ABSTRACT
Translation of muscarinic acetylcholine receptor (mAChR) agonists into clinically used therapeutic agents has been difficult due to their poor subtype selectivity. M4 mAChR subtype-selective positive allosteric modulators (PAMs) may provide better therapeutic outcomes, hence investigating their detailed pharmacological properties is crucial to advancing them into the clinic. Herein, we report the synthesis and comprehensive pharmacological evaluation of M4 mAChR PAMs structurally related to 1e, Me-C-c, [11C]MK-6884 and [18F]12. Our results show that small structural changes to the PAMs can result in pronounced differences to baseline, potency (pEC50) and maximum effect (Emax) measures in cAMP assays when compared to the endogenous ligand acetylcholine (ACh) without the addition of the PAMs. Eight selected PAMs were further assessed to determine their binding affinity and potential signalling bias profile between cAMP and ß-arrestin 2 recruitment. These rigorous analyses resulted in the discovery of the novel PAMs, 6k and 6l, which exhibit improved allosteric properties compared to the lead compound, and probative in vivo exposure studies in mice confirmed that they maintain the ability to cross the blood-brain barrier, making them more suitable for future preclinical assessment.
Subject(s)
Acetylcholine , Receptors, Muscarinic , Mice , Animals , Cricetinae , Allosteric Regulation , Receptors, Muscarinic/metabolism , Acetylcholine/metabolism , Pyridines/pharmacology , Pyridines/chemistry , Signal Transduction , CHO CellsABSTRACT
Allosteric modulation of G protein-coupled receptors (GPCRs) is a major paradigm in drug discovery. Despite decades of research, a molecular-level understanding of the general principles that govern the myriad pharmacological effects exerted by GPCR allosteric modulators remains limited. The M4 muscarinic acetylcholine receptor (M4 mAChR) is a validated and clinically relevant allosteric drug target for several major psychiatric and cognitive disorders. In this study, we rigorously quantified the affinity, efficacy, and magnitude of modulation of two different positive allosteric modulators, LY2033298 (LY298) and VU0467154 (VU154), combined with the endogenous agonist acetylcholine (ACh) or the high-affinity agonist iperoxo (Ipx), at the human M4 mAChR. By determining the cryo-electron microscopy structures of the M4 mAChR, bound to a cognate Gi1 protein and in complex with ACh, Ipx, LY298-Ipx, and VU154-Ipx, and applying molecular dynamics simulations, we determine key molecular mechanisms underlying allosteric pharmacology. In addition to delineating the contribution of spatially distinct binding sites on observed pharmacology, our findings also revealed a vital role for orthosteric and allosteric ligand-receptor-transducer complex stability, mediated by conformational dynamics between these sites, in the ultimate determination of affinity, efficacy, cooperativity, probe dependence, and species variability. There results provide a holistic framework for further GPCR mechanistic studies and can aid in the discovery and design of future allosteric drugs.
Subject(s)
Receptor, Muscarinic M4 , Receptors, Muscarinic , Humans , Acetylcholine/metabolism , Allosteric Regulation , Allosteric Site , Cryoelectron Microscopy , Ligands , Receptor, Muscarinic M4/agonists , Receptor, Muscarinic M4/metabolismABSTRACT
BACKGROUND: Selective serotonin reuptake inhibitors (SSRIs) are the most common class of medicines used for the treatment of major depression. Recent studies have reported an association between depression and inflammation and suggested the significant effects of SSRIs on inflammatory processes. METHODS: The current study aimed to evaluate the effects of fluoxetine, an SSRI, on the level of inflammatory cytokines, including interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), in the rat serum and RAW264.7 mouse macrophage cell line, using ELISA sandwich assays. Also, the expression of inflammatory genes, including JAK/STAT3 and TLR4/JNK, was examined in macrophages, using real-time quantitative reverse transcription PCR to determine the potential mechanism of fluoxetine in inflammation. The rats received fluoxetine (10, 20, and 40 mg/kg) 30 min before lipopolysaccharide (LPS) treatment for 90 min. The cells received different doses of fluoxetine (5, 10, and 20 µg/mL) before stimulation with LPS for 24 or 48 h. RESULTS: The serum concentrations of IL-1ß, IL-6, and TNF-α were reduced in rats and cells treated with fluoxetine. Following fluoxetine administration, the expression of JAK/STAT3 and TLR4/JNK genes was significantly decreased in the RAW264.7 cells treated with LPS for 24 h. However, after 48 h of treatment with LPS, fluoxetine failed to diminish the elevated expression of JAK and JNK genes, while it significantly decreased the expression of STAT3 and TLR4 genes. CONCLUSION: The findings revealed that fluoxetine has anti-inflammatory properties, mainly due to the reduction of inflammatory cytokines and inhibition of JAK/STAT3 and TLR4/JNK gene expression in macrophages.
Subject(s)
Fluoxetine , Tumor Necrosis Factor-alpha , Animals , Mice , Rats , Cytokines/metabolism , Fluoxetine/pharmacology , Gene Expression , Inflammation/drug therapy , Inflammation/genetics , Inflammation/metabolism , Inflammation Mediators , Interleukin-6/genetics , Interleukin-6/metabolism , Lipopolysaccharides/pharmacology , Selective Serotonin Reuptake Inhibitors/pharmacology , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/metabolism , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Many dementias are propagated through the spread of "prion-like" misfolded proteins. This includes prion diseases themselves (such as Creutzfeldt-Jakob disease) and Alzheimer's disease (AD), for which no treatments are available to slow or stop progression. The M1 acetylcholine muscarinic receptor (M1 receptor) is abundant in the brain, and its activity promotes cognitive function in preclinical models and in patients with AD. Here, we investigated whether activation of the M1 receptor might slow the progression of neurodegeneration associated with prion-like misfolded protein in a mouse model of prion disease. Proteomic and transcriptomic analysis of the hippocampus revealed that this model had a molecular profile that was similar to that of human neurodegenerative diseases, including AD. Chronic enhancement of the activity of the M1 receptor with the positive allosteric modulator (PAM) VU0486846 reduced the abundance of prion-induced molecular markers of neuroinflammation and mitochondrial dysregulation in the hippocampus and normalized the abundance of those associated with neurotransmission, including synaptic and postsynaptic signaling components. PAM treatment of prion-infected mice prolonged survival and maintained cognitive function. Thus, allosteric activation of M1 receptors may reduce the severity of neurodegenerative diseases caused by the prion-like propagation of misfolded protein.
Subject(s)
Alzheimer Disease , Neurodegenerative Diseases , Prion Diseases , Prions , Humans , Animals , Mice , Prions/genetics , Neurodegenerative Diseases/genetics , Pathology, Molecular , Proteomics , Prion Diseases/genetics , Alzheimer Disease/genetics , Alzheimer Disease/pathology , Receptor, Muscarinic M1/genetics , Receptor, Muscarinic M1/metabolismABSTRACT
This study investigated the structure-activity relationships of 4-phenylpyridin-2-one and 6-phenylpyrimidin-4-one M1 muscarinic acetylcholine receptor (M1 mAChRs) positive allosteric modulators (PAMs). The presented series focuses on modifications to the core and top motif of the reported leads, MIPS1650 (1) and MIPS1780 (2). Profiling of our novel analogues showed that these modifications result in more nuanced effects on the allosteric properties compared to our previous compounds with alterations to the biaryl pendant. Further pharmacological characterisation of the selected compounds in radioligand binding, IP1 accumulation and ß-arrestinâ 2 recruitment assays demonstrated that, despite primarily acting as affinity modulators, the PAMs displayed different pharmacological properties across the two cellular assays. The novel PAM 7 f is a potential lead candidate for further development of peripherally restricted M1 PAMs, due to its lower blood-brain-barrier (BBB) permeability and improved exposure in the periphery compared to lead 2.
Subject(s)
Pyridones/chemistry , Receptor, Muscarinic M1/metabolism , Allosteric Regulation/drug effects , Animals , Blood-Brain Barrier/drug effects , Blood-Brain Barrier/metabolism , Half-Life , Humans , Mice , Permeability/drug effects , Pyridones/metabolism , Pyridones/pharmacology , Receptor, Muscarinic M1/chemistry , Structure-Activity RelationshipABSTRACT
Designer receptors exclusively activated by designer drugs (DREADDs) have been successfully employed to activate signaling pathways associated with specific muscarinic acetylcholine receptor (mAChR) subtypes. The M1 DREADD mAChR displays minimal responsiveness to the endogenous agonist acetylcholine (ACh) but responds to clozapine-N-oxide (CNO), an otherwise pharmacologically inert ligand. We have previously shown that benzyl quinolone carboxylic acid (BQCA), an M1 mAChR positive allosteric modulator (PAM), can rescue ACh responsiveness at these receptors. However, whether this effect is chemotype specific or applies to next-generation M1 PAMs with distinct scaffolds is unknown. Here, we reveal that new M1 PAMs restore ACh function at the M1 DREADD while modulating ACh binding at the M1 wild-type mAChR. Importantly, we demonstrate that the modulation of ACh function by M1 PAMs is translated in vivo using transgenic M1 DREADD mice. Our data provide important insights into mechanisms that define allosteric ligand modulation of agonist affinity vs efficacy and how these effects play out in the regulation of in vivo responses.
Subject(s)
Acetylcholine , Receptor, Muscarinic M1 , Allosteric Regulation , Animals , CHO Cells , Cricetinae , Cricetulus , Mice , Receptor, Muscarinic M1/geneticsABSTRACT
BACKGROUND AND PURPOSE: Negative allosteric modulators (NAMs) that target the calcium-sensing receptor (CaS receptor) were originally developed for the treatment of osteoporosis by stimulating the release of endogenous parathyroid hormone, but failed in human clinical trials. Several chemically and structurally distinct NAM scaffolds have been described, but it is not known how these different scaffolds interact with the CaS receptor to inhibit receptor signalling in response to agonists. EXPERIMENTAL APPROACH: In the present study, we used a mutagenesis approach combined with analytical pharmacology and computational modelling to probe the binding sites of four distinct NAM scaffolds. KEY RESULTS: Although all four scaffolds bind to the 7-transmembrane and/or extracellular or intracellular loops, they occupy distinct regions, as previously shown for positive allosteric modulators of the CaS receptor. Furthermore, different NAM scaffolds mediate negative allosteric modulation via distinct amino acid networks. CONCLUSION AND IMPLICATIONS: These findings aid our understanding of how different NAMs bind to and inhibit the CaS receptor. Elucidation of allosteric binding sites in the CaS receptor has implications for the discovery of novel allosteric modulators.
Subject(s)
Receptors, Calcium-Sensing , Allosteric Regulation , Allosteric Site , Binding Sites , HEK293 Cells , HumansABSTRACT
Targeting allosteric sites of the M1 muscarinic acetylcholine receptor (mAChR) is an enticing approach to overcome the lack of receptor subtype selectivity observed with orthosteric ligands. This is a promising strategy for obtaining novel therapeutics to treat cognitive deficits observed in Alzheimer's disease and schizophrenia, while reducing the peripheral side effects such as seen in the current treatment regimes, which are non-subtype selective. We previously described compound 2, the first positive allosteric modulator (PAM) of the M1 mAChR based on a 6-phenylpyrimidin-4-one scaffold, which has been further developed in this study. Herein, we present the synthesis, characterization, and pharmacological evaluation of a series of 6-phenylpyrimidin-4-ones with modifications to the 4-(1-methylpyrazol-4-yl)benzyl pendant. Selected compounds, BQCA, 1, 2, 9i, 13, 14b, 15c, and 15d, were further profiled in terms of their allosteric affinity, cooperativity with acetylcholine (ACh), and intrinsic efficacy. Additionally, 2 and 9i were tested in mouse primary cortical neurons, displaying various degrees of intrinsic agonism and potentiation of the acetylcholine response. Overall, the results suggest that the pendant moiety is important for allosteric binding affinity and the direct agonistic efficacy of the 6-phenylpyrimidin-4-one based M1 mAChR PAMs.
Subject(s)
Pyrimidines/chemical synthesis , Pyrimidines/pharmacology , Quinolines/chemical synthesis , Quinolines/pharmacology , Receptor, Muscarinic M1/agonists , Receptor, Muscarinic M1/physiology , Allosteric Regulation/drug effects , Allosteric Regulation/physiology , Animals , CHO Cells , Cells, Cultured , Cricetinae , Cricetulus , Crystallography, X-Ray/methods , MiceABSTRACT
Subtype-selective allosteric modulation of the M1 muscarinic acetylcholine (ACh) receptor (M1 mAChR) is an attractive approach for the treatment of numerous disorders, including cognitive deficits. The discovery of benzyl quinolone carboxylic acid, BQCA, a selective M1 mAChR positive allosteric modulator (PAM), spurred the subsequent development of newer generation M1 PAMs representing diverse chemical scaffolds, different pharmacodynamic properties and, in some instances, improved pharmacokinetics. Key exemplar molecules from such efforts include PF-06767832 (N-((3R,4S)-3-hydroxytetrahydro-2H-pyran-4-yl)-5-methyl-4-(4-(thiazol-4-yl)benzyl)pyridine-2-carboxamide), VU6004256 (4,6-difluoro-N-(1S,2S)-2-hydroxycyclohexyl-1-((6-(1-methyl-1H-pyrazol-4-yl)pyridine-3-yl)methyl)-1H-indole-3-carboxamide) and MIPS1780 (3-(2-hydroxycyclohexyl)-6-(2-((4-(1-methyl-1H-pyrazol-4-yl)-benzyl)oxy)phenyl)pyrimidin-4(3H)-one). Given these diverse scaffolds and pharmacodynamics, the current study combined pharmacological analysis and site-directed mutagenesis to explore the potential binding site and function of newer M1 mAChR PAMs relative to BQCA. Interestingly, the mechanism of action of the novel PAMs was consistent with a common model of allostery, as previously described for BQCA. Key residues involved in the activity of BQCA, including Y179 in the second extracellular loop (ECL) and W4007.35 in transmembrane domain (TM) 7, were critical for the activity of all PAMs tested. Overall, our data indicate that structurally distinct PAMs share a similar binding site with BQCA, specifically, an extracellular allosteric site defined by residues in TM2, TM7 and ECL2. These findings provide valuable insights into the structural basis underlying modulator binding, cooperativity and signaling at the M1 mAChR, which is essential for the rational design of PAMs with tailored pharmacological properties.
Subject(s)
Acetylcholine/metabolism , Muscarinic Agonists/metabolism , Receptor, Muscarinic M1/genetics , Receptor, Muscarinic M1/metabolism , Acetylcholine/pharmacology , Allosteric Regulation/drug effects , Allosteric Regulation/physiology , Allosteric Site/drug effects , Allosteric Site/physiology , Animals , Binding Sites/drug effects , Binding Sites/physiology , CHO Cells , Cholinergic Agonists/metabolism , Cholinergic Agonists/pharmacology , Cricetinae , Cricetulus , Dose-Response Relationship, Drug , Humans , Muscarinic Agonists/pharmacology , Receptor, Muscarinic M1/agonistsABSTRACT
Positive allosteric modulators (PAMs) that target the M1 muscarinic acetylcholine (ACh) receptor (M1 mAChR) are potential treatments for cognitive deficits in conditions such as Alzheimer disease and schizophrenia. We recently reported novel 4-phenylpyridine-2-one and 6-phenylpyrimidin-4-one M1 mAChR PAMs with the potential to display different modes of positive allosteric modulation and/or agonism but whose molecular mechanisms of action remain undetermined. The current study compared the pharmacology of three such novel PAMs with the prototypical first-generation PAM, benzyl quinolone carboxylic acid (BQCA), in a recombinant Chinese hamster ovary (CHO) cell line stably expressing the human M1 mAChR. Interactions between the orthosteric agonists and the novel PAMs or BQCA suggested their allosteric effects were solely governed by modulation of agonist affinity. The greatest degree of positive co-operativity was observed with higher efficacy agonists, whereas minimal potentiation was observed when the modulators were tested against the lower efficacy agonist, xanomeline. Each PAM was investigated for its effects on the endogenous agonist ACh on three different signaling pathways [extracellular signal-regulated kinases 1/2 phosphorylation, inositol monophosphate (IP1) accumulation, and ß-arrestin-2 recruitment], revealing that the allosteric potentiation generally tracked with the efficiency of stimulus-response coupling, and that there was little pathway bias in the allosteric effects. Thus, despite the identification of novel allosteric scaffolds targeting the M1 mAChR, the molecular mechanism of action of these compounds is largely consistent with a model of allostery previously described for BQCA, suggesting that this may be a more generalized mechanism for M1 mAChR PAM effects than previously appreciated.
Subject(s)
Allosteric Regulation/drug effects , Pyridones/pharmacology , Receptor, Muscarinic M1/metabolism , Acetylcholine/metabolism , Animals , CHO Cells , Cholinergic Agonists/pharmacology , Cricetulus , Humans , Inositol Phosphates/metabolism , Pyridines/pharmacology , Quinolones/pharmacology , Signal Transduction/drug effects , Thiadiazoles/pharmacologyABSTRACT
Targeting allosteric sites at M1 muscarinic acetylcholine receptors is a promising strategy for the treatment of Alzheimer's disease. Positive allosteric modulators not only may potentiate binding and/or signaling of the endogenous agonist acetylcholine (ACh) but also may possess direct agonist activity (thus referred to as PAM-agonists). Recent studies suggest that PAM-agonists with robust intrinsic efficacy are more likely to produce adverse effects in vivo. Herein we present the synthesis and pharmacological evaluation of a series of pyrrole-3-carboxamides with a diverse range of allosteric profiles. We proposed structural modifications at top, core, or pendant moieties of a prototypical molecule. Although generally there was a correlation between the degree of agonist activity and the modulatory potency of the PAMs, some derivatives displayed weak intrinsic efficacy yet maintained strong allosteric modulation. We also identified molecules with the ability to potentiate mainly the affinity or both affinity and efficacy of ACh.
Subject(s)
Heterocyclic Compounds/chemical synthesis , Heterocyclic Compounds/pharmacology , Muscarinic Agonists/chemical synthesis , Muscarinic Agonists/pharmacology , Receptor, Muscarinic M1/agonists , Acetylcholine/pharmacology , Allosteric Regulation , Animals , CHO Cells , Cricetinae , Cricetulus , Drug Design , Humans , Inositol Phosphates/metabolism , Molecular Conformation , Structure-Activity RelationshipABSTRACT
Chemogenetic tools such as designer receptors exclusively activated by designer drugs (DREADDs) are routinely used to modulate neuronal and non-neuronal signaling and activity in a relatively noninvasive manner. The first generation of DREADDs were templated from the human muscarinic acetylcholine receptor family and are relatively insensitive to the endogenous agonist acetylcholine but instead are activated by clozapine-N-oxide (CNO). Despite the undisputed success of CNO as an activator of muscarinic DREADDs, it has been known for some time that CNO is subject to a low rate of metabolic conversion to clozapine, raising the need for alternative chemical actuators of muscarinic-based DREADDs. Here we show that DREADD agonist 21 (C21) (11-(1-piperazinyl)-5H-dibenzo[b,e][1,4]diazepine) is a potent and selective agonist at both excitatory (hM3Dq) and inhibitory (hM4Di) DREADDs and has excellent bioavailability, pharmacokinetic properties, and brain penetrability. We also show that C21-induced activation of hM3Dq and hM4Di in vivo can modulate bidirectional feeding in defined circuits in mice. These results indicate that C21 represents an alternative to CNO for in vivo studies where metabolic conversion of CNO to clozapine is a concern.
ABSTRACT
Negative allosteric modulators (NAMs) of the human calcium-sensing receptor (CaSR) have previously failed to show efficacy in human osteoporosis clinical trials, but there is now significant interest in repurposing these drugs for hypocalcemic disorders and inflammatory lung diseases. However, little is known about how CaSR NAMs inhibit the response to endogenous activators. An improved understanding of CaSR negative allosteric modulation may afford the opportunity to develop therapeutically superior CaSR-targeting drugs. In an attempt to elucidate the mechanistic and structural basis of allosteric modulation mediated by the previously reported NAM, calhex231, we herein demonstrate that calhex231 actually potentiates or inhibits the activity of multiple CaSR agonists depending on whether it occupies one or both protomers in a CaSR dimer. These findings reveal a novel mechanism of mode-switching at a Class C G protein-coupled receptor that has implications for drug discovery and potential clinical utility.
ABSTRACT
Drugs that allosterically target the human calcium-sensing receptor (CaSR) have substantial therapeutic potential, but are currently limited. Given the absence of high-resolution structures of the CaSR, we combined mutagenesis with a novel analytical approach and molecular modeling to develop an "enriched" picture of structure-function requirements for interaction between Ca(2+)o and allosteric modulators within the CaSR's 7 transmembrane (7TM) domain. An extended cavity that accommodates multiple binding sites for structurally diverse ligands was identified. Phenylalkylamines bind to a site that overlaps with a putative Ca(2+)o-binding site and extends towards an extracellular vestibule. In contrast, the structurally and pharmacologically distinct AC-265347 binds deeper within the 7TM domains. Furthermore, distinct amino acid networks were found to mediate cooperativity by different modulators. These findings may facilitate the rational design of allosteric modulators with distinct and potentially pathway-biased pharmacological effects.
Subject(s)
Pharmaceutical Preparations/chemistry , Pharmaceutical Preparations/metabolism , Receptors, Calcium-Sensing/chemistry , Receptors, Calcium-Sensing/metabolism , Allosteric Regulation , Amines/chemistry , Amino Acid Sequence , Amino Acids/genetics , Binding Sites , Calcium/metabolism , HEK293 Cells , Humans , Mutation/genetics , Receptors, Calcium-Sensing/genetics , Signal Transduction , Structure-Activity RelationshipABSTRACT
CB1 cannabinoid receptors (CB1Rs) are attractive therapeutic targets for numerous central nervous system disorders. However, clinical application of cannabinoid ligands has been hampered owing to their adverse on-target effects. Ligand-biased signaling from, and allosteric modulation of, CB1Rs offer pharmacological approaches that may enable the development of improved CB1R drugs, through modulation of only therapeutically desirable CB1R signaling pathways. There is growing evidence that CB1Rs are subject to ligand-biased signaling and allosterism. Therefore, in the present study, we quantified ligand-biased signaling and allosteric modulation at CB1Rs. Cannabinoid agonists displayed distinct biased signaling profiles at CB1Rs. For instance, whereas 2-arachidonylglycerol and WIN55,212-2 [(R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone] showed little preference for inhibition of cAMP and phosphorylation of extracellular signal-regulated kinase 1/2 (pERK1/2), N-arachidonoylethanolamine (anandamide), methanandamide, CP55940 [2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]-5-(2-methyloctan-2-yl)phenol], and HU-210 [11-hydroxy-Δ(8)-THC-dimethylheptyl] were biased toward cAMP inhibition. The small-molecule allosteric modulator Org27569 [5-chloro-3-ethyl-1H-indole-2-carboxylic acid [2-(4-piperidin-1-yl-phenyl)ethyl]amide] displayed biased allosteric effects by blocking cAMP inhibition mediated by all cannabinoid ligands tested, at the same time having little or no effect on ERK1/2 phosphorylation mediated by a subset of these ligands. Org27569 also displayed negative binding cooperativity with [(3)H]SR141716A [5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide]; however, it had minimal effects on binding of cannabinoid agonists. Furthermore, we highlight the need to validate the reported allosteric effects of the endogenous ligands lipoxin A4 and pregnenolone at CB1Rs. Pregnenolone but not lipoxin A4 displaced [(3)H]SR141716A, but there was no functional interaction between either of these ligands and cannabinoid agonists. This study demonstrates an approach to validating and quantifying ligand-biased signaling and allosteric modulation at CB1Rs, revealing ligand-biased "fingerprints" that may ultimately allow the development of improved CB1R-targeted therapies.
Subject(s)
Cannabinoid Receptor Agonists/pharmacology , Indoles/pharmacology , Piperidines/pharmacology , Pyrazoles/pharmacology , Receptor, Cannabinoid, CB1/metabolism , Allosteric Regulation , Animals , Benzoxazines/pharmacology , CHO Cells , Cricetulus , Cyclohexanols/pharmacology , Dronabinol/analogs & derivatives , Dronabinol/pharmacology , Humans , MAP Kinase Signaling System/drug effects , Morpholines/pharmacology , Naphthalenes/pharmacology , Phosphorylation , Receptor, Cannabinoid, CB1/chemistry , Rimonabant , Signal Transduction/drug effectsABSTRACT
The aim of present study was to assess the effects of chronic administration of calcium-magnesium soft gels on the development of morphine tolerance and dependence in mice. Tolerance was assessed using the tail-pinch test and withdrawal signs of morphine were precipitated by injecting naloxone 2 h after the final morphine injection. CalMag capsules were given in final doses of 50/25, 25/12.5 and 12.5/6.25 mg/kg based on calcium/magnesium ratio. Similar doses of Ca and/or Mg were prepared, separately. CalMag at 50/25, 25/12.5 mg/kg and the mixture of calcium and magnesium (Ca + Mg) at 50/25, 25/12.5, 12.5/6.25 mg/kg and calcium at 50, 25 mg/kg significantly reduced the number of jumps. The number of standings was only reduced after the administration of CalMag at 50/25 mg/kg and Ca + Mg at 25/12.5 mg/kg. The development of morphine tolerance was prevented in all drug-treated groups, except the one which received 6.25 mg/kg Mg. The data suggested that combination of calcium and magnesium at 50/25 and 25/12.5 mg/kg prevented the development of tolerance and dependence. It seems that other ingredients of CalMag capsules do not have an important effect on preventing tolerance and withdrawal signs. Compared to the acute effects, chronic administration of Cal-Mag allowed the effective dose to be reduced. Unlike the acute treatment, chronic administration of calcium alone was effective in reducing morphine tolerance and dependence, and magnesium had no significant effect on withdrawal signs, suggestive of some pharmacological adaptations.