Cannabidiol and it fluorinate analog PECS-101 reduces hyperalgesia and allodynia in trigeminal neuralgia via TRPV1 receptors.

Trigeminal neuralgia (TN) is an intense and debilitating orofacial pain. The gold standard treatment for TN is carbamazepine. This antiepileptic drug provides pain relief with limited efficacy and side effects. To study the antinociceptive potential of cannabidiol (CBD) and its fluorinated analog PECS-101 (former HUF-101), we induced unilateral chronic constriction injury of the infraorbital nerve (IoN-CCI) in male Wistar rats. Seven days of treatment with CBD (30 mg/kg), PECS-101 (3, 10, and 30 mg/kg), or carbamazepine (10 and 30 mg/kg) reduced allodynia and hyperalgesia responses. Unlike carbamazepine, CBD and PECS-101 did not impair motor activity. The relief of the hypersensitive reactions has been associated with transient receptor potential vanilloid type 1 (TRPV1) modulation in the trigeminal spinal nucleus. CBD (30 mg/kg) and PECS-101 (10 and 30 mg/kg) reversed the increased expression of TRPV1 induced by IoN-CCI in this nucleus. Using a pharmacological strategy, the combination of the selective TRPV1 antagonist (capsazepine-CPZ - 5 mg/kg) with sub-effective doses of CBD (3 and 10 mg/kg) is also able to reverse the IoN-CCI-induced allodynia and hyperalgesia responses. This effect was accompanied by reduced TRPV1 protein expression in the trigeminal spinal nucleus. Our results suggest that CBD and PECS-101 may benefit trigeminal neuralgia without motor coordination impairments. PECS-101 is more potent against the hypernociceptive and motor impairment induced by TN compared to CBD and carbamazepine. The antinociceptive effect of these cannabinoids is partially mediated by TRPV1 receptors in the caudal part of the trigeminal spinal nucleus, the first central station of orofacial pain processing.

The two synthetic cannabinoid compounds 4'-F-CBD and HU-910 efficiently restrain inflammatory responses of brain microglia and astrocytes.

To study the anti-inflammatory potential of the two synthetic cannabinoids 4'-F-CBD and HU-910, we used post-natal brain cultures of mouse microglial cells and astrocytes activated by reference inflammogens. We found that 4'-F-CBD and HU-910 efficiently curtailed the release of TNF-α, IL-6, and IL-1ß in microglia and astrocytes activated by the bacterial Toll-Like Receptor (TLR)4 ligand LPS. Upon LPS challenge, 4'-F-CBD and HU-910 also prevented the activation of phenotypic activation markers specific to microglia and astrocytes, that is, Iba-1 and GFAP, respectively. In microglial cells, the two test compounds also efficiently restrained LPS-stimulated release of glutamate, a non-cytokine inflammation marker for these cells. The immunosuppressive effects of the two cannabinoid compounds were concentration-dependent and observable between 1 and 10 µM. These effects were not dependent on cannabinoid or cannabinoid-like receptors. Both 4'-F-CBD and HU-910 were also capable of restraining the inflammogenic activity of Pam3CSK4, a lipopeptide that activates TLR2, and of BzATP, a prototypic agonist of P2X7 purinergic receptors, suggesting that these two cannabinoids could exert immunosuppressive effects against a variety of inflammatory stimuli. Using LPS-stimulated microglia and astrocytes, we established that the immunosuppressive action of 4'-F-CBD and HU-910 resulted from the inhibition of ROS produced by NADPH oxidase and subsequent repression of NF-κB-dependent signaling events. Our results suggest that 4'-F-CBD and HU-910 may have therapeutic utility in pathological conditions where neuroinflammatory processes are prominent.

Doxycycline attenuates l-DOPA-induced dyskinesia through an anti-inflammatory effect in a hemiparkinsonian mouse model.

The pharmacological manipulation of neuroinflammation appears to be a promising strategy to alleviate l-DOPA-induced dyskinesia (LID) in Parkinson's disease (PD). Doxycycline (Doxy), a semisynthetic brain-penetrant tetracycline antibiotic having interesting anti-inflammatory properties, we addressed the possibility that this compound could resolve LID in l-DOPA-treated C57BL/6 mice presenting either moderate or intermediate lesions of the mesostriatal dopaminergic pathway generated by intrastriatal injections of 6-OHDA. Doxy, when given subcutaneously before l-DOPA at doses of 20 mg kg-1 and 40 mg kg-1, led to significant LID reduction in mice with moderate and intermediate dopaminergic lesions, respectively. Importantly, Doxy did not reduce locomotor activity improved by l-DOPA. To address the molecular mechanism of Doxy, we sacrificed mice with mild lesions 1) to perform the immunodetection of tyrosine hydroxylase (TH) and Fos-B and 2) to evaluate a panel of inflammation markers in the striatum, such as cyclooxygenase-2 and its downstream product Prostaglandin E2 along with the cytokines TNF-α, IL-1ß and IL-6. TH-immunodetection revealed that vehicle and Doxy-treated mice had similar striatal lesions, excluding that LID improvement by Doxy could result from neurorestorative effects. Importantly, LID inhibition by Doxy was associated with decreased Fos-B and COX-2 expression and reduced levels of PGE2, TNF-α, and IL-1ß in the dorsolateral striatum of dyskinetic mice. We conclude 1) that Doxy has the potential to prevent LID regardless of the intensity of dopaminergic lesioning and 2) that the anti-inflammatory effects of Doxy probably account for LID attenuation. Overall, the present results further indicate that Doxy might represent an attractive and alternative treatment for LID in PD.

Caffeine Improves GABA Transport in the Striatum of Spontaneously Hypertensive Rats (SHR).

The spontaneously hypertensive rat (SHR) is an excellent animal model that mimics the behavioral and neurochemical phenotype of attention-deficit/hyperactivity disorder (ADHD). Here, we characterized the striatal GABA transport of SHR and investigated whether caffeine, a non-selective antagonist of adenosine receptors, could influence GABAergic circuitry. For this purpose, ex vivo striatal slices of SHR and Wistar (control strain) on the 35th postnatal day were dissected and incubated with [3H]-GABA to quantify the basal levels of uptake and release. SHR exhibited a reduced [3H]-GABA uptake and release, suggesting a defective striatal GABAergic transport system. GAT-1 appears to be the primary transporter for [3H]-GABA uptake in SHR striatum, as GAT-1 selective blocker, NO-711, completely abolished it. We also verified that acute exposure of striatal slices to caffeine improved [3H]-GABA uptake and release in SHR, whereas Wistar rats were not affected. GABA-uptake increase and cAMP accumulation promoted by caffeine was reverted by A1R activation with N6-cyclohexyl adenosine (CHA). As expected, the pharmacological blockade of cAMP-PKA signaling by H-89 also prevented caffeine-mediated [3H]-GABA uptake increment. Interestingly, a single caffeine exposure did not affect GAT-1 or A1R protein density in SHR, which was not different from Wistar protein levels, suggesting that the GAT-1-dependent transport in SHR has a defective functional activity rather than lower protein expression. The current data support that caffeine regulates GAT-1 function and improves striatal GABA transport via A1R-cAMP-PKA signaling, specifically in SHR. These results reinforce that caffeine may have therapeutic use in disorders where the GABA transport system is impaired.

Cannabidiol has therapeutic potential for myofascial pain in female and male parkinsonian rats.

The musculoskeletal orofacial pain is a complex symptom of Parkinson's disease (PD) resulting in stomatognathic system dysfunctions aggravated by the disease rigidity and postural instability. We tested the effect of cannabidiol (CBD), a non-psychotomimetic constituent of Cannabis sativa, in PD-related myofascial pain. Wistar adult female and male rats orofacial allodynic and hyperalgesic responses were tested by Von Frey and formalin tests, before and 21 days past 6-OHDA lesion. Algesic response was tested after masseter muscle injection of CBD (10, 50, 100 µg in 10 µL) or vehicle. Males compared to females in all estrous cycles' phases presented reduced orofacial allodynia and hyperalgesia. According to the estrous cycle's phases, females presented distinct orofacial nociceptive responses, being the estrus phase well-chosen for nociceptive analysis after 6-OHDA lesion (phase with fewer hormone alterations and adequate length). Dopaminergic neuron lesion decreased mechanical and inflammatory nociceptive thresholds in females and males in a higher proportion in females. CBD local treatment reduced the increased orofacial allodynia and hyperalgesia, in males and females. The female rats were more sensitive to CBD effect considering allodynia, responding to the lowest dose. Although females and males respond to the effect of three doses of CBD in the formalin test, males showed a superior reduction in the hyperalgesic response. These results indicate that hemiparkinsonian female in the estrus phase and male answer differently to the different doses of CBD therapy and nociceptive tests. CBD therapy is effective for parkinsonism-induced orofacial nociception.

CMT-3 targets different α-synuclein aggregates mitigating their toxic and inflammogenic effects.

Parkinson's disease (PD) is a neurodegenerative disorder for which only symptomatic treatments are available. Repurposing drugs that target α-synuclein aggregation, considered one of the main drivers of PD progression, could accelerate the development of disease-modifying therapies. In this work, we focused on chemically modified tetracycline 3 (CMT-3), a derivative with reduced antibiotic activity that crosses the blood-brain barrier and is pharmacologically safe. We found that CMT-3 inhibited α-synuclein amyloid aggregation and led to the formation of non-toxic molecular species, unlike minocycline. Furthermore, CMT-3 disassembled preformed α-synuclein amyloid fibrils into smaller fragments that were unable to seed in subsequent aggregation reactions. Most interestingly, disaggregated species were non-toxic and less inflammogenic on brain microglial cells. Finally, we modelled the interactions between CMT-3 and α-synuclein aggregates by molecular simulations. In this way, we propose a mechanism for fibril disassembly. Our results place CMT-3 as a potential disease modifier for PD and possibly other synucleinopathies.

Single Cocaine Exposure Inhibits GABA Uptake via Dopamine D1-Like Receptors in Adolescent Mice Frontal Cortex.

Cocaine (COC) is a psychostimulant that acts by increasing catecholaminergic neurotransmission mainly due to its effects on the dopamine transporter (DAT). However, other neurotransmitter systems may also be regulated by COC, including the GABAergic system. Since the effect of COC in modulating gamma-aminobutyric acid (GABA) reuptake is not defined, we investigated the molecular mechanisms related to the increase in GABA uptake induced by acute COC exposure and its effects on locomotor activity in adolescent mice. Behavioral experiments showed that COC increased locomotor activity and decreased immobilization time in mice. A single COC exposure reduced both GABA uptake and GAT-1 protein levels. On the other hand, cyclic adenosine monophosphate (cAMP) levels increased after a COC challenge. The major changes induced by acute COC on behavioral and neurochemical assays were avoided by previous treatment with the selective D1 receptor antagonist SCH-23390 (0.5 mg/kg). Our findings suggest that GABA uptake naturally decreases during mice development from preadolescence until adulthood and that dopamine (DA) D1-like receptors are key players in the regulation of GABA uptake levels following a single COC exposure in adolescent mice.

Cannabidiol and Cannabinoid Compounds as Potential Strategies for Treating Parkinson's Disease and L-DOPA-Induced Dyskinesia.

Parkinson's disease (PD) and L-DOPA-induced dyskinesia (LID) are motor disorders with significant impact on the patient's quality of life. Unfortunately, pharmacological treatments that improve these disorders without causing severe side effects are not yet available. Delay in initiating L-DOPA is no longer recommended as LID development is a function of disease duration rather than cumulative L-DOPA exposure. Manipulation of the endocannabinoid system could be a promising therapy to control PD and LID symptoms. In this way, phytocannabinoids and synthetic cannabinoids, such as cannabidiol (CBD), the principal non-psychotomimetic constituent of the Cannabis sativa plant, have received considerable attention in the last decade. In this review, we present clinical and preclinical evidence suggesting CBD and other cannabinoids have therapeutic effects in PD and LID. Here, we discuss CBD pharmacology, as well as its neuroprotective effects and those of other cannabinoids. Finally, we discuss the modulation of several pro- or anti-inflammatory factors as possible mechanisms responsible for the therapeutic/neuroprotective potential of Cannabis-derived/cannabinoid synthetic compounds in motor disorders.

Contributive Role of TNF-α to L-DOPA-Induced Dyskinesia in a Unilateral 6-OHDA Lesion Model of Parkinson's Disease.

Our present objective was to better characterize the mechanisms that regulate striatal neuroinflammation in mice developing L-DOPA-induced dyskinesia (LID). For that, we used 6-hydroxydopamine (6-OHDA)-lesioned mice rendered dyskinetic by repeated intraperitoneal injections of 3,4-dihydroxyphenyl-L-alanine (L-DOPA) and quantified ensuing neuroinflammatory changes in the dopamine-denervated dorsal striatum. LID development was associated with a prominent astrocytic response, and a more moderate microglial cell reaction restricted to this striatal area. The glial response was associated with elevations in two pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-1ß. Treatment with the phytocannabinoid cannabidiol and the transient receptor potential vanilloid-1 (TRPV-1) channel antagonist capsazepine diminished LID intensity and decreased TNF-α levels without impacting other inflammation markers. To possibly reproduce the neuroinflammatory component of LID, we exposed astrocyte and microglial cells in culture to candidate molecules that might operate as inflammatory cues during LID development, i.e., L-DOPA, dopamine, or glutamate. Neither L-DOPA nor dopamine produced an inflammatory response in glial cell cultures. However, glutamate enhanced TNF-α secretion and GFAP expression in astrocyte cultures and promoted Iba-1 expression in microglial cultures. Of interest, the antidyskinetic treatment with cannabidiol + capsazepine reduced TNF-α release in glutamate-activated astrocytes. TNF-α, on its own, promoted the synaptic release of glutamate in cortical neuronal cultures, whereas cannabidiol + capsazepine prevented this effect. Therefore, we may assume that the release of TNF-α by glutamate-activated astrocytes may contribute to LID by exacerbating corticostriatal glutamatergic inputs excitability and maintaining astrocytes in an activated state through a self-reinforcing mechanism.

Repurposing an established drug: an emerging role for methylene blue in L-DOPA-induced dyskinesia.

The nitric oxide (NO) system has been proven to be a valuable modulator of L-DOPA-induced dyskinesia in Parkinsonian rodents. NO activates the enzyme soluble guanylyl cyclase and elicits the synthesis of the second-messenger cGMP. Although we have previously described the anti-dyskinetic potential of NO synthase inhibitors on L-DOPA-induced dyskinesia, the effect of soluble guanylyl cyclase inhibitors remains to be evaluated. The aim of this study was to analyze whether the clinically available non-selective inhibitor methylene blue, or the selective soluble guanylyl cyclase inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), could mitigate L-DOPA-induced dyskinesia in 6-hydroxydopamine-lesioned rats. Here, we demonstrated that methylene blue was able to reduce L-DOPA-induced dyskinesia incidence when chronically co-administered with L-DOPA during 3 weeks. Methylene blue chronic (but not acute) administration (2 weeks) was effective in attenuating L-DOPA-induced dyskinesia in rats rendered dyskinetic by a previous course of L-DOPA chronic treatment. Furthermore, discontinuous methylene blue treatment (e.g., co-administration of methylene blue and L-DOPA for 2 consecutive days followed by vehicle and L-DOPA co-administration for 5 days) was effective in attenuating dyskinesia. Finally, we demonstrated that microinjection of methylene blue or ODQ into the lateral ventricle effectively attenuated L-DOPA-induced dyskinesia. Taken together, these results demonstrate an important role of NO-soluble guanylyl cyclase-cGMP signaling on L-DOPA-induced dyskinesia. The clinical implications of this discovery are expected to advance the treatment options for patients with Parkinson's disease.

Beta-adrenergic receptor activation increases GABA uptake in adolescent mice frontal cortex: Modulation by cannabinoid receptor agonist WIN55,212-2.

GABA transporters regulate synaptic GABA levels and dysfunctions in this system might result in psychiatric disorders. The endocannabinoid system (ECS) is the main circuit breaker in the nervous system and may alter noradrenaline (NA) communication, which in turn modulates the release of GABA. However, a close relationship between these systems has not been recognized. We asked whether NA and ECS might control extracellular GABA levels in slices of frontal cortex (FC) of adolescent Swiss mice with 40 days after birth (PN40). Here we show that NA and isoproterenol (ISO), a beta-adrenergic agonist, increased [3H]-GABA uptake in mice FC, while alpha1-adrenergic agonist phenylephrine had no effect. As GAT-1 is expressed and fully functional at the FC, addition of NO-711, a GAT-1 inhibitor, dose dependently blocked [3H]-GABA uptake. The increase of [3H]-GABA uptake induced by ISO was also blocked by NO-711. [3H]-GABA release induced by 80 mM KCl was reduced by NO-711, but not by removal of Ca2+. ISO also increased cyclic AMP (cAMP) levels and addition of WIN 55,212-2, a mixed CB1/CB2 receptor agonist, inhibited the effect of ISO in GABA uptake increase, GAT-1 expression and cAMP levels compared to control. Our data show that GABA transport increased by NA and ISO is negatively regulated by cannabinoid receptor agonist WIN55,212-2.

The role of striatum and prefrontal cortex in the prevention of amphetamine-induced schizophrenia-like effects mediated by nitric oxide compounds.

Pharmacological manipulation of nitric oxide (NO) has been suggested as a promising treatment for schizophrenia symptoms. A single infusion of sodium nitroprusside, a NO donor with short half-life, was found to improve schizophrenia symptoms. However, an increasing number of preclinical studies have demonstrated the potential beneficial effects of both NO donors and inhibitors. We investigated the potential synergistic effect of sub-effective doses of the NO donor sodium nitroprusside or the NO inhibitor 7-Nitroindazole (7NI) combined with clozapine, a standard atypical antipsychotic, on counteracting amphetamine or MK-801-induced psychosis-like behaviors. The impact of sodium nitroprusside and 7NI on cAMP regulation in the prefrontal cortex and striatum was also evaluated. Confirming previous results, we found that both NO donors and inhibitors prevented amphetamine-induced effects (prepulse inhibition [PPI] disruption and hyperlocomotion). In addition, we observed a synergistic effect of sodium nitroprusside and clozapine on antagonizing the disruptive effects of amphetamine, but not MK-801, in the PPI test. The sub-effective dose of 7NI tested did not prevent amphetamine or MK-induced PPI effects when combined with clozapine. Interestingly, cAMP levels were significantly decreased in the prefrontal cortex after treatment with sodium nitroprusside. In the striatum, both sodium nitroprusside and 7NI blocked the amphetamine-induced increase of cAMP. Our data corroborate previous findings on the dopaminergic mechanisms involved in the action of sodium nitroprusside. It is likely that the differential effects of sodium nitroprusside are related to its ability to modify cAMP levels in the prefrontal cortex.

l-DOPA-induced dyskinesia in Parkinson's disease: Are neuroinflammation and astrocytes key elements?

Inflammation in Parkinson's disease (PD) is a new concept that has gained ground due to the potential of mitigating dopaminergic neuron death by decreasing inflammation. The solution to this question is likely to be complex. We propose here that the significance of inflammation in PD may go beyond the nigral cell death. The pathological process that underlies PD requires years to reach its full extent. A growing body of evidence has been accumulated on the presence of multiple inflammatory signs in the brain of PD patients even in very late stages of the disease. Because neuron-microglia-astrocyte interactions play a major role in the plasticity of neuronal response to l-DOPA in post-synaptic neurons, we focused this review on our recent results of l-DOPA-induced dyskinesia in rodents correlating it to significant findings regarding glial cells and neuroinflammation. We showed that in the rat model of PD/l-DOPA-induced dyskinesia there was an increased expression of inflammatory markers, such as the enzymes COX2 in neurons and iNOS in glial cells, in the dopamine-denervated striatum. The gliosis commonly seem in PD was associated with modifications in astrocytes and microglia that occur after chronic treatment with l-DOPA. Either as a cause, consequence, or promoter of progression of neuronal degeneration, inflammation plays a role in PD. The key aims of current PD research ought to be to elucidate (a) the time sequence in which the inflammatory factors act in PD patient brain and (b) the mechanisms by which neuroinflammatory response contributes to the collateral effects of l-DOPA treatment.

Antidyskinetic Effect of 7-Nitroindazole and Sodium Nitroprusside Associated with Amantadine in a Rat Model of Parkinson's Disease.

Amantadine is the noncompetitive antagonist of N-methyl-D-aspartate, receptor activated by the excitatory neurotransmitter glutamate. It is the only effective medication used to alleviate dyskinesia induced by L-3,4-dihydroxyphenylalanine (L-DOPA) in Parkinson's disease patients. Unfortunately, adverse effects as abnormal involuntary movements (AIMs) known as L-DOPA-induced dyskinesia limit its clinical utility. Combined effective symptomatic treatment modalities may lessen the liability to undesirable events. Likewise drugs known to interfere with nitrergic system reduce AIMs in animal models of Parkinson's disease. We aimed to analyze an interaction between amantadine, neuronal nitric oxide synthase inhibitor (7-nitroindazole, 7NI), and nitric oxide donor (sodium nitroprusside, SNP) in 6-hydroxydopamine-(6-OHDA)-lesioned rats (microinjection in the medial forebrain bundle) presenting L-DOPA-induced dyskinesia (20 mg/kg, gavage, during 21 days). We confirm that 7NI-30 mg/kg, SNP-2/4 mg/kg and amantadine-40 mg/kg, individually reduced AIMs. Our results revealed that co-administration of sub-effective dose of amantadine (10 mg/kg) plus sub-effective dose of 7NI (20 mg/kg) potentiates the effect of reducing AIMs scores when compared to the effect of the drugs individually. No superior benefit on L-DOPA-induced AIMs was observed with the combination of amantadine and SNP. The results revealed that combination of ineffective doses of amantadine and 7NI represents a new strategy to increase antidyskinetic effect in L-DOPA-induced AIMs. It may provide additional therapeutic benefits to Parkinson's disease patients from these disabling complications at lower and thus safer and more tolerable doses than required when either drug is used alone. To close, we discuss the paradox of both nitric oxide synthase inhibitor and/or donor produced AIMs reduction by targeting nitric oxide synthase.

Long Withdrawal of Methylphenidate Induces a Differential Response of the Dopaminergic System and Increases Sensitivity to Cocaine in the Prefrontal Cortex of Spontaneously Hypertensive Rats.

Methylphenidate (MPD) is one of the most prescribed drugs for alleviating the symptoms of Attention Deficit/Hyperactivity Disorder (ADHD). However, changes in the molecular mechanisms related to MPD withdrawal and susceptibility to consumption of other psychostimulants in normal individuals or individuals with ADHD phenotype are not completely understood. The aims of the present study were: (i) to characterize the molecular differences in the prefrontal dopaminergic system of SHR and Wistar strains, (ii) to establish the neurochemical consequences of short- (24 hours) and long-term (10 days) MPD withdrawal after a subchronic treatment (30 days) with Ritalin® (Methylphenidate Hydrochloride; 2.5 mg/kg orally), (iii) to investigate the dopaminergic synaptic functionality after a cocaine challenge in adult MPD-withdrawn SHR and Wistar rats. Our results indicate that SHR rats present reduced [3H]-Dopamine uptake and cAMP accumulation in the prefrontal cortex (PFC) and are not responsive to dopaminergic stimuli in when compared to Wistar rats. After a 24-hour withdrawal of MPD, SHR did not present any alterations in [3H]-Dopamine Uptake, [3H]-SCH 23390 binding and cAMP production; nonetheless, after a 10-day MPD withdrawal, the results showed a significant increase of [3H]-Dopamine uptake, of the quantity of [3H]-SCH 23390 binding sites and of cAMP levels in these animals. Finally, SHR that underwent a 10-day MPD withdrawal and were challenged with cocaine (10 mg/kg i.p.) presented reduced [3H]-Dopamine uptake and increased cAMP production. Wistar rats were affected by the 10-day withdrawal of MPD in [3H]-dopamine uptake but not in cAMP accumulation; in addition, cocaine was unable to induce significant modifications in [3H]-dopamine uptake and in cAMP levels after the 10-day withdrawal of MPD. These results indicate a mechanism that could explain the high comorbidity between ADHD adolescent patients under methylphenidate treatment and substance abuse in adult life.