Efficacy In Rodent Models Research Articles

Targeting peripheral ϰ-opioid receptors for the non-addictive treatment of pain.

Drug addiction to prescription mu-opioid agonists used in the setting of pain is a major public health threat, affecting millions of Americans. Kappa opioid agonists (KOAs) may serve as a possible solution. KOAs have demonstrated indistinguishable analgesic activity relative to mu-opioid agonists in models of acute and chronic pain; however, conventional KOAs suffer from central nervous system-mediated psychoactive side-effects. In this review, we discuss our efforts, as well as other's efforts, in developing peripherally-restricted kappa opioid agonists with retained or improved efficacy in rodent models of pain. Results indicate that our lead compound JT09 acts as efficacious as morphine in alleviating peripheral pain, while failing to produce undesired central nervous system-mediated side-effects. In this review, we discuss our former results and future directions.

Beneficial Effects of Trehalose on Striatal Dopaminergic Deficits in Rodent and Primate Models of Synucleinopathy in Parkinson's Disease.

Disease modification in Parkinson's disease (PD) is an unmet medical need. In the current study, we evaluated trehalose, a safe and well-tolerated disaccharide that has previously demonstrated efficacy in rodent models of neurodegenerative diseases, including PD. In a rat model of PD, based on delivery of adeno-associated virus serotype 1/2 containing the mutated human A53T α-synuclein gene (AAV1/2-hourA53T-aSyn) to the substantia nigra (SN), we showed that rats administered trehalose (2.67 g/kg per day, by mouth) for 6 weeks had less forelimb asymmetry (93% reduction) and higher striatal dopamine (54% increase) compared with rats receiving vehicle. In a pharmacokinetic study, we determined that efficacy was associated with plasma C max of 8900 ng/ml and area under the curve from time 0 to infinity (AUC0-inf) of 11,136 hour⋅ng/ml. We then showed, in macaques, that oral administration of trehalose (2.67 g/kg per day) produced plasma exposures of similar magnitude, with plasma C max of 10,918 ng/ml and AUC0-inf of 27,445 hour⋅ng/ml. In a macaque model of PD, also based on delivery of AAV1/2-hourA53T-aSyn to the SN, trehalose (2.67 g/kg per day, by mouth), administered for 142 days, produced higher striatal dopamine (by 39%) and dopamine transporter levels (by 50%), compared with macaques receiving vehicle. In neither model did trehalose treatment prevent loss of tyrosine hydroxylase (TH) positive (TH+ve) cells in the SN or alter α-synuclein levels in the striatum. These studies demonstrated that trehalose reduces striatal dopaminergic deficits in a rodent and macaque model of synucleinopathy in PD. Furthermore, we have determined the pharmacokinetic parameters associated with efficacy, and thus defined exposures to target in future clinical trials.

Selective allosteric modulation of muscarinic acetylcholine receptors for the treatment of schizophrenia and substance use disorders.

Muscarinic acetylcholine receptor (mAChRs) subtypes represent exciting new targets for the treatment of schizophrenia and substance use disorder (SUD). Recent advances in the development of subtype-selective allosteric modulators have revealed promising effects in preclinical models targeting the different symptoms observed in schizophrenia and SUD. M1 PAMs display potential for addressing the negative and cognitive symptoms of schizophrenia, while M4 PAMs exhibit promise in treating preclinical models predictive of antipsychotic-like activity. In SUD, there is increasing support for modulation of mesocorticolimbic dopaminergic circuitry involved in SUD with selective M4 mAChR PAMs or M5 mAChR NAMs. Allosteric modulators of these mAChR subtypes have demonstrated efficacy in rodent models of cocaine and ethanol seeking, with indications that these ligand may also be useful for other substances of abuse, as well as in various stages in the cycle of addiction. Importantly, allosteric modulators of the different mAChR subtypes may provide viable treatment options, while conferring greater subtype specificity and corresponding enhanced therapeutic index than orthosteric muscarinic ligands and maintaining endogenous temporo-spatial ACh signaling. Overall, subtype specific mAChR allosteric modulators represent important novel therapeutic mechanisms for schizophrenia and SUD.

PBF509, an Adenosine A2A Receptor Antagonist With Efficacy in Rodent Models of Movement Disorders.

Adenosine A2A receptor (A2AR) antagonists have emerged as complementary non-dopaminergic drugs to alleviate Parkinson’s disease (PD) symptomatology. Here, we characterize a novel non-xhantine non-furan A2AR antagonist, PBF509, as a potential pro-dopaminergic drug for PD management. First, PBF509 was shown to be a highly potent ligand at the human A2AR, since it antagonized A2AR agonist-mediated cAMP accumulation and impedance responses with KB values of 72.8 ± 17.4 and 8.2 ± 4.2 nM, respectively. Notably, these results validated our new A2AR-based label-free assay as a robust and sensitive approach to characterize A2AR ligands. Next, we evaluated the efficacy of PBF509 reversing motor impairments in several rat models of movement disorders, including catalepsy, tremor, and hemiparkinsonism. Thus, PBF509 (orally) antagonized haloperidol-mediated catalepsy, reduced pilocarpine-induced tremulous jaw movements and potentiated the number of contralateral rotations induced by L-3,4-dihydroxyphenylalanine (L-DOPA) in unilaterally 6-OHDA-lesioned rats. Moreover, PBF509 (3 mg/kg) inhibited L-DOPA-induced dyskinesia (LID), showing not only its efficacy on reversing parkinsonian motor impairments but also acting as antidyskinetic agent. Overall, here we describe a new orally selective A2AR antagonist with potential utility for PD treatment, and for some of the side effects associated to the current pharmacotherapy (i.e., dyskinesia).

Novel agonist of α4β2* neuronal nicotinic receptor with antinociceptive efficacy in rodent models of acute and chronic pain.

ObjectiveTo demonstrate the antinociceptive and antihypersensitivity mechanisms of Cris-104 (1-{2-[5-(4-fluorophenyl)–1H-pyrazol-4-yl]ethyl}piperidine), a novel selective α4β2* nicotinic acetylcholine receptor (nAChR) agonist, in rodent acute/inflammatory and chronic pain models.Materials and methodsHot-plate and formalin tests in mice were used to examine Cris-104-induced antinociceptive effects on thermal/inflammatory pain. Cris-104 effects on hypersensitivity, norepinephrine (NE) release in the spinal dorsal horn, and neuronal activity in the locus coeruleus (LC) were examined in rats with lumbar spinal nerve ligation using behavioral, microdialysis, and extracellular recording methods. Cris-104 effects on spontaneous locomotion were examined in an open-field test.ResultsCris-104 induced dose-dependent antinociception effects in hot-plate and formalin tests, and these effects were blocked by the general nAChR antagonist mecamylamine, the selective α4β2* nAChR antagonist dihydro-beta-erythroidine, and the α2-adrenoceptor antagonist yohimbine, but not by the α1-adrenoceptor antagonist prazosin. Systemic and spinally perfused Cris-104 increased NE concentrations in microdialysates from the spinal cord in both normal and SNL rats. Systemic Cris-104 increased neuronal activity in the LC of normal rats. Mecamylamine blocked the effects of Cris-104 on spinal NE release and LC neuronal activity. Systemic Cris-104 did not affect locomotor activity significantly.ConclusionThe α4β2 neuronal nAChR agonist, Cris-104, was effective for treatment of pain via descending noradrenergic inhibition of pain signaling.

Assessment of systemic administration of PEGylated IGF-1 in a mouse model of traumatic brain injury.

Traumatic brain injury can result in lasting cognitive dysfunction due to degeneration of mature hippocampal neurons as well as the loss of immature neurons within the dentate gyrus. While endogenous neurogenesis affords a partial recovery of the immature neuron population, hippocampal neurogenesis may be enhanced through therapeutic intervention. Insulin-like growth factor-1 (IGF-1) has the potential to improve cognitive function and promote neurogenesis after TBI, but its short half-life in the systemic circulation makes it difficult to maintain a therapeutic concentration. IGF-1 modified with a polyethylene glycol moiety (PEG-IGF-1) exhibits improved stability and half-life while retaining its ability to enter the brain from the periphery, increasing its viability as a translational approach. The goal of this study was to evaluate the ability of systemic PEG-IGF-1 administration to attenuate acute neuronal loss and stimulate the recovery of hippocampal immature neurons in brain-injured mice. In a series of studies utilizing a well-established contusion brain injury model, PEG-IGF-1 was administered subcutaneously after injury. Serum levels of PEG were verified using ELISA and histological staining was used to investigate numbers of degenerating neurons and cortical contusion size at 24 h after injury. Immunofluorescent staining was used to evaluate numbers of immature neurons at 10 d after injury. Although subcutaneous injections of PEG-IGF-1 increased serum IGF-1 levels in a dose-dependent manner, no effects were observed on cortical contusion size, neurodegeneration within the dentate gyrus, or recovery of hippocampal immature neuron numbers. In contrast to its efficacy in rodent models of neurodegenerative diseases, PEG- IGF-1 was not effective in ameliorating early neuronal loss after contusion brain trauma.

Prevention of colonic neoplasia with polyethylene glycol: A short term randomized placebo-controlled double-blinded trial.

Chemoprevention represents an attractive modality against colorectal cancer (CRC) although widespread clinical implementation of promising agents (e.g. aspirin/NSAIDS) have been stymied by both suboptimal efficacy and concerns over toxicity. This highlights the need for better agents. Several groups, including our own, have reported that the over-the-counter laxative polyethylene glycol (PEG) has remarkable efficacy in rodent models of colon carcinogenesis. In this study, we undertook the first randomized human trial to address the role of PEG in prevention of human colonic neoplasia. This was a double-blind, placebo-controlled, three-arm trial where eligible subjects were randomized to 8g PEG-3350 (n = 27) or 17g PEG-3350 (n = 24), or placebo (n = 24; maltodextrin) orally for a duration of six months. Our initial primary endpoint was rectal aberrant crypt foci (ACF) but this was changed during protocol period to rectal mucosal epidermal growth factor receptor (EGFR). Of the 87 patients randomized, 48 completed study primary endpoints and rectal EGFR unchanged PEG treatment. Rectal ACF had a trend suggesting potentially reduction with PEG treatment (pre-post change 1.7 in placebo versus -0.3 in PEG 8+ 17g doses, p = 0.108). Other endpoints (proliferation, apoptosis, expression of SNAIL and E-cadherin), previously noted to be modulated in rodent models, appeared unchanged with PEG treatment in this clinical trial. We conclude that PEG was generally well tolerated with the trial failing to meet primary efficacy endpoints. However, rectal ACFs demonstrated a trend (albeit statistically insignificant) for suppression with PEG. Moreover, all molecular assays including EGFR were unaltered with PEG underscoring issues with lack of translatability of biomarkers from preclinical to clinical trials. This data may provide the impetus for future clinical trials on PEG using more robust biomarkers of chemoprevention.Trial registration: ClinicalTrials.gov NCT00828984

Efficacy of negative allosteric modulators of mGlu2 and mGlu3 in a rodent model of Major depressive disorder

IntroductionCurrent first‐line medications for Major depressive disorder (MDD) exhibit an undesirable delay in onset and poor efficacy in the general population, and there remains a great unmet need for novel fast‐acting antidepressant medications that retain efficacy in treatment‐resistant populations. The rapid reversal of depressive‐like symptoms by ketamine and related antidepressants is thought to be mediated by acutely enhancing glutamate signaling within the prefrontal cortex (PFC). Likewise, non‐selective antagonists that inhibit metabotropic glutamate receptor subtypes 2 (mGlu2) and 3 (mGlu3), exemplified by LY341495, modulate PFC glutamate transmission and have shown efficacy in preclinical models of MDD. Previous research has relied heavily on molecules that display similar potency for inhibiting mGlu2 and mGlu3, thus the specific contributions of each individual mGlu subtype remain unclear. Recently, our lab has developed highly selective and centrally penetrant negative allosteric modulators (NAMs) for both mGlu2 and mGlu3. These exciting new tools have allowed us to test the hypothesis that selectively inhibiting mGlu2 or mGlu3 will confer rapid antidepressant efficacy in rodent models of depressive‐like behavior.MethodsWe modeled MDD symptomology in adult C57Bl/6J mice through chronic (4‐week) treatment with the stress hormone corticosterone (CORT). Anxiogenic, anhedonic, and depressive outcomes were assessed using a battery of behavioral assays. To interrogate the effects of CORT treatment on PFC function, we evaluated basal membrane and synaptic properties of layer 5 pyramidal neurons and we assessed mGlu function through a variety of pharmacological approaches.ResultsChronic CORT treatment induced anhedonic‐ and depressive‐like behaviors in adult mice while no effects on anxiety‐like behavior were observed. Acute treatment with either the mGlu2 NAM VU6001966 or the mGlu3 NAM VU0650786 reversed CORT‐induced anhedonia as assessed with the sucrose preference test. In contrast, VU6001966 and VU0650786 exhibited differential effects on despair‐like behavior in the tail suspension and forced swim tests. In slices prepared from the PFC, we found that acute application of LY341495 or VU6001966 enhanced glutamate transmission, while VU0650786 was without effect.ConclusionsWe have validated that chronic CORT treatment induces several behavioral and synaptic changes consistent with a depressive‐like phenotype. Together, our data suggest that NAMs of both mGlu2 or mGlu3 reverse stress‐induced anhedonia and should continue to be explored as potential treatments for MDD. However, the mechanisms of action of these two drug classes is distinct and warrants further study. We are continuing to explore these mechanistic differences as well as the specific neurocircuits affected by CORT exposure and mGlu2 and mGlu3 NAM treatment.Support or Funding InformationThis work was supported by National Institutes of Health (NIH) grants R01MH062646 and R37NS031373 (P.J.C.). M.E.J. was supported by NIH grant T32MH093366 and a postdoctoral fellowship through the Pharmaceutical Research and Manufacturers of America Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

FRI-077 - Combination of an FXR agonist and an ACC inhibitor increases anti-fibrotic efficacy in rodent models of NASH

FRI-077 - Combination of an FXR agonist and an ACC inhibitor increases anti-fibrotic efficacy in rodent models of NASH

43.1 GENETIC INSIGHTS LEAD TO DISCOVERY OF SELECTIVE ACTIVATORS OF MGLU1 AND MGLU3 METABOTROPIC GLUTAMATE RECEPTORS AS POTENTIAL TREATMENTS FOR SCHIZOPHRENIA

BackgroundA large number of clinical and preclinical studies suggest that dysfunction at synapses for the excitatory neurotransmitter glutamate may play a critical role in the pathophysiological changes that underlie each of the major symptom clusters observed in schizophrenia patients. Interestingly, recent genetic studies identified multiple nonsynonymous single nucleotide polymorphisms (SNPs) in the human genes encoding two specific subtypes of metabotropic glutamate (mGlu) receptors that are associated with schizophrenia. These include GRM1 and GRM3, the genes encoding for the mGlu1 and mGlu3 receptor subtypes respectively. Furthermore, postmortem studies suggest that expression of these mGlu receptor subtypes is altered brains of schizophrenia patients compared to controls. Mutations in GRM1 were identified a range of schizophrenia patients, whereas SNPs in the human gene encoding mGlu3 (GRM3) are selectively associated with poor performance on cognitive tests that are dependent on function of the prefrontal cortex (PFC) and hippocampus. These studies raise the possibility that disrupted signaling of mGlu1 and/or mGlu3 could contribute to the symptoms of schizophrenia and that selective modulators of these receptors could provide a novel approach to treatment of this disorder.MethodsWild-type and mutant forms of mGlu receptors were expressed in cell lines and used for discovery and optimization of highly selective positive allosteric modulators (PAMs) of mGlu1 and mGlu3. Optimized mGlu1 and mGlu3 PAMs were then used along with mouse genetic studies to evaluate the roles of these receptors in specific basal ganglia and forebrain circuits that have been implicated in schizophrenia. Finally, these compounds were used in animal models to assess potential efficacy in rodent models that are relevant for reducing positive, negative, and cognitive symptoms that are observed in schizophrenia patients.ResultsGRM1 mutations associated with schizophrenia were found to reduce mGlu1 signaling, suggesting that loss of function of this receptor could contribute to symptoms associated with schizophrenia. Furthermore, we found that highly selective mGlu1 PAMs reverse deficits in mGlu1 signaling observed in these mutant receptors, induced a profound reduction in dopamine release in striatal areas implicated in schizophrenia, and have robust antipsychotic-like effects that are mediated by localized inhibition of dopamine release in striatum. In contrast to existing antipsychotic medications, selective mGlu1 PAMs also improve motivation and reduce anhedonia in animal models. Interestingly, selective mGlu3 PAMs have multiple effects in the prefrontal cortex and hippocampus that would be expected to improve cognitive function. Consistent with this, highly selective mGlu3 PAMs have robust cognition-enhancing effects in rodent models that are relevant for the cognitive deficits observed in schizophrenia patients.DiscussionThese studies provide exciting new evidence that highly selective activators of two glutamate receptors identified in human genetic studies have potential utility in treatment of positive (mGLu1), negative (mGlu1), and cognitive (mGlu3) symptoms of schizophrenia patients. Furthermore, the novel mGlu1 and mGlu3 PAMs discovered in these studies provide excellent drug leads for further optimization and ultimate clinical testing.

A Human Biofilm-Disrupting Monoclonal Antibody Potentiates Antibiotic Efficacy in Rodent Models of both Staphylococcus aureus and Acinetobacter baumannii Infections.

Many serious bacterial infections are antibiotic refractory due to biofilm formation. A key structural component of biofilm is extracellular DNA, which is stabilized by bacterial proteins, including those from the DNABII family. TRL1068 is a high-affinity human monoclonal antibody against a DNABII epitope conserved across both Gram-positive and Gram-negative bacterial species. In the present study, the efficacy of TRL1068 for the disruption of biofilm was demonstrated in vitro in the absence of antibiotics by scanning electron microscopy. The in vivo efficacy of this antibody was investigated in a well-characterized catheter-induced aortic valve infective endocarditis model in rats infected with a methicillin-resistant Staphylococcus aureus (MRSA) strain with the ability to form thick biofilms, obtained from the blood of a patient with persistent clinical infection. Animals were treated with vancomycin alone or in combination with TRL1068. MRSA burdens in cardiac vegetations and within intracardiac catheters, kidneys, spleen, and liver showed significant reductions in the combination arm versus vancomycin alone (P < 0.001). A trend toward mortality reduction was also observed (P = 0.09). In parallel, the in vivo efficacy of TRL1068 against a multidrug-resistant clinical Acinetobacter baumannii isolate was explored by using an established mouse model of skin and soft tissue catheter-related biofilm infection. Catheter segments infected with A. baumannii were implanted subcutaneously into mice; animals were treated with imipenem alone or in combination with TRL1068. The combination showed a significant reduction of catheter-adherent bacteria versus the antibiotic alone (P < 0.001). TRL1068 shows excellent promise as an adjunct to standard-of-care antibiotics for a broad range of difficult-to-treat bacterial infections.

Small molecule-mediated inhibition of myofibroblast transdifferentiation for the treatment of fibrosis

Fibrosis, a disease in which excessive amounts of connective tissue accumulate in response to physical damage and/or inflammatory insult, affects nearly every tissue in the body and can progress to a state of organ malfunction and death. A hallmark of fibrotic disease is the excessive accumulation of extracellular matrix-secreting activated myofibroblasts (MFBs) in place of functional parenchymal cells. As such, the identification of agents that selectively inhibit the transdifferentiation process leading to the formation of MFBs represents an attractive approach for the treatment of diverse fibrosis-related diseases. Herein we report the development of a high throughput image-based screen using primary hepatic stellate cells that identified the antifungal drug itraconazole (ITA) as an inhibitor of MFB cell fate in resident fibroblasts derived from multiple murine and human tissues (i.e., lung, liver, heart, and skin). Chemical optimization of ITA led to a molecule (CBR-096-4) devoid of antifungal and human cytochrome P450 inhibitory activity with excellent pharmacokinetics, safety, and efficacy in rodent models of lung, liver, and skin fibrosis. These findings may serve to provide a strategy for the safe and effective treatment of a broad range of fibrosis-related diseases.

Translating Myocardial Remuscularization.

Myocardial remuscularization can be achieved by the implantation of cardiomyocyte-containing grafts. Critical steps toward clinical translation of the first generation cardiac remuscularization therapies include the choice of animal models with high predictive validity for therapeutic outcome in clinical trials. Clinically relevant remuscularization of the failing human heart remains unachieved to date. Proof-of-concept studies in small and large animal models provide compelling evidence as to the principle feasibility of myocardial remuscularization with human cardiomyocyte grafts.1,2 Clinical trials suggest myocardial protection and preservation by cell therapeutics.3,4 The mechanisms underlying the reported therapeutic effects remain under debate, but likely include the release of protective factors (growth factors, noncoding RNA) and modulation of the disease-related inflammatory response. Further studies are needed to define the paracrine milieu hypothesis and unleash its full therapeutic potential, aiming at delaying or preventing disease progression in patients with acute and subacute myocardial injury. The implantation of cardiomyocyte grafts follows a different strategy, namely remuscularization by integration of exogenously produced cardiomyocytes. Patients with chronically scarred myocardium presenting clinically with end-stage heart failure are the primary target. The suggested plug-and-play mechanism of direct cardiac remuscularization seems straight forward, but similar as for the paracrine milieu hypothesis there is a clear need for a better understanding of how, when, and where to integrate exogenous cardiomyocytes into the failing heart to achieve optimal results. Moreover, the path toward clinical application as off-the-shelf cardiomyocyte allograft therapeutics is less defined as for point-of-care autograft formulations and small chemical or biological compounds. A challenge to direct cardiac remuscularization was for many years the limited availability of bona fide cardiomyocytes. Cardiac biopsies can be used to harvest proliferative mesenchymal cells with progenitor cell properties, but without the capacity to spontaneously convert/transdifferentiate into terminally differentiated cardiomyocytes at a therapeutically relevant scale. Induced …

Rho-kinase inhibitor prevents acute injury against transient focal cerebral ischemia by enhancing the expression and function of GABA receptors in rats

Studies have shown hydroxyfasudil-mediated inhibition of Rho-kinase (ROCK) has efficacy in rodent models of focal ischemia and in in vitro systems that recapitulate stroke conditions. However, the mechanisms underlying the neuroprotective effects of the ROCK inhibitor on stroke are not well understood. In this study, we examined the role of γ-aminobutyric acid (GABA) interneurons in the effects of hydroxyfasudil on transient middle cerebral artery occlusion (tMCAO) induced acute ischemia-reperfusion injury to explore the mechanisms. tMCAO rats developed marked neurological deficits and impaired long-term potentiation; these effects were attenuated by hydroxyfasudil. Expression of GABAA and GABAB receptors after ischemia was decreased to various extents in three brain regions: the cortex, hippocampus, and striatum. Hydroxyfasudil up-regulated expression in the cortex and hippocampus but not in the striatum. Moreover, hydroxyfasudil could suppress the activation of nuclear factor-κB (NF-κB) in these regions during the acute ischemia. We further found that in tMCAO rats, hydroxyfasudil was able to abolish the decrease in GABAA and GABAB receptor phosphorylation triggered by high-frequency stimulation as well as the activation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). In summary, our results suggest ROCK inhibition improved neurological function and mitigated synaptic plasticity deficits caused by transient focal cerebral ischemia through the inactivation of two distinct pathways: (1) a ROCK-NF-κB pathway that leads to the suppression of GABAA and GABAB expression, and (2) a ROCK-PTEN pathway that decreases the phosphorylation of GABAA and GABAB, thereby affecting receptor function by triggering receptor endocytosis and the degradation of internalized receptors.

Diverse Effects on M1 Signaling and Adverse Effect Liability within a Series of M1 Ago-PAMs.

Both historical clinical and recent preclinical data suggest that the M1 muscarinic acetylcholine receptor is an exciting target for the treatment of Alzheimer's disease and the cognitive and negative symptom clusters in schizophrenia; however, early drug discovery efforts targeting the orthosteric binding site have failed to afford selective M1 activation. Efforts then shifted to focus on selective activation of M1 via either allosteric agonists or positive allosteric modulators (PAMs). While M1 PAMs have robust efficacy in rodent models, some chemotypes can induce cholinergic adverse effects (AEs) that could limit their clinical utility. Here, we report studies aimed at understanding the subtle structural and pharmacological nuances that differentiate efficacy from adverse effect liability within an indole-based series of M1 ago-PAMs. Our data demonstrate that closely related M1 PAMs can display striking differences in their in vivo activities, especially their propensities to induce adverse effects. We report the discovery of a novel PAM in this series that is devoid of observable adverse effect liability. Interestingly, the molecular pharmacology profile of this novel PAM is similar to that of a representative M1 PAM that induces severe AEs. For instance, both compounds are potent ago-PAMs that demonstrate significant interaction with the orthosteric site (either bitopic or negative cooperativity). However, there are subtle differences in efficacies of the compounds at potentiating M1 responses, agonist potencies, and abilities to induce receptor internalization. While these differences may contribute to the differential in vivo profiles of these compounds, the in vitro differences are relatively subtle and highlight the complexities of allosteric modulators and the need to focus on in vivo phenotypic screening to identify safe and effective M1 PAMs.

Integrated Strategy for Use of Positron Emission Tomography in Nonhuman Primates to Confirm Multitarget Occupancy of Novel Psychotropic Drugs: An Example with AZD3676.

Positron emission tomography (PET) is widely applied in central nervous system (CNS) drug development for assessment of target engagement in vivo. As the majority of PET investigations have addressed drug interaction at a single binding site, findings of multitarget engagement have been less frequently reported and have often been inconsistent with results obtained in vitro. AZD3676 [N,N-dimethyl-7-(4-(2-(pyridin-2-yl)ethyl)piperazin-1-yl) benzofuran-2-carboxamide] is a novel combined serotonin (5-hydroxytryptamine) 5-HT1A and 5-HT1B receptor antagonist that was developed for the treatment of cognitive impairment in Alzheimer's disease. Here, we evaluated the properties of AZD3676 as a CNS drug by combining in vitro and ex vivo radioligand binding techniques, behavioral pharmacology in rodents, and PET imaging in nonhuman primates. Target engagement in the nonhuman primate brain was assessed in PET studies by determination of drug-induced occupancy using receptor-selective radioligands. AZD3676 showed preclinical properties consistent with CNS drug potential, including nanomolar receptor affinity and efficacy in rodent models of learning and memory. In PET studies of the monkey brain, AZD3676 inhibited radioligand binding in a dose-dependent manner with similar affinity at both receptors. The equally high affinity at 5-HT1A and 5-HT1B receptors as determined in vivo was not predicted from corresponding estimates obtained in vitro, suggesting more than 10-fold selectivity for 5-HT1A versus 5-HT1B receptors. These findings support the further integrated use of PET for confirmation of multitarget occupancy of CNS drugs. Importantly, earlier introduction of PET studies in nonhuman primates may reduce future development costs and the requirement for animal experiments in preclinical CNS drug development programs.

Development and Pharmacological Characterization of Selective Blockers of 2-Arachidonoyl Glycerol Degradation with Efficacy in Rodent Models of Multiple Sclerosis and Pain

We report the discovery of compound 4a, a potent β-lactam-based monoacylglycerol lipase (MGL) inhibitor characterized by an irreversible and stereoselective mechanism of action, high membrane permeability, high brain penetration evaluated using a human in vitro blood-brain barrier model, high selectivity in binding and affinity-based proteomic profiling assays, and low in vitro toxicity. Mode-of-action studies demonstrate that 4a, by blocking MGL, increases 2-arachidonoylglycerol and behaves as a cannabinoid (CB1/CB2) receptor indirect agonist. Administration of 4a in mice suffering from experimental autoimmune encephalitis ameliorates the severity of the clinical symptoms in a CB1/CB2-dependent manner. Moreover, 4a produced analgesic effects in a rodent model of acute inflammatory pain, which was antagonized by CB1 and CB2 receptor antagonists/inverse agonists. 4a also relieves the neuropathic hypersensitivity induced by oxaliplatin. Given these evidence, 4a, as MGL selective inhibitor, could represent a valuable lead for the future development of therapeutic options for multiple sclerosis and chronic pain.

The concentration game: differential effects of bioactive signaling in 2D and 3D culture.

Traumatic injuries to the central nervous system, such as traumatic brain injury, spinal cord injury and stroke, have a high prevalence, enormous financial costs and lack clinical treatments that restore neurological function (Ma et al., 2014). These injuries trigger a series of secondary biochemical and cellular responses that ultimately lead to cellular death and the maintenance of an unsupportive extracellular matrix (ECM) for tissue regeneration (Silva et al., 2014). Artificial ECM or scaffolds represent a way to alter this unsupportive environment to improve the efficacy of stem cell therapies and enhance neural tissue regeneration (Figure 1). Scaffold use could lead to greater improvements in neurological function (such as improved bladder control, increased dexterity and body control, etc.) than observed with the implantation of cells alone. To date, the inclusion of basic scaffolds with stem cell therapy treatments have shown increased efficacy in rodent models (Yasuda et al., 2010). More advanced scaffolds could better mimic the chemical, physical and mechanical properties of the ECM to promote cellular survival, adhesion, proliferation and differentiation. Altering the injured ECM to mitigate the barriers to axon invasion, myelination and cellular maturation further and lead to even greater gains in neurological function.

Hyperglycemia Does Not Affect Iron Mediated Toxicity of Cultured Endothelial and Renal Tubular Epithelial Cells: Influence of L-Carnosine.

Iron has been suggested to affect the clinical course of type 2 diabetes (T2DM) as accompanying increased intracellular iron accumulation may provide an alternative source for reactive oxygen species (ROS). Although carnosine has proven its therapeutic efficacy in rodent models of T2DM, little is known about its efficacy to protect cells from iron toxicity. We sought to assess if high glucose (HG) exposure makes cultured human umbilical vein endothelial cells (HUVECs) and renal proximal tubular epithelial cells (PTECs) more susceptible to metal induced toxicity and if this is ameliorated by L-carnosine. HUVECs and PTECs, cultured under normal glucose (5 mM, NG) or HG (30 mM), were challenged for 24 h with FeCl3. Cell viability was not impaired under HG conditions nor did HG increase susceptibility to FeCl3. HG did not change the expression of divalent metal transporter 1 (DMT1), ferroportin (IREG), and transferrin receptor protein 1 (TFRC). Irrespective of glucose concentrations L-carnosine prevented toxicity in a dose-dependent manner, only if it was present during the FeCl3 challenge. Hence our study indicates that iron induced cytotoxicity is not enhanced under HG conditions. L-Carnosine displayed a strong protective effect, most likely by chelation of iron mediated toxicity.

Quetiapine and its metabolite norquetiapine: translation from in vitro pharmacology to in vivo efficacy in rodent models.

Quetiapine has a range of clinical activity distinct from other atypical antipsychotic drugs, demonstrating efficacy as monotherapy in bipolar depression, major depressive disorder and generalized anxiety disorder. The neuropharmacological mechanisms underlying this clinical profile are not completely understood; however, the major active metabolite, norquetiapine, has been shown to have a distinct in vitro pharmacological profile consistent with a broad therapeutic range and may contribute to the clinical profile of quetiapine. We evaluated quetiapine and norquetiapine, using in vitro binding and functional assays of targets known to be associated with antidepressant and anxiolytic drug actions and compared these activities with a representative range of established antipsychotics and antidepressants. To determine how the in vitro pharmacological properties translate into in vivo activity, we used preclinical animal models with translational relevance to established antidepressant-like and anxiolytic-like drug action. Norquetiapine had equivalent activity to established antidepressants at the noradrenaline transporter (NET), while quetiapine was inactive. Norquetiapine was active in the mouse forced swimming and rat learned helplessness tests. In in vivo receptor occupancy studies, norquetiapine had significant occupancy at NET at behaviourally relevant doses. Both quetiapine and norquetiapine were agonists at 5-HT1A receptors, and the anxiolytic-like activity of norquetiapine in rat punished responding was blocked by the 5-HT1A antagonist, WAY100635. Quetiapine and norquetiapine have multiple in vitro pharmacological actions, and results from preclinical studies suggest that activity at NET and 5-HT1A receptors contributes to the antidepressant and anxiolytic effects in patients treated with quetiapine.