RESUMO
The disposition of a novel kynurenine monooxygenase inhibitor, CHDI-340246, was investigated in vitro and in animals.In vitro, there was minimal metabolic turnover of CHDI-340246 in all species. The protein binding was higher in human plasma (99.7%) relative to other species.In all species, blood clearance was low (<20% of liver blood flow) and volume of distribution was small (<0.5 L/kg). The terminal half-life was longer in monkeys (9 hr) than in mice, rats, or dogs (1-2 hr). CHDI-340246 was orally bioavailable (>60%) in all species.In rats, [14C]CHDI-340246 showed wide distribution of radioactivity in all tissues except brain and testes. In rats, the parent drug was the major circulating moiety with minor amounts of a sulphate conjugate of an O-dealkylated metabolite. The elimination occurred via the urinary route and to a lesser extent by biliary route, but mostly as metabolites. In cynomolgus monkeys, the parent drug predominated in plasma with only trace amounts of metabolites detected.Acyl glucuronide conjugate of CHDI-340246 was not detected in plasma of rats or monkeys.Overall, the ADME profile of CHDI-340246 was favourable in rats and monkeys for potential evaluation of KMO inhibition in humans.
Assuntos
Cinurenina , Pirimidinas , Animais , Animais de Laboratório , Cães , Camundongos , Oxigenases de Função Mista , Ratos , Especificidade da EspécieRESUMO
Huntington's disease (HD) is a neurodegenerative disease caused by polyglutamine expansion in the huntingtin protein. For drug candidates targeting HD, the ability to cross the blood-brain barrier (BBB) and reach the site of action in the central nervous system (CNS) is crucial for achieving pharmacological activity. To assess the permeability of selected compounds across the BBB, we utilized a two-dimensional model composed of primary porcine brain endothelial cells and rat astrocytes. Our objective was to use this in vitro model to rank and prioritize compounds for in vivo pharmacokinetic and brain penetration studies. The model was first characterized using a set of validation markers chosen based on their functional importance at the BBB. It was shown to fulfill the major BBB characteristics, including functional tight junctions, high transendothelial electrical resistance, expression, and activity of influx and efflux transporters. The in vitro permeability of 54 structurally diverse known compounds was determined and shown to have a good correlation with the in situ brain perfusion data in rodents. We used this model to investigate the BBB permeability of a series of new HD compounds from different chemical classes, and we found a good correlation with in vivo brain permeation, demonstrating the usefulness of the in vitro model for optimizing CNS drug properties and for guiding the selection of lead compounds in a drug discovery setting.
Assuntos
Barreira Hematoencefálica/metabolismo , Fármacos do Sistema Nervoso Central/uso terapêutico , Descoberta de Drogas/métodos , Doença de Huntington/tratamento farmacológico , Modelos Biológicos , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Astrócitos/metabolismo , Permeabilidade Capilar/fisiologia , Células Cultivadas , Córtex Cerebral/citologia , Técnicas de Cocultura , Impedância Elétrica , Células Endoteliais/metabolismo , Permeabilidade , Ratos , Ratos Sprague-Dawley , Proteínas Carreadoras de Solutos/metabolismo , Suínos , Junções Íntimas/metabolismoRESUMO
Background: Dysregulation of the kynurenine metabolic pathway has been reported in several neurological conditions. Methods & results: Sensitive and selective LC-MS/MS methods have been validated for six kynurenine pathway metabolites in human cerebrospinal fluid and plasma. For each matrix, we validated three methods - one for the simultaneous determination of kynurenine, kynurenic acid, anthranilic acid and 3-hydroxy-kynurenine (four-analyte assay), one for quinolinic acid and one for tryptophan - using stable-isotopically labeled internal standards. The dynamic range and quantitation limits were based on endogenous concentrations for each analyte. Conclusion: The use of validated methods for kynurenine pathway metabolites in human cerebrospinal fluid and plasma will provide definitive information in neurological diseases.
Assuntos
Cinurenina , Espectrometria de Massas em Tandem , Humanos , Cromatografia Líquida , Espectrometria de Massas em Tandem/métodos , Triptofano , Plasma/metabolismo , Ácido Quinolínico/líquido cefalorraquidianoRESUMO
Therapeutic interventions are being developed for Huntington's disease (HD), a hallmark of which is mutant huntingtin protein (mHTT) aggregates. Following the advancement to human testing of two [11C]-PET ligands for aggregated mHTT, attributes for further optimization were identified. We replaced the pyridazinone ring of CHDI-180 with a pyrimidine ring and minimized off-target binding using brain homogenate derived from Alzheimer's disease patients. The major in vivo metabolic pathway via aldehyde oxidase was blocked with a 2-methyl group on the pyrimidine ring. A strategically placed ring-nitrogen on the benzoxazole core ensured high free fraction in the brain without introducing efflux. Replacing a methoxy pendant with a fluoro-ethoxy group and introducing deuterium atoms suppressed oxidative defluorination and accumulation of [18F]-signal in bones. The resulting PET ligand, CHDI-650, shows a rapid brain uptake and washout profile in non-human primates and is now being advanced to human testing.
Assuntos
Doença de Huntington , Tomografia por Emissão de Pósitrons , Animais , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Ligantes , Tomografia por Emissão de Pósitrons/métodos , Doença de Huntington/diagnóstico por imagem , Doença de Huntington/tratamento farmacológico , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismoRESUMO
Huntington's disease (HD) is caused by an expanded CAG trinucleotide repeat in exon 1 of the huntingtin (HTT) gene. We report the design of a series of HTT pre-mRNA splicing modulators that lower huntingtin (HTT) protein, including the toxic mutant huntingtin (mHTT), by promoting insertion of a pseudoexon containing a premature termination codon at the exon 49-50 junction. The resulting transcript undergoes nonsense-mediated decay, leading to a reduction of HTT mRNA transcripts and protein levels. The starting benzamide core was modified to pyrazine amide and further optimized to give a potent, CNS-penetrant, and orally bioavailable HTT-splicing modulator 27. This compound reduced canonical splicing of the HTT RNA exon 49-50 and demonstrated significant HTT-lowering in both human HD stem cells and mouse BACHD models. Compound 27 is a structurally diverse HTT-splicing modulator that may help understand the mechanism of adverse effects such as peripheral neuropathy associated with branaplam.
RESUMO
AMPA receptors (AMPARs) are an important therapeutic target in the CNS. A series of substituted benzobistriazinone, benzobispyrimidinone and related derivatives have been prepared with high potency and selectivity for the allosteric binding site of AMPARs. Further improvements have been made to previously reported series of positive AMPAR modulators and these compounds exhibit excellent in vivo activity and improved in vivo metabolic stability with up to 100% oral bioavailability in rat.
Assuntos
Compostos Heterocíclicos/química , Receptores de AMPA/química , Triazinas/química , Administração Oral , Regulação Alostérica , Animais , Sítios de Ligação , Ratos , Receptores de AMPA/metabolismo , Triazinas/síntese química , Triazinas/farmacocinéticaRESUMO
AMPA receptors (AMPARs) have been demonstrated to be an important therapeutic CNS target. A series of substituted benzotriazinone and benzopyrimidinone derivatives were prepared with the aim to improve in vivo activity over the previously reported bis-benzoxazinone based AMPAKINE series from our laboratory. These compounds were shown to be potent, positive allosteric AMPAR modulators that have better in vivo activity and improved metabolic stability over the analogous benzoxazinone derivatives.
Assuntos
Pirimidinonas/química , Pirimidinonas/farmacologia , Receptores de AMPA/metabolismo , Triazinas/química , Triazinas/farmacologia , Regulação Alostérica , Animais , Desenho de Fármacos , Hipocampo/efeitos dos fármacos , Humanos , Ratos , Relação Estrutura-AtividadeRESUMO
The expanded polyglutamine-containing mutant huntingtin (mHTT) protein is implicated in neuronal degeneration of medium spiny neurons in Huntington's disease (HD) for which multiple therapeutic approaches are currently being evaluated to eliminate or reduce mHTT. Development of effective and orthogonal biomarkers will ensure accurate assessment of the safety and efficacy of pharmacologic interventions. We have identified and optimized a class of ligands that bind to oligomerized/aggregated mHTT, which is a hallmark in the HD postmortem brain. These ligands are potentially useful imaging biomarkers for HD therapeutic development in both preclinical and clinical settings. We describe here the optimization of the benzo[4,5]imidazo[1,2-a]pyrimidine series that show selective binding to mHTT aggregates over Aß- and/or tau-aggregates associated with Alzheimer's disease pathology. Compound [11C]-2 was selected as a clinical candidate based on its high free fraction in the brain, specific binding in the HD mouse model, and rapid brain uptake/washout in nonhuman primate positron emission tomography imaging studies.
Assuntos
Encéfalo/diagnóstico por imagem , Compostos Heterocíclicos com 3 Anéis/química , Proteína Huntingtina/metabolismo , Agregados Proteicos/fisiologia , Piridinas/química , Compostos Radiofarmacêuticos/química , Doença de Alzheimer , Animais , Biomarcadores/metabolismo , Encéfalo/metabolismo , Radioisótopos de Carbono/química , Feminino , Compostos Heterocíclicos com 3 Anéis/síntese química , Compostos Heterocíclicos com 3 Anéis/farmacocinética , Humanos , Macaca fascicularis , Masculino , Camundongos Endogâmicos C57BL , Estrutura Molecular , Tomografia por Emissão de Pósitrons , Piridinas/síntese química , Piridinas/farmacocinética , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/farmacocinética , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
The blood-brain barrier (BBB) is responsible for the homeostasis between the cerebral vasculature and the brain and it has a key role in regulating the influx and efflux of substances, in healthy and diseased states. Stem cell technology offers the opportunity to use human brain-specific cells to establish in vitro BBB models. Here, we describe the establishment of a human BBB model in a two-dimensional monolayer culture, derived from human induced pluripotent stem cells (hiPSCs). This model was characterized by a transendothelial electrical resistance (TEER) higher than 2000 Ωâcm2 and associated with negligible paracellular transport. The hiPSC-derived BBB model maintained the functionality of major endothelial transporter proteins and receptors. Some proprietary molecules from our central nervous system (CNS) programs were evaluated revealing comparable permeability in the human model and in the model from primary porcine brain endothelial cells (PBECs).
Assuntos
Transporte Biológico/efeitos dos fármacos , Barreira Hematoencefálica/citologia , Barreira Hematoencefálica/metabolismo , Células Endoteliais/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Animais , Astrócitos/metabolismo , Transporte Biológico/fisiologia , Encéfalo/citologia , Diferenciação Celular/fisiologia , Células Cultivadas , Sistema Nervoso Central/química , Sistema Nervoso Central/metabolismo , Criopreservação/métodos , Humanos , Imuno-Histoquímica , Permeabilidade , SuínosRESUMO
Mutant huntingtin (mHTT) protein carrying the elongated N-terminal polyglutamine (polyQ) tract misfolds and forms protein aggregates characteristic of Huntington's disease (HD) pathology. A high-affinity ligand specific for mHTT aggregates could serve as a positron emission tomography (PET) imaging biomarker for HD therapeutic development and disease progression. To identify such compounds with binding affinity for polyQ aggregates, we embarked on systematic structural activity studies; lead optimization of aggregate-binding affinity, unbound fractions in brain, permeability, and low efflux culminated in the discovery of compound 1, which exhibited target engagement in autoradiography (ARG) studies in brain slices from HD mouse models and postmortem human HD samples. PET imaging studies with 11C-labeled 1 in both HD mice and WT nonhuman primates (NHPs) demonstrated that the right-hand-side labeled ligand [11C]-1R (CHDI-180R) is a suitable PET tracer for imaging of mHTT aggregates. [11C]-1R is now being advanced to human trials as a first-in-class HD PET radiotracer.
Assuntos
Proteína Huntingtina/análise , Doença de Huntington/diagnóstico por imagem , Tomografia por Emissão de Pósitrons/métodos , Agregação Patológica de Proteínas/diagnóstico por imagem , Animais , Modelos Animais de Doenças , Cães , Feminino , Humanos , Proteína Huntingtina/genética , Doença de Huntington/genética , Ligantes , Células Madin Darby de Rim Canino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Peptídeos/genética , Agregação Patológica de Proteínas/genética , Compostos Radiofarmacêuticos/análise , Ratos Sprague-DawleyRESUMO
Neuroactive metabolites in the kynurenine pathway of tryptophan catabolism are associated with neurodegenerative disorders. Tryptophan is transported across the blood-brain barrier and converted via the kynurenine pathway to N-formyl-L-kynurenine, which is further degraded to L-kynurenine. This metabolite can then generate a group of metabolites called kynurenines, most of which have neuroactive properties. The association of tryptophan catabolic pathway alterations with various central nervous system (CNS) pathologies has raised interest in analytical methods to accurately quantify kynurenines in body fluids. We here describe a rapid and sensitive reverse-phase HPLC-MS/MS method to quantify L-kynurenine (KYN), kynurenic acid (KYNA), 3-hydroxy-L-kynurenine (3HK) and anthranilic acid (AA) in rat plasma. Our goal was to quantify these metabolites in a single run; given their different physico-chemical properties, major efforts were devoted to develop a chromatography suitable for all metabolites that involves plasma protein precipitation with acetonitrile followed by chromatographic separation by C18 RP chromatography, detected by electrospray mass spectrometry. Quantitation range was 0.098-100 ng/ml for 3HK, 9.8-20,000 ng/ml for KYN, 0.49-1000 ng/ml for KYNA and AA. The method was linear (r>0.9963) and validation parameters were within acceptance range (calibration standards and QC accuracy within ±30%).