasting analgesic activity in mice and rats. This was reversibly antagonized by naloxone, a pure opioid antagonist, and by the noradrenaline synthesis inhibitors, α-methyl-p-thyrosine (α-MPT) and diethyldithiocarbamate. Furthermore cathine has been shown to enhance the analgesic effect of morphine in hot plate and formalin test in mice (Feyissa and Kelly, 2008). Cathinone shares analgesic properties with other psychostimulant

substances, like amphetamine and cocaine. It reduces the motor reaction aroused by painful thermal or irritative stimuli, hence showing an inhibitory effect on the pain perception. Cathinone's analgesic effects are antagonized by either monoaminergic depletion or adrenergic receptors blockade or by the opiate antagonist naloxone (Giannini et al., 1992). Cannabinoids can be used for the treatment of neuropathic pain, inflammatory and cancer pain. Cannabinoids have been studied in various types of neuropathic pain. Systemic administration of both WIN55,212-2 and HU-210 suppressed mechanical allodynia and thermal hyperalgesia in a rat model of trigeminal neuralgia. WIN-55,212-2 also provided antinociception in a model of sciatic nerve injury, with enhanced action if administered pre-emptively. Intrathecal JWH-133, a CB2 agonist, also significantly improved mechanical allodynia after sciatic nerve injury (Guerrero et al., 2008; Potenzieri et al., 2008). Both CB1 and CB2 receptors are involved in the mediation of inflammatory pain. WIN-55,212-2 has been shown to attenuate the delayed phase of oro-facial pain induced by formalin injection in rats. Systemic HU-308, a novel CB2 agonist also attenuated inflammatory pain during hot plate test in mice. Both endogenous and exogenous cannabinoids are being investigated for a role in cancer pain management. Cannabinoids have been found effective in increasing the threshold at which pain is perceived in tumor-afflicted mice. Mechanical hyperalgesia in a murine model of bone cancer pain is associated with decreased AEA levels in the affected area and was alleviated by local injection of AEA. Hyperalgesia in this model was tested by measuring the paw withdrawal frequency in mice injected with fibrosarcoma cells into the calcaneum. The cannabinoid agonist WIN-55,212-2 has also been shown to attenuate tumor induced hyeralgesia in mice, through peripheral action on CB1 and CB2 receptors, rather than by central action (Guerrero et al., 2008; Potenzieri et al., 2008).

4.8. Motor activity The stimulatory effect of khat is perceived as an increase in alertness and energy and relief from fatigue. Indeed, these effects have been reproduced in rats after oral administration of different concentrations of khat extract in which higher doses of the plant increased motor activity (Hassan et al., 2007). Once cathinone was identified as an active constituent of khat, there have been investigations of its effect on animal behavior, particularly on locomotor activity. Subcutaneous administration of cathinone in rats markedly increased spontaneous locomotor activity of the animals. It was reported that the potency of cathinone was almost comparable with (+)-amphetamine (Banjaw et al., 2003; Kimani and Nyongesa, 2008). The occurrence of strong behavioral sensitization after repeated intermittent oral administration of C. edulis leaves or cathinone in rats was demonstrated recently. The rats developed sensitization for locomotor activity, rearing, upward and downward sniffing, and turning after oral administration of the extract which was also observed with cathinone and amphetamine (Connor et al., 2002; Banjaw and Schmidt, 2005). Recently Geresu and Engidawork (2010) reported that, acute and sub-acute administration of khat at 200 and 300 mg/kg dose as well as amphetamine (50 mg/kg) produced a consistent improvement in motor performance in mice. The effect of khat on motor activity is modulated by the neurotransmitters dopamine and serotonin. Cathinone, the active compound of khat is associated directly and/or indirectly with dopamine or serotonin release, by its action on dopamine or serotonin transporter function (Banjaw et al., 2003). In addition, cathinone is regarded as a dopamine releaser and acts through D1, type dopamine receptors in mediating its reinforcing effects (Kalix, 1992). It has also been documented that cathinone and its close analog amphetamine, increase the efflux of [3H] dopamine from slices of rat striatum. Further, it has been demonstrated that the motor activities induced by S – (−) cathinone in

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experimental animals are associated with dopamine release (Calcagnetti and Schecter, 1992). It is now well accepted that the control of movement is one of the more relevant physiological role of the endocannabinoids in the brain. Synthetic, plant-derived and endogenous cannabinoids have powerful actions on motor activity in animals. In fact, these effects are bidirectional, depending on the dose. Large doses of cannabinoid reduced motor activity in a variety of behavioral tests and even produced strong catalepsy, whereas low doses stimulated motor activity as indicated by hyperlocomotion in intact animals, and ipsilateral circling in rats with unilateral 6-hydroxydopamine (6-OH-DA) lesion of the substantia nigra (Chaperon and Thiébot, 1999; Fernández-Ruiz et al., 2002). Likewise, low doses (0.01 mg/kg) of AEA enhanced, and moderate or high doses (10–100 mg/kg) reduced motor activity in rodents. However, although the overall pharmacological activity of endocannabinoids is similar to that of exogenous cannabinoids, there are also differences, and it is clear that AEA has partial effects for some behavioral components (Sulcova et al., 1998). Moreover, when different routes of AEA administration were compared, a complex pattern of full and partial agonist activities was observed. Additional behavioral differences include the inhibition by very low doses (0.0001–0.01 mg/kg) of AEA and docosahexaenyl ethanolamide, a synthetic endocannabinoid-like compound, but not Δ9-THC, of the pharmacological effects of conventional doses of Δ9-THC. There also evidence that, rimonabant prevented the motor effects of CB receptor agonists (Chaperon and Thiébot, 1999). 4.9. Cognition and memory Kimani and Nyongesa (2008) reported that khat extract has differential effects on learning and memory task in mice depending on dose. Moderate and high doses (120 and 360 mg/kg body weight) of khat extract significantly impaired while low dose (40 mg/kg body weight) of khat extract did not have a significant effect on CBA mice acquisition learning. The high dose of khat extract significantly improved while moderate and low doses impaired accuracy for spatial memory of the platform location in the Morris water maze experiment. This study has shown that khat extract has selective effect on spatial learning and memory, with low dose having no effect on learning but impairing memory, whereas high dose impairs learning but improves memory. Administration of both synthetic and phytocannabinoids, including Δ9-THC, WIN 55,212-2, and CP 55,940, impair working memory and short-term memory through a CB1 receptor-mediated mechanism in rats (Braida and Sala, 2000; Egashira et al., 2002). The cellular and molecular mechanisms underlying learning and memory deficits produced by cannabinoids, and the role of endocannabinoids in such mechanisms, have been investigated in a number of studies using in vivo and in vitro systems. In vitro experiments indicated that cannabinoids and endocannabinoids produce persistent changes in memory-related neuronal activity. The dense localization of CB1 receptors in the hippocampus and amygdala, which play an important role in learning and memory, may represent the anatomical substrate for cannabinoids to influence mnemonic processes (Gerdeman and Lovinger, 2003). Δ9-THC impairs memory in rodents and monkeys tested in a variety of experimental procedures (radial maze, instrumental discrimination tasks, Morris water maze). The effects exerted by CB receptor agonists, including Δ9-THC, WIN-55,212-2, CP 55,940, and AEA, are reversed by rimonabant, providing evidence for the involvement of CB1-related mechanisms. Although endocannabinoids mimic the pharmacological effects of cannabinoids, experiments carried out by the latter group have shown that AEA impairs memory consolidation in random bred mice, and exerts genotype-dependent influences on memory in inbred strains of mice). The mechanism by which cannabinoids and endocannabinoids influence learning and memory may be by directly acting on the cannabinoid CB1 receptors in the hippocampus or through the modulation of the release of other neurotransmitters, such as glutamate and acetylcholine (Castellano et al., 2003).

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5. Concluding remarks From the previous and ongoing evidence, the joint consumption of khat and cannabinoids may produce a body of parallel and contrasting behavioral effects in human beings as well as in animals. Most of the behavioral effects depend on different factors like dose of the compound, animal species under investigation, test paradigm and the specific drug used. Concurrent consumption of khat and cannabinoids may increase the risk of getting or precipitating psychosis, has rewarding and motivational effect, increases the threshold of pain perception and impairs learning and memory. The combined use of these compounds should be discouraged as the rewarding and motivational effects of the two compounds can increase drug seeking behavior and may escalate the risk of psychosis. On the other hand, the joint use of khat and cannabinoids reduce pain perception and hence the possible role this combination should be critically evaluated in the management of pain. The action of cannabis to enhance food intake is likely to reduce khat's appetite suppressant effects and this would certainly be considered a disadvantage by those who value chewing khat as an aid to weight control. Research in the last decade has considerably increased our knowledge of the complexities and peculiarities of the endocannabinoid system. The ability of this system to interact with multiple other systems provides limitless signaling capabilities of cross talk between receptors. This opens the arena for the search of drug targets that can be modulated, by administration of exogenous compounds, for possible therapeutic use. This review necessitates further research in animals as well as human subjects to be done to clarify the interactions between khat and cannabinoids as well as to enumerate the underlying mechanisms.

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