New serotonin 5-HT(1A) receptor agonists with neuroprotective effect against ischemic cell damage

We report the synthesis of new compounds 4-35 based on structural modifications of different moieties of previously described lead UCM-2550. The new nonpiperazine derivatives, representing second-generation agonists, were assessed for binding affinity, selectivity, and functional activity at the 5-HT(1A) receptor (5-HT(1A)R). Computational β(2)-based homology models of the ligand-receptor complexes were used to explain the observed structure-affinity relationships. Selected candidates were also evaluated for their potential in vitro and in vivo neuroprotective properties. Interestingly, compound 26 (2-{6-[(3,4-dihydro-2H-chromen-2-ylmethyl)amino]hexyl}tetrahydro-1H-pyrrolo[1,2-c]imidazole-1,3(2H)-dione) has been characterized as a high-affinity and potent 5-HT(1A)R agonist (K(i) = 5.9 nM, EC(50) = 21.8 nM) and exhibits neuroprotective effect in neurotoxicity assays in primary cell cultures from rat hippocampus and in the MCAO model of focal cerebral ischemia in rats.     

Cannabidiol reduced the striatal atrophy caused 3-nitropropionic acid in vivo by mechanisms independent of the activation of cannabinoid, vanilloid TRPV1 and adenosine A2A receptors

The neuroprotective potential of cannabinoids has been examined in rats with striatal lesions caused by 3-nitropropionic acic (3NP), an inhibitor of mitochondrial complex II. We used the CB1 agonist arachidonyl-2-chloroethylamide (ACEA), the CB2 agonist HU-308, and cannabidiol (CBD), an antioxidant phytocannabinoid with negligible affinity for cannabinoid receptors. The administration of 3NP reduced GABA contents and also mRNA levels for several markers of striatal GABAergic projection neurons, including proenkephalin (PENK), substance P (SP) and neuronal-specific enolase (NSE). We also found reductions in mRNA levels for superoxide dismutase-1 (SOD-1) and -2 (SOD-2), which indicated that 3NP reduced the endogenous antioxidant defences. The administration of CBD, but not ACEA or HU-308, completely reversed 3NP-induced reductions in GABA contents and mRNA levels for SP, NSE and SOD-2, and partially attenuated those found in SOD-1 and PENK. This indicates that CBD is neuroprotective but acted preferentially on striatal neurons that project to the substantia nigra. The effects of CBD were not reversed by the CB1 receptor antagonist SR141716. The same happened with the TRPV1 receptor antagonist capsazepine, in concordance with the observation that capsaicin, a TRPV1 receptor agonist, failed to reproduce the CBD effects. The effects of CBD were also independent of adenosine signalling as they were not attenuated by the adenosine A2A receptor antagonist MSX-3. In summary, this study demonstrates that CBD provides neuroprotection against 3NP-induced striatal damage, which may be relevant for Huntington's disease, a disorder characterized by the preferential loss of striatal projection neurons. This capability seems to be based exclusively on the antioxidant properties of CBD.     

Therapeutic potential of the endocannabinoid system in the brain

Cannabinoids have been predominantly considered as the substances responsible of the psychoactive properties of marijuana and other derivatives of Cannabis sativa. However, these compounds are now being also considered for their therapeutic potential, since the term "cannabinoid" includes much more compounds than those present in Cannabis sativa derivatives. Among them, there are numerous synthetic cannabinoids obtained by modifications from plant-derived cannabinoids, but also from the compounds that behave as endogenous ligands for the different cannabinoid receptor subtypes. Within the family of "cannabinoid-related compounds", one should also include some prototypes of selective antagonists for these receptors, and also the recently developed inhibitors of the mechanism of finalization of the biological action of endocannabinoids (transporter + FAAH). All this boom of the cannabinoid pharmacology has, therefore, an explanation in the recent discovery and characterization of the endocannabinoid signaling system, which plays a modulatory role mainly in the brain but also in the periphery. The objective of the present article will be to review, from pharmacological and biochemical points of view, the more recent advances in the study of the endocannabinoid system and their functions in the brain, as well as their alterations in a variety of pathologies and the proposed therapeutic benefits of novel cannabinoid-related compounds that improve the pharmacokinetic and pharmacodynamic properties of classic cannabinoids.     

Phase I and pharmacokinetic study of gemcitabine administered at fixed-dose rate, combined with docetaxel/melphalan/carboplatin, with autologous hematopoietic progenitor-cell support, in patients with advanced refractory tumors.

The purpose of this trial was to define the maximum tolerated duration (MTD), dose-limiting toxicity (DLT), regimen-related toxicities (RRT), and pharmacokinetics of gemcitabine infused at a fixed dose rate (FDR) of 10 mg/m2/min, combined with docetaxel/melphalan/carboplatin, using autologous stem cell transplantation (ASCT). The duration of gemcitabine infusion was incrementally escalated as a single treatment on day -6 or as 4 daily infusions on days -5 to -2. Gemcitabine was followed by docetaxel (300 or 350 mg/m2) on day -5, and then melphalan (50 mg/m2/day) and carboplatin (333 mg/m2/day) on days -4 to -2. Fifty-two patients with refractory tumors were accrued with a median age of 40 (range: 6-66), a median of 3 (1-6) prior chemotherapy regimens, and 3 (1-7) organs involved. The gemcitabine MTD was defined at 20 hours (total dose 12,000 mg/m2) on both schedules. The DLT was enteritis. Three patients died from aspiration, catheter-related sepsis, and enteritis, respectively. The tumor response rate was 91%, with 50% complete responses. At current 2-year median follow-up, the event-free and overall survival (EFS, OS) rates are 54% (median 26 months) and 79% (median not reached), respectively. Gemcitabine area under the curve (AUC), but not clearance, increased linearly with infusion duration, and correlated with grade 3 RRT. Docetaxel showed a linear increase of its AUC and similar clearance compared with prior reports at lower doses. In conclusion, ASCT-supported infusions of gemcitabine at FDR could be prolonged up to 20 hours. The resulting gemcitabine/docetaxel/melphalan/carboplatin combination was highly active in refractory cancers and should be further tested in disease-specific trials.     

A restricted population of CB1 cannabinoid receptors with neuroprotective activity

The CB₁ cannabinoid receptor, the main molecular target of endocannabinoids and cannabis active components, is the most abundant G protein-coupled receptor in the mammalian brain. Of note, CB₁ receptors are expressed at the synapses of two opposing (i.e., GABAergic/inhibitory and glutamatergic/excitatory) neuronal populations, so the activation of one and/or another receptor population may conceivably evoke different effects. Despite the widely reported neuroprotective activity of the CB₁ receptor in animal models, the precise pathophysiological relevance of those two CB₁ receptor pools in neurodegenerative processes is unknown. Here, we first induced excitotoxic damage in the mouse brain by (i) administering quinolinic acid to conditional mutant animals lacking CB₁ receptors selectively in GABAergic or glutamatergic neurons, and (ii) manipulating corticostriatal glutamatergic projections remotely with a designer receptor exclusively activated by designer drug pharmacogenetic approach. We next examined the alterations that occur in the R6/2 mouse, a well-established model of Huntington disease, upon (i) fully knocking out CB₁ receptors, and (ii) deleting CB₁ receptors selectively in corticostriatal glutamatergic or striatal GABAergic neurons. The data unequivocally identify the restricted population of CB₁ receptors located on glutamatergic terminals as an indispensable player in the neuroprotective activity of (endo)cannabinoids, therefore suggesting that this precise receptor pool constitutes a promising target for neuroprotective therapeutic strategies.Endocannabinoids are a family of neuron-communication messengers that act by engaging CB₁ cannabinoid receptors, which are also targeted by Δ⁹-tetrahydrocannabinol (THC), the main bioactive component of cannabis. Endocannabinoid signaling serves as a pivotal feedback mechanism to prevent excessive presynaptic activity, thereby tuning the functionality and plasticity of many synapses (1, 2). The CB₁ receptor is the most abundant G protein-coupled receptor in the brain, and is highly expressed in GABAergic terminals of the forebrain (particularly in cholecystokinin-positive and parvalbumin-negative interneurons) (3), where it inhibits GABA release. Functional CB₁ receptors reside as well on terminals of glutamatergic neurons in several brain regions, where they inhibit glutamate release (4). In concert with this well-established neuromodulatory function, the CB₁ receptor protects neurons in many different animal models of acute brain damage and chronic neurodegeneration, which, during recent years, has raised hope about the possible clinical use of cannabinoids as neuroprotective drugs, especially in still unexplored conditions such as Alzheimer’s disease, Huntington disease (HD), amyotrophic lateral sclerosis, and stroke (5–7). However, the assessment of the physiological relevance and therapeutic potential of the CB₁ receptor in neurological diseases is hampered, at least in part, by the lack of knowledge of the neuron-population specificity of CB₁ receptor action. Here, by using various genetic models of CB₁ receptor loss of function, together with pharmacological and pharmacogenetic tools, we show that a unique population of CB₁ receptors, namely that located on glutamatergic terminals, plays an indispensable role in the neuroprotective activity of the endocannabinoid system in the mouse brain. This finding opens a new conceptual view on how the CB₁ receptor evokes neuroprotection, and provides preclinical support for improving the development of cannabinoid-based neuroprotective therapies.     

Cannabidiol for neurodegenerative disorders: important new clinical applications for this phytocannabinoid?

Cannabidiol (CBD) is a phytocannabinoid with therapeutic properties for numerous disorders exerted through molecular mechanisms that are yet to be completely identified. CBD acts in some experimental models as an anti-inflammatory, anticonvulsant, anti-oxidant, anti-emetic, anxiolytic and antipsychotic agent, and is therefore a potential medicine for the treatment of neuroinflammation, epilepsy, oxidative injury, vomiting and nausea, anxiety and schizophrenia, respectively. The neuroprotective potential of CBD, based on the combination of its anti-inflammatory and anti-oxidant properties, is of particular interest and is presently under intense preclinical research in numerous neurodegenerative disorders. In fact, CBD combined with Δ⁹-tetrahydrocannabinol is already under clinical evaluation in patients with Huntington''s disease to determine its potential as a disease-modifying therapy. The neuroprotective properties of CBD do not appear to be exerted by the activation of key targets within the endocannabinoid system for plant-derived cannabinoids like Δ⁹-tetrahydrocannabinol, i.e. CB₁ and CB₂ receptors, as CBD has negligible activity at these cannabinoid receptors, although certain activity at the CB₂ receptor has been documented in specific pathological conditions (i.e. damage of immature brain). Within the endocannabinoid system, CBD has been shown to have an inhibitory effect on the inactivation of endocannabinoids (i.e. inhibition of FAAH enzyme), thereby enhancing the action of these endogenous molecules on cannabinoid receptors, which is also noted in certain pathological conditions. CBD acts not only through the endocannabinoid system, but also causes direct or indirect activation of metabotropic receptors for serotonin or adenosine, and can target nuclear receptors of the PPAR family and also ion channels.     

Cannabinoid CB2 receptor agonists protect the striatum against malonate toxicity: Relevance for Huntington's disease

Cannabinoid agonists might serve as neuroprotective agents in neurodegenerative disorders. Here, we examined this hypothesis in a rat model of Huntington's disease (HD) generated by intrastriatal injection of the mitochondrial complex II inhibitor malonate. Our results showed that only compounds able to activate CB₂ receptors were capable of protecting striatal projection neurons from malonate‐induced death. That CB₂ receptor agonists are neuroprotective was confirmed by using the selective CB₂ receptor antagonist, SR144528, and by the observation that mice deficient in CB₂ receptor were more sensitive to malonate than wild‐type animals. CB₂ receptors are scarce in the striatum in healthy conditions, but they are markedly upregulated after the lesion with malonate. Studies of double immunostaining revealed a significant presence of CB₂ receptors in cells labeled with the marker of reactive microglia OX‐42, and also in cells labeled with GFAP (a marker of astrocytes). We further showed that the activation of CB₂ receptors significantly reduced the levels of tumor necrosis factor‐α (TNF‐α) that had been increased by the lesion with malonate. In summary, our results demonstrate that stimulation of CB₂ receptors protect the striatum against malonate toxicity, likely through a mechanism involving glial cells, in particular reactive microglial cells in which CB₂ receptors would be upregulated in response to the lesion. Activation of these receptors would reduce the generation of proinflammatory molecules like TNF‐α. Altogether, our results support the hypothesis that CB₂ receptors could constitute a therapeutic target to slowdown neurodegeneration in HD. © 2008 Wiley‐Liss, Inc.     

Sativex-like Combination of Phytocannabinoids is Neuroprotective in Malonate-Lesioned Rats, an Inflammatory Model of Huntington's Disease: Role of CB(1) and CB(2) Receptors

We have investigated whether a 1:1 combination of botanical extracts enriched in either Δ(9)-tetrahydrocannabinol (Δ(9)-THC) or cannabidiol (CBD), which are the main constituents of the cannabis-based medicine Sativex, is neuroprotective in Huntington's disease (HD), using an experimental model of this disease generated by unilateral lesions of the striatum with the mitochondrial complex II inhibitor malonate. This toxin damages striatal neurons by mechanisms that primarily involve apoptosis and microglial activation. We monitored the extent of this damage and the possible preservation of the striatal parenchyma by treatment with a Sativex-like combination of phytocannabinoids using different histological and biochemical markers. Results were as follows: (i) malonate increased the volume of edema measured by in vivo NMR imaging and the Sativex-like combination of phytocannabinoids partially reduced this increase; (ii) malonate reduced the number of Nissl-stained cells, while enhancing the number of degenerating cells stained with FluoroJade-B, and the Sativex-like combination of phytocannabinoids reversed both effects; (iii) malonate caused a strong glial activation (i.e., reactive microglia labeled with Iba-1, and astrogliosis labeled with GFAP) and the Sativex-like combination of phytocannabinoids attenuated both responses; and (iv) malonate increased the expression of inducible nitric oxide synthase and the neurotrophin IGF-1, and both responses were attenuated after the treatment with the Sativex-like combination of phytocannabinoids. We also wanted to establish whether targets within the endocannabinoid system (i.e., CB(1) and CB(2) receptors) are involved in the beneficial effects induced in this model by the Sativex-like combination of phytocannabinoids. This we did using selective antagonists for both receptor types (i.e., SR141716 and AM630) combined with the Sativex-like phytocannabinoid combination. Our results indicated that the effects of this combination are blocked by these antagonists and hence that they do result from an activation of both CB(1) and CB(2) receptors. In summary, this study provides preclinical evidence in support of a beneficial effect of the cannabis-based medicine Sativex as a neuroprotective agent capable of delaying signs of disease progression in a proinflammatory model of HD, which adds to previous data obtained in models priming oxidative mechanisms of striatal injury. However, the interest here is that, in contrast with these previous data, we have now obtained evidence that both CB(1) and CB(2) receptors appear to be involved in the effects produced by a Sativex-like phytocannabinoid combination, thus stressing the broad-spectrum properties of Sativex that may combine activity at the CB(1) and/or CB(2) receptors with cannabinoid receptor-independent actions.