ABSTRACT
After the publication of the report titled Toxicity Testing in the 21st Century - A Vision and a Strategy, many initiatives started to foster a major paradigm shift for toxicity testing - from apical endpoints in animal-based tests to mechanistic endpoints through delineation of pathways of toxicity (PoT) in human cell based systems. The US EPA has funded an important project to develop new high throughput technologies based on human cell based in vitro technologies. These methods are currently being incorporated into the chemical risk assessment process. In the pharmaceutical industry, the efficacy and toxicity of new drugs are evaluated during preclinical investigations that include drug metabolism, pharmacokinetics, pharmacodynamics and safety toxicology studies. The results of these studies are analyzed and extrapolated to predict efficacy and potential adverse effects in humans. However, due to the high failure rate of drugs during the clinical phases, a new approach for a more predictive assessment of drugs both in terms of efficacy and adverse effects is getting urgent. The food industry faces the challenge of assessing novel foods and food ingredients for the general population, while using animal safety testing for extrapolation purposes is often of limited relevance. The question is whether the latest paradigm shift proposed by the Tox21c report for chemicals may provide a useful tool to improve the risk assessment approach also for drugs and food ingredients.
Subject(s)
Drug Evaluation/methods , Risk Assessment , Toxicity Tests/methods , Animal Testing Alternatives/methods , Animals , Drug Approval , Drug Industry , Humans , In Vitro Techniques , Safety/standards , United States , United States Environmental Protection AgencyABSTRACT
Our knowledge regarding the molecular pathophysiology underlying anxiety disorders remains incomplete. Increasing evidence points to a role of glutamate in anxiety. The group III metabotropic glutamate receptors (mGlu4, mGlu6, mGlu7 and mGlu8 receptors) remain the least investigated glutamate receptor subtypes partially due to a delay in the development of specific pharmacological tools. Early work using knockout animals and pharmacological tools aimed at investigating the role of mGlu7 receptor in the pathophysiology of anxiety disorders has yielded exciting yet not always consistent results. To further investigate the role this receptor plays in anxiety-like behaviour, we knocked down mGlu7 receptor mRNA levels in the adult mouse brain using siRNA delivered via an osmotic minipump. This reduced anxiety-like behaviour in the light-dark box coupled with an attenuation of stress-induced hyperthermia (SIH) and a reduction of the acoustic startle response (ASRs) in the fear-potentiated startle paradigm (FPS). These effects on anxiety-like behaviour were independent of any impairment of locomotor activity and surprisingly, no behavioural changes were observed in the forced swim test (FST), which is in contrast to mGlu7 receptor knockout animals. Furthermore, the previously reported epilepsy-prone phenotype seen in mGlu7 receptor knockout animals was not observed following siRNA-induced knockdown of the receptor. These data suggest targeting mGlu7 receptors with selective antagonist drugs may be an effective and safe strategy for the treatment of anxiety disorders.
Subject(s)
Anxiety/drug therapy , Anxiety/metabolism , RNA, Small Interfering/therapeutic use , Receptors, Metabotropic Glutamate/metabolism , Adaptation, Ocular/drug effects , Animals , Disease Models, Animal , Dose-Response Relationship, Drug , Fear/drug effects , Hyperthermia, Induced/psychology , Male , Mice , Mice, Inbred BALB C , Motor Activity/drug effects , Pentylenetetrazole/toxicity , Receptors, Metabotropic Glutamate/genetics , Reflex, Startle/drug effects , Seizures/chemically induced , Seizures/drug therapy , Stress, Physiological/physiology , Swimming/psychologyABSTRACT
Quinazoline-2,4-diones with a sulfonamide group attached to the N(3) ring atom constitute a novel class of competitive AMPA receptor antagonists. One of the synthesized compounds, 28, shows nanomolar receptor affinity, whereas other examples of the series display oral anticonvulsant activity in animal models.
Subject(s)
Quinazolinones/chemical synthesis , Receptors, AMPA/antagonists & inhibitors , Sulfonamides/chemical synthesis , Administration, Oral , Animals , Anticonvulsants/pharmacology , Binding, Competitive/drug effects , Crystallography, X-Ray , Mice , Molecular Structure , Quinazolinones/chemistry , Quinazolinones/pharmacology , Rats , Structure-Activity Relationship , Sulfonamides/chemistry , Sulfonamides/pharmacologyABSTRACT
PURPOSE: To evaluate the anticonvulsant profile and behavioral toxicity of rufinamide in animal seizure models compared to the established antiepileptic drugs (AEDs): phenytoin, phenobarbital, valproate, and ethosuximide, or vehicle. METHODS: In acute studies of anticonvulsant efficacy, the AEDs were administered via oral (CF1 mice and Sprague-Dawley rats) and intraperitoneal (CF1 mice) routes. The AEDs were assessed for their ability to inhibit seizures induced by maximal electroshock (MES) or subcutaneous pentylenetetrazol, and ability to block seizures induced by subcutaneous strychnine, bicuculline, or picrotoxin. Tolerance of oral rufinamide was assessed in rats following 5-day (versus single-dose) treatment with oral rufinamide using the dose equivalent necessary to achieve a 50% decrease in seizure frequency (ED(50)). Metabolic tolerance was also evaluated using an in vitro liver microsomal assay. RESULTS: Oral rufinamide suppressed pentylenetetrazol-induced seizures in mice (ED(50) 45.8 mg/kg) but not rats, and was active against MES-induced tonic seizures in mice (ED(50) 23.9 mg/kg) and rats (ED(50) 6.1 mg/kg). Intraperitoneal rufinamide suppressed pentylenetetrazol-, bicuculline-, and picrotoxin-induced clonus in mice (ED(50) 54.0, 50.5, and 76.3 mg/kg, respectively). Rufinamide was partially effective in the mouse strychnine test. The behavioral toxicity of rufinamide was similar to or better than established AEDs tested in this study. In general, the protective index of rufinamide was greater than that of the other AEDs. CONCLUSIONS: The efficacy and behavioral toxicity profiles in these animal models suggest that rufinamide may be effective in the treatment of generalized and partial seizures.
Subject(s)
Anticonvulsants/toxicity , Anticonvulsants/therapeutic use , Seizures/prevention & control , Triazoles/toxicity , Triazoles/therapeutic use , Administration, Oral , Animals , Disease Models, Animal , Injections, Intraperitoneal , Mice , Phenobarbital/pharmacology , Phenobarbital/therapeutic use , Phenytoin/pharmacology , Phenytoin/therapeutic use , Rats , Rats, Sprague-Dawley , Seizures/drug therapy , Severity of Illness Index , Valproic Acid/pharmacology , Valproic Acid/therapeutic useABSTRACT
RNA interference (RNAi) is a natural mechanism for regulating gene expression, which exists in plants, invertebrates, and mammals. We investigated whether non-viral infusion of short interfering RNA (siRNA) by the intracerebroventricular route would enable a sequence-specific gene knockdown in the mouse brain and whether the knockdown translates into disease-relevant behavioral changes. Initially, we targeted enhanced green fluorescent protein (EGFP) in mice overexpressing EGFP. A selective knockdown of both EGFP protein and mRNA was observed throughout the brain, with lesser down-regulation in regions distal to the infusion site. We then targeted endogenous genes, encoding the dopamine (DAT) and serotonin transporters (SERT). DAT-siRNA infusion in adult mice produced a significant down-regulation of DAT mRNA and protein and elicited hyperlocomotion similar, but delayed, to that produced on infusion of GBR-12909, a potent and selective DAT inhibitor. Similarly, SERT-siRNA infusion resulted in significant knockdown of SERT mRNA and protein and elicited reduced immobility in the forced swim test similar to that obtained on infusion of citalopram, a very selective and potent SSRI. Application of this non-viral RNAi approach may accelerate target validation for neuropsychiatric disorders that involve a complex interplay of gene(s) from various brain regions.
Subject(s)
Brain/drug effects , Gene Expression Regulation/drug effects , Plasma Membrane Neurotransmitter Transport Proteins/genetics , RNA Interference/drug effects , Receptors, G-Protein-Coupled/genetics , Animals , Brain/metabolism , Dopamine Plasma Membrane Transport Proteins/antagonists & inhibitors , Dopamine Plasma Membrane Transport Proteins/genetics , Down-Regulation/genetics , Green Fluorescent Proteins/genetics , Locomotion/drug effects , Mental Disorders/drug therapy , Mice , Nervous System Diseases/drug therapy , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/pharmacology , Serotonin Plasma Membrane Transport Proteins/drug effects , Serotonin Plasma Membrane Transport Proteins/geneticsABSTRACT
The current review will focus on the recent patents for AMPA receptor antagonists and their claims, evidence for their therapeutic effectiveness in the treatment of epilepsy and their potential role in psychiatric and neurodegenerative disorders. It will also highlight the proposed mechanisms of action and the implications thereof for our current understanding of the biomolecular basis of these pathologies. It will conclude with a summary of what we know, but also point out the remaining uncertainties, especially as this relates to the claims in the patent under discussion.
Subject(s)
Receptors, AMPA/antagonists & inhibitors , Animals , Anxiety Disorders/drug therapy , Epilepsy/drug therapy , Humans , Multiple Sclerosis/drug therapy , Neuroprotective Agents/pharmacology , Pain/drug therapy , Patents as Topic , Schizophrenia/drug therapyABSTRACT
GABAB receptors mediate slow synaptic inhibition in the nervous system. In transfected cells, functional GABAB receptors are usually only observed after coexpression of GABAB(1) and GABAB(2) subunits, which established the concept of heteromerization for G-protein-coupled receptors. In the heteromeric receptor, GABAB(1) is responsible for binding of GABA, whereas GABAB(2) is necessary for surface trafficking and G-protein coupling. Consistent with these in vitro observations, the GABAB(1) subunit is also essential for all GABAB signaling in vivo. Mice lacking the GABAB(1) subunit do not exhibit detectable electrophysiological, biochemical, or behavioral responses to GABAB agonists. However, GABAB(1) exhibits a broader cellular expression pattern than GABAB(2), suggesting that GABAB(1) could be functional in the absence of GABAB(2). We now generated GABAB(2)-deficient mice to analyze whether GABAB(1) has the potential to signal without GABAB(2) in neurons. We show that GABAB(2)-/- mice suffer from spontaneous seizures, hyperalgesia, hyperlocomotor activity, and severe memory impairment, analogous to GABAB(1)-/- mice. This clearly demonstrates that the lack of heteromeric GABAB(1,2) receptors underlies these phenotypes. To our surprise and in contrast to GABAB(1)-/- mice, we still detect atypical electrophysiological GABAB responses in hippocampal slices of GABAB(2)-/- mice. Furthermore, in the absence of GABAB(2), the GABAB(1) protein relocates from distal neuronal sites to the soma and proximal dendrites. Our data suggest that association of GABAB(2) with GABAB(1) is essential for receptor localization in distal processes but is not absolutely necessary for signaling. It is therefore possible that functional GABAB receptors exist in neurons that naturally lack GABAB(2) subunits.
Subject(s)
Hippocampus/physiopathology , Hyperalgesia/genetics , Hyperkinesis/genetics , Memory Disorders/genetics , Receptors, GABA-B/metabolism , Seizures/genetics , Animals , Brain/metabolism , Brain/pathology , Brain Chemistry , Dimerization , Electroencephalography , GABA Agonists/pharmacology , Hippocampus/drug effects , Hippocampus/metabolism , Hyperalgesia/pathology , Hyperkinesis/pathology , Memory Disorders/pathology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Neurons/drug effects , Neurons/metabolism , Neurons/pathology , Pain Measurement , Patch-Clamp Techniques , Potassium Channels/drug effects , Protein Subunits/genetics , Protein Subunits/metabolism , Protein Transport/genetics , Protein Transport/physiology , Radioligand Assay , Receptors, GABA-B/genetics , Seizures/pathology , Signal Transduction/genetics , Signal Transduction/physiologyABSTRACT
gamma-Hydroxybutyrate (GHB), a metabolite of gamma-aminobutyric acid (GABA), is proposed to function as a neurotransmitter or neuromodulator. gamma-Hydroxybutyrate and its prodrug, gamma-butyrolactone (GBL), recently received increased public attention as they emerged as popular drugs of abuse. The actions of GHB/GBL are believed to be mediated by GABAB and/or specific GHB receptors, the latter corresponding to high-affinity [3H]GHB-binding sites coupled to G-proteins. To investigate the contribution of GABAB receptors to GHB actions we studied the effects of GHB in GABAB(1)-/- mice, which lack functional GABAB receptors. Autoradiography reveals a similar spatial distribution of [3H]GHB-binding sites in brains of GABAB(1)-/- and wild-type mice. The maximal number of binding sites and the KD values for the putative GHB antagonist [3H]6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene acetic acid (NCS-382) appear unchanged in GABAB(1)-/- compared with wild-type mice, demonstrating that GHB- are distinct from GABAB-binding sites. In the presence of the GABAB receptor positive modulator 2,6-di-tert-butyl-4-(3-hydroxy-2,2-dimethyl-propyl)-phenol GHB induced functional GTPgamma[35S] responses in brain membrane preparations from wild-type but not GABAB(1)-/- mice. The GTPgamma[35S] responses in wild-type mice were blocked by the GABAB antagonist [3-[[1-(S)-(3,4dichlorophenyl)ethyl]amino]-2-(S)-hydroxy-propyl]-cyclohexylmethyl phosphinic acid hydrochloride (CGP54626) but not by NCS-382. Altogether, these findings suggest that the GHB-induced GTPgamma[35S] responses are mediated by GABAB receptors. Following GHB or GBL application, GABAB(1)-/- mice showed neither the hypolocomotion, hypothermia, increase in striatal dopamine synthesis nor electroencephalogram delta-wave induction seen in wild-type mice. It, therefore, appears that all studied GHB effects are GABAB receptor dependent. The molecular nature and the signalling properties of the specific [3H]GHB-binding sites remain elusive.
Subject(s)
Binding, Competitive , Receptors, GABA-B/metabolism , Sodium Oxybate/pharmacology , 4-Butyrolactone/pharmacokinetics , Adjuvants, Anesthesia/pharmacology , Animals , Anticonvulsants/pharmacokinetics , Autoradiography , Baclofen/pharmacology , Behavior, Animal/drug effects , Benzocycloheptenes/pharmacokinetics , Body Weight/drug effects , Brain/anatomy & histology , Brain/drug effects , Brain/metabolism , Cell Membrane/drug effects , Cell Membrane/metabolism , Chromatography, High Pressure Liquid , Dihydroxyphenylalanine/metabolism , Dose-Response Relationship, Drug , Drug Interactions , Electrochemistry , Electroencephalography , GABA-B Receptor Agonists , Guanosine 5'-O-(3-Thiotriphosphate)/pharmacokinetics , Mice , Mice, Inbred BALB C , Mice, Knockout , Motor Activity/drug effects , Organophosphorus Compounds/pharmacokinetics , Phenols/pharmacokinetics , Radioligand Assay , Receptors, GABA-B/deficiency , Receptors, GABA-B/genetics , Time Factors , gamma-Aminobutyric Acid/pharmacologyABSTRACT
NMDA antagonists derived from 5-phosphonomethyl-1,4-dihydroquinoxaline-2,3-dione (3a) are potent anticonvulsant agents, and display strong protective effects in the electroshock-induced convulsion assay in mice. Their preference for the human NMDAR 1A/2A over 1A/2B subunit composition was optimized, leading to (1RS,1'S)-PEAQX (9r), which shows a >100-fold selectivity.