Preclinical Assessment of the Abuse Potential of Purified Botanical Cannabidiol: Self-Administration, Drug Discrimination, and Physical Dependence.

Cannabidiol (CBD) is a constituent of the cannabis plant with a diverse array of pharmacological activities as well as potential therapeutic uses. An oral formulation of CBD (Epidiolex in the US; Epidyolex in Europe) is approved for treating seizures associated with rare and severe forms of epilepsy. These studies, which supported the approval of the medication, investigated abuse-related effects of CBD in rats and nonhuman primates (NHPs) using drug self-administration, drug discrimination, and physical dependence procedures and characterized its pharmacokinetics. In NHPs (n = 5) that self-administered midazolam (0.01 or 0.032 mg/kg/infusion), CBD (0.1-3.2 mg/kg/infusion) failed to maintain responding above vehicle levels. CBD maintained very modest levels of self-administration in rats (n = 7-8) that self-administered heroin (0.015 mg/kg/infusion) and did not increase drug-lever responding, up to a dose of 150 mg/kg (by mouth), in rats (n = 6) trained to discriminate 0.5 mg/kg (i.p.) midazolam. In juvenile (5-6 weeks old) and adult (10-11 weeks old) male and female rats, discontinuation of chronic treatment (twice daily for 20 days) with an oral formulation of CBD (20 or 100 mg/kg, by mouth) did not reliably produce signs of withdrawal. Pharmacokinetic studies confirmed that the dosing regimens used in these studies resulted in therapeutically relevant plasma levels. Taken together, the lack of reliable self-administration, the failure to increase drug-lever responding in rats trained to discriminate midazolam, and the absence of withdrawal signs upon discontinuation of chronic treatment indicate that CBD has very low abuse potential and is unlikely to produce physical dependence. SIGNIFICANCE STATEMENT: Legalization of cannabis across the United States and elsewhere has led to intense investigation into the safety and therapeutic potential of cannabis and its constituent materials, including cannabidiol (CBD). Results of these preclinical abuse potential studies on CBD indicate no rewarding properties, physical dependence potential, or similarity to a benzodiazepine. Together with data from in vitro pharmacology and human abuse potential studies, the abuse potential of Epidiolex in humans is likely to be negligible.

Kinetic characterization and substrate requirement for the Ca2+ uptake system in platelet membrane.

An ATP-dependent mechanism for Ca2+ uptake in human platelet membrane fractions has been identified and characterized. Ca2+ uptake into a membrane fraction is shown to be stimulated at low concentrations of ATP and Ca2+ and to require magnesium ions. Initial rate kinetics, using Eadie-Scatchard analysis, indicated a single class of calcium uptake sites in the presence of ATP, with a Kd for free [Ca2+] of 0.145 microM. Ca2+ uptake in the presence of several ATP concentrations demonstrates that ATP binds to at least two sites, representing high and low affinities of 3.21 and 80.1 microM, respectively. The neuroleptic drug fluphenazine inhibited ATP-stimulated calcium uptake (IC50 = 55 microM), suggesting this ATP-dependent Ca2+ uptake system may provide a useful ion-transport model with which to study neuroleptic therapy in humans.

Physiological and behavioral effects of amphetamine in BACE1(-/-) mice.

ß-Site APP-cleaving Enzyme 1 (BACE1) is a protease that has been linked to schizophrenia, a severe mental illness that is potentially characterized by enhanced dopamine (DA) release in the striatum. Here, we used acute amphetamine administration to stimulate neuronal activity and investigated the neurophysiological and locomotor-activity response in BACE1-deficient (BACE1(-/-) ) mice. We measured locomotor activity at baseline and after treatment with amphetamine (3.2 and 10 mg/kg). While baseline locomotor activity did not vary between groups, BACE1(-/-) mice exhibited reduced sensitivity to the locomotor-enhancing effects of amphetamine. Using high-performance liquid chromatography (HPLC) to measure DA and DA metabolites in the striatum, we found no significant differences in BACE1(-/-) compared with wild-type mice. To determine if DA neuron excitability is altered in BACE1(-/-) mice, we performed patch-clamp electrophysiology in putative DA neurons from brain slices that contained the substantia nigra. Pacemaker firing rate was slightly increased in slices from BACE1(-/-) mice. We next measured G protein-coupled potassium currents produced by activation of D2 autoreceptors, which strongly inhibit firing of these neurons. The maximal amplitude and decay times of D2 autoreceptor currents were not altered in BACE1(-/-) mice, indicating no change in D2 autoreceptor-sensitivity and DA transporter-mediated reuptake. However, amphetamine (30 µm)-induced potassium currents produced by efflux of DA were enhanced in BACE1(-/-) mice, perhaps indicating increased vesicular DA content in the midbrain. This suggests a plausible mechanism to explain the decreased sensitivity to amphetamine-induced locomotion, and provides evidence that decreased availability of BACE1 can produce persistent adaptations in the dopaminergic system.

Characterization of the effect of the adenosine agonist cyclohexyladenosine on platelet activating factor-induced increases in [Ca2+]i in human platelets in vitro.

Previous studies have shown that adenosine agonists acting at A-2 receptors inhibit platelet aggregation. Since an increase in cytosolic Ca2+ concentration (delta [Ca2+]i) is closely associated with the time frame of platelet aggregation, we have examined the effect of adenosine receptor function on induced increases of [Ca2+]i by a potent platelet activator, platelet activating factor (PAF). We loaded washed platelets with Fura-2, then induced increases in [Ca2+]i with various concentrations of PAF, and then determined EC50 values (PAF concentration at half-maximal response) and values for maximal response of delta[Ca2+]i (max-delta[Ca2+]i). The EC50 for PAF-delta[Ca2+]i was 112 +/- 37 (SD) pM and the max-delta[Ca2+]i was 284 +/- 138 (SD) nM. Our results show that PAF-delta[Ca2+]i was inhibited in a non-competitive manner by the adenosine receptor agonist cyclohexyladenosine (CHA) with an IC50 of 14.9 microM. This inhibition was partially reversed by theophylline, an adenosine receptor antagonist, with an IC50 of 19 microM. Based on the results of these studies together with evidence from other research groups that platelets do not possess A-1 receptors, our results suggest that CHA inhibited PAF-delta[Ca2+]i in platelets through an activation of A-2 receptors.

Calcium function in affective disorders and healthy controls.

Calcium metabolism has been reported to be disturbed in some forms of affective disorder. We studied concurrently a battery of calcium measures in 29 unipolar, 14 bipolar depressed, 11 manic, and 10 healthy control subjects. In addition to measures of extracellular calcium, we studied intracellular calcium concentration in platelets and measures that reflect cellular capability to maintain a low intracellular Ca++ concentration in red blood cells (RBCs) and platelets. Plasma calcium was lower in unipolar and manic patients than in control subjects. Platelet calcium concentration was lower in unipolar than bipolar depressed patients. RBC Ca++ adenosine triphosphatase (ATPase) was lower in unipolar and control subjects than in bipolar depressed and manic patients. Platelet Ca++ ATPase and Ca++ uptake were inversely correlated with severity of illness in unipolar patients. In bipolar depressed patients, RBC Ca++ ATPase and platelet Ca++ uptake were inversely correlated with severity. In addition to indicating abnormalities in calcium activity in affective disorders, the data suggest that unipolar and bipolar patients differ in several measures and may have different pathophysiological disturbances in calcium metabolism.

Studies of catecholamine metabolism in schizophrenia/psychosis--II.

Acutely psychotic schizophrenic patients were maintained on debrisoquin (DBQ) throughout 5 weeks of treatment with haloperidol. Treatment with haloperidol caused initial increases in urinary homovanillic acid (HVA) output that returned toward baseline by the 5th week. During haloperidol treatment, plasma levels of HVA tended to decrease, concurrent with increased renal clearance of HVA. Plasma 3-methoxy-4-hydroxyphenylglycol (MHPG) levels and urinary MHPG output both decreased over the course of treatment. The differences in HVA and MHPG metabolism suggest differential effects of treatment on dopamine and norepinephrine systems. Neuroleptic treatment also abolished the marked morning decreases in plasma HVA concentrations (reported in part I).

Studies of catecholamine metabolism in schizophrenia/psychosis--I.

Acutely psychotic schizophrenic patients not taking antipsychotic medications and control subjects were studied before and during treatment with debrisoquin (DBQ), an inhibitor of monoamine oxidase, which does not penetrate into brain. Homovanillic acid (HVA) and 3-methoxy-4-hydroxyphenylglycol (MHPG) were measured in plasma, urine, and cerebrospinal fluid (CSF). Significant differences between patients and control subjects were more easily discerned during treatment with DBQ. In patients, HVA was increased in plasma but not in urine or CSF, although MHPG was increased in all three fluids. There were many significant correlations between plasma MHPG and HVA levels and clinical ratings of psychoticism. Plasma MHPG correlated positively with both the severity of positive and negative symptoms and plasma HVA correlated only with positive symptom severity. These data suggest that both dopamine and norepinephrine (NE) metabolism are disturbed in acutely psychotic schizophrenic patients; disturbed NE metabolism may relate to negative symptoms as well.

Non-specific neuroleptic isomer effect on calmodulin-stimulated calcium transport in erythrocyte membrane.

Calmodulin-stimulated calcium transport by everted erythrocyte membrane vesicles was inhibited by equivalent doses of active and inactive isomers of the neuroleptic drugs butaclamol and chlorprothixene. However, the trans-isomer of flupenthixol, which lacks neuroleptic properties, inhibited calcium transport more than did the active cis-isomer. The drug concentrations required for inhibition greatly exceeded those serum levels necessary for neuroleptic activity. These results are consistent with a non-specific inhibition of calmodulin-activation of calcium transport by neuroleptic drugs and provide further evidence that this effect is unrelated to clinical antipsychotic properties of these drugs. Effects on basal calcium transport activity indicate binding of neuroleptics at a site other than cytosolic calmodulin.

Thrombin-stimulated increases in cytosolic Ca2+ level and gonadotropin-releasing hormone release in GT1-7 neurons.

The effects of thrombin on cytosolic calcium levels ([Ca2+]cyt), and on gonadotropin-releasing hormone (GnRH) release, were characterized in cultured GT1-7 neurons. GnRH release from GT1-7 neurons was pulsatile with an average pulse amplitude of 14.3+/-5.8 pg x min x ml(-1) and an average pulse duration of 21.3+/-4.2 min. The [Ca2+]cyt response to 0.005 to 0.2 U/ml thrombin was saturable and concentration dependent (EC50 = 0.0268 U/ml). Ethyleneglycotetraacetic acid (EGTA) chelation of extracellular Ca2+ resulted in an approximately 70% attenuation of thrombin-stimulated increase in [Ca2+]cyt. By use of a special superfusion system, a 5-min exposure to 0.1 U/ml thrombin significantly increased the amplitude (193.2+/-67.8 pg x min x ml(-1); P = 0.001) but not the duration (22.5+/-2.4 min; P = 0.8) of GnRH release. These results suggest that thrombin increases [Ca2+]cyt and GnRH release from GT1-7 neurons via specific membrane-bound receptors.

Neurochemistry and child and adolescent psychiatry.

This article reviews some of the neurochemistry and neurophysiology of three neurotransmitters: dopamine, norepinephrine, and serotonin. These neurotransmitters are selected because they appear to be involved in the regulation of several important behavioral systems that help regulate the interaction of the organism with its external environment, because many of the psychotropic drugs' modes of action may be result from their effects on these neurotransmitter systems, and because the majority of neurochemical studies in child psychiatry have focused on these three neurotransmitters. After the review of the neurotransmitter systems, neurochemical studies in several child psychiatric disorders are reviewed to illustrate possible biochemical/behavioral relationships in child psychiatry.

Urinary catecholamines in attention-deficit hyperactivity disorder with and without comorbid anxiety.

OBJECTIVE: To determine whether there are differences in noradrenergic or adrenergic functioning in children with attention-deficit hyperactivity disorder (ADHD) with and without anxiety. METHOD: ADHD children with and without a comorbid overanxious (ANX) disorder were compared to each other and to normal controls in terms of 2-hour urinary excretion of norepinephrine (NE), epinephrine (EPI), and their metabolites. All subjects performed a fixed series of mentally stressful tasks during the collection period. RESULTS: Children with ADHD, regardless of comorbid anxiety, excreted more normetanephrine (NMN), the chief extracellular metabolite of NE, than controls, as well as more vanillylmandelic acid. Children with ADHD alone had lower NE/NMN and EPI/metanephrine ratios compared to controls. Children with ADHD/ANX excreted more EPI than ADHD children without anxiety. CONCLUSIONS: Children with ADHD may have a higher tonic activity of the noradrenergic system than controls, while children with comorbid ADHD/ANX may be differentiated from those with ADHD alone by higher adrenergic activity.

Catecholamines and diagnoses in children.

Theoretically, noradrenergic (NA) function may be lower in subjects with undersocialized conduct disorder (CDU) and higher in subjects with anxiety/depressive disorder. To test this hypothesis, diagnostic and 24-hour urine catecholamine measures were compared between subjects with plasma dopamine-beta-hydroxylase (D beta H) activities less than 6 mumoles/min/L (low D beta H group) and greater than 15 mumoles/min/L (high D beta H group). Several measures relating to norepinephrine metabolism were lower in the low D beta H group, and the low D beta H group had more diagnoses of CDU and fewer anxiety and depressive disorder diagnoses. Comparisons between clinical and biological measures within each of the D beta H groups were also consistent with the hypothesized relationship between NA function, CDU, and anxiety/depressive disorder.

Partial characterization of K(+)-induced increase in [Ca2+]cyt and GnRH release in GT1-7 neurons.

Secretion of pituitary gonadotropins is regulated centrally by the hypothalamic decapeptide gonadotropin releasing hormone (GnRH). Using the immortalized hypothalamic GT1-7 neuron, we characterized pharmacologically the dynamics of cytosolic Ca2+ and GnRH release in response to K+-induced depolarization of GT1-7 neurons. Our results showed that K+ concentrations from 7.5 to 60 mM increased [Ca2+]cyt in a concentration-dependent manner. Resting [Ca2+]cyt in GT1 -7 cells was determined to be 69.7 +/- 4.0 nM (mean +/- S.E.M.; n = 69). K+-induced increases in [Ca2+]cyt ranged from 58.2 nM at 7.5 mM [K+] to 347 nM at 60 mM [K+]. K+-induced GnRH release ranged from about 10 pg/ml at 7.5 mM [K+] to about 60 pg/ml at 45 mM [K+]. K+-induced increases in (Ca2+]cyt and GnRH release were enhanced by 1 microM BayK 8644, an L-type Ca2+ channel agonist. The BayK enhancement was completely inhibited by 1 microM nimodipine, an L-type Ca2+ channel antagonist. Nimodipine (1 microM) alone partially inhibited K+-induced increases in [Ca2+]cyt and GnRH release. Conotoxin (1 microM) alone had no effect on K+-induced GnRH release or [Ca2+]cyt, but the combination of conotoxin (1 microM) and nimodipine (1 microM) inhibited K+-induced increase in [Ca2+]cyt significantly more (p < 0.02) than nimodipine alone, suggesting that N-type Ca2+ channels exist in GT1-7 neurons and may be part of the response to K+. The response of [Ca2+]cyt to K+ was linear with increasing [K+] whereas the response of GnRH release to increasing [K+] appeared to be saturable. K+-induced increase in [Ca2+]cyt and GnRH release required extracellular [Ca2+]. These experiments suggest that voltage dependent N- and L-type Ca2+ channels are present in immortalized GT1-7 neurons and that GnRH release is, at least in part, dependent on these channels for release of GnRH.

Characterization of chloride efflux from GT1-7 neurons: lack of effect of ethanol on GABAA response.

The purpose of this study of GT1-7 neurons was to partially characterize basal Cl- transport and GABAA mediated Cl- efflux and to test the effect of ethanol on a GABAA receptor that lacks a gamma subunit. We measured GABAA function and Cl- transport with 36Cl-. Our results show that basal 36Cl- efflux varied with temperature at 4 degrees C, 23 degrees C, and 37 degrees C. At 23 degrees C, DIDS, an inhibitor of anion exchange, reduced basal 36Cl- efflux maximally by 79.6% with an IC50 of 42.1 microM, whereas bumetanide, an inhibitor of (Na-K-Cl) cotransport, had no effect on basal 36Cl- efflux at concentrations up to 150 microM. At 4 degrees C, muscimol, a GABAA receptor agonist, stimulated 36Cl- efflux with an EC50 of 1.47 microM. Bicuculline, a GABAA receptor antagonist, completely reversed the effect of 20 microM muscimol with an IC50 of 6.08 microM. Ethanol, at concentrations up to 87 mM (0.4% (w/v)), had no effect on muscimol-induced 36Cl- efflux at 4 degrees C or at 32 degrees C. Our results indicate that stimulation of GABAA receptors causes an efflux of Cl- from GT1-7 neurons. This finding is consistent with the concept that stimulation of GABAA receptors produces depolarization of the plasma membrane, increase in cytosolic [Ca2+], and GnRH release. Our results represent the first description of chloride transport in GT1-7 neurons and suggest the presence of a Cl- exchange, but not (Na-K-Cl), transporter mechanism. Furthermore, the lack of an effect of ethanol observed in this study is consistent with the idea that a gamma 2L subunit may be necessary for the effects of low concentrations of ethanol at GABAA receptors.

Concentration-dependent effects of muscimol to enhance pulsatile GnRH release from GT1-7 neurons in vitro.

Immortalized GT1-7 neurons were used to characterize the effect of muscimol, a GABAA receptor agonist, to enhance pulsatile gonadotropin-releasing hormone (GnRH) release. GT1-7 neurons were grown on Cytodex-3 beads and placed in special superfusion microchambers. The cells were superfused at a rate of 6.2 ml x h-1 with Media 199 (pH 7.35) using a commercially available perfusion system. After a pre-muscimol period of 120 min, the cells were exposed for 5 min to 0.35, 1, 5 or 10 microM muscimol or 5 microM muscimol+20 microM of the GABAA receptor antagonist, bicuculline. Following removal of the muscimol (and bicuculline, in the case of the latter experiment), the superfusion was continued for another 115 min. Sample fractions were collected at 5 min intervals throughout the perfusion. Basal GnRH release from the GT1-7 neurons was pulsatile with an average interpulse interval of 45.4+/-0.5 min and an average pulse amplitude of 191.5+/-22.6 pg x min x ml-1. Our results also demonstrated that the GABAA receptor agonist, muscimol, enhances pulsatile GnRH release from GT1-7 neurons in culture. The response to muscimol was saturable and concentration-dependent with an EC50 of 0.47 microM. The effects of 5 microM muscimol to increase GnRH pulsatility were blocked by co-exposure to the GABAA receptor antagonist, bicuculline. The average GnRH interpulse intervals were 41.7+/-1.8 min, 32.5+/-2.9 min, 30.6+/-0.7 min and 25.5+/-0.4 min in the period following exposure to 0.35, 1, 5 and 10 microM of muscimol, respectively (post-muscimol period). GnRH pulse amplitude (mean-area for each pulse) was increased during exposure to muscimol but not during the pre- or post-muscimol periods. The GABAA receptor antagonist, bicuculline, itself had no effect on pulsatile GnRH release. These results are consistent with previously published reports suggesting that activation of the GABAA receptor stimulates hypothalamic GnRH release in embryonic and neonatal animals.

Schizophrenia, psychosis, and cerebral spinal fluid homovanillic acid concentrations.

Neuroleptic drugs block brain dopamine receptors and are effective in treating psychoses of diverse origins. This finding has become a cornerstone of the dopamine theory of schizophrenia, but clinical studies relating schizophrenia, per se, to brain dopamine metabolism have ranged from controversial to negative. This article presents new evidence that cerebrospinal fluid levels of the dopamine metabolite homovanillic acid are related to the severity of psychosis in schizophrenia. These results support the concept that homovanillic acid levels in cerebrospinal fluid vary as a function of psychosis rather than being related to the diagnosis of schizophrenia per se.

Effects of suramin on human platelet aggregation and Ca2+ mobilization induced by thrombin and other agonists.

The purpose of this study was to investigate the effect of suramin, a polyanionic napthalene sulfonic acid, on human platelet aggregation and Ca2+ mobilization induced by various agonists. Our results show that suramin completely inhibited aggregation by thrombin, platelet activating factor (PAF), alkyllysophosphatidic acid (ALPA), or arachidonic acid in a concentration-dependent manner. The IC50 values of suramin for inhibition of aggregation by PAF, arachidonic acid, and thrombin were 76.7, 239, and 1.49 microg/ml, respectively. Ca2+ mobilization induced by thrombin was inhibited by suramin with an approximate IC50 value of 20 microg/ml. This concentration of suramin had no effect on PAF or oleic acid-induced Ca2+ mobilization. The mechanism by which suramin inhibits aggregation is not clear, but our results suggest that suramin inhibits the ligand-receptor interaction.

Effects of fire ant venom alkaloids on platelet and neutrophil function.

About 95% of venom of the imported fire ant Solenopsis invicta is composed of dialkyl piperidines. These alkaloids produce a distinct pustule at the site of injection. The formation of this pustule may involve the activation of platelets and neutrophils. The purpose of this paper was to characterize the effects of fire ant venom alkaloids (FAVA) on certain physiological and biochemical functions of human platelets and neutrophils. In platelets, FAVA caused a rise in intracellular [Ca2+], secretion of dense granules as measured by ATP release, and aggregation as measured by light transmission through a suspension of platelets. Aggregation response was less complete with FAVA than with thrombin or PAF. However, secretion response was greater with FAVA than thrombin. One of our most significant findings was that pretreatment of platelets with subthreshold concentrations of FAVA produced enhanced PAF-induced increase in [Ca2+]cyt, suggesting that synergism between the two agonists might play an important role in the physiological response to FAVA. In neutrophils, FAVA produced a rise in intracellular [Ca2+] and aggregation, although the responses were more moderate than those observed in platelets. These results suggest that FAVA activation of platelets and neutrophils may occur in vivo as a response to stings by red fire ants.

The effect of debrisoquin on MAO A and MAO B activities.

To examine the mode of action of debrisoquin (DEB), we studied the effect of this drug in vitro on MAO A and MAO B enzyme activities. DEB was shown to be a competitive inhibitor of highly purified human MAO A and MAO B enzyme activities. DEB inhibited placental MAO A with a Ki value of 0.5 microM and liver MAO B with a Ki value of 8.8 microM, 18-fold greater effect on the A form. Kynuramine was used as substrate for both enzymes. Additional studies using a dilution technique showed that DEB was a reversible inhibitor of both forms of the enzyme. The results of this study show that DEB is a potent competitive and reversible inhibitor of both MAO A and MAO B enzymes.

Effects of lithium on platelet ionic intracellular calcium concentration in patients with bipolar (manic-depressive) disorder and healthy controls.

Lithium is an effective drug in the treatment of both manic and depressive episodes of bipolar disorder. Lithium has been shown to block the metabolism of the intracellular second messenger inositol-1,4,5-trisphosphate which is involved in the rise in ionic intracellular calcium [( Ca++]i) which triggers neurotransmitter release and other cellular changes in secretory cells. We have measured the effect of lithium on [Ca++]i dynamics in platelets from bipolar patients stabilized with lithium treatment, and from healthy controls. Both resting [Ca++]i and the thrombin stimulated increase in [Ca++]i were higher in bipolar patients than in controls. Lithium added in vitro tended to increase the thrombin-stimulated rise in [Ca++]i. The use of the fluorescent Ca++ probe fura-2 in human platelets provides a useful method to investigate the mechanism of lithium's action in bipolar disorder and to study Ca++ related systems which may be abnormal in bipolar disorders.