RESUMEN
Phosphodiesterase 2A (PDE2A) inhibitors have been reported to demonstrate in vivo activity in preclinical models of cognition. To more fully explore the biology of PDE2A inhibition, we sought to identify potent PDE2A inhibitors with improved brain penetration as compared to current literature compounds. Applying estimated human dose calculations while simultaneously leveraging synthetically enabled chemistry and structure-based drug design has resulted in a highly potent, selective, brain penetrant compound 71 (PF-05085727) that effects in vivo biochemical changes commensurate with PDE2A inhibition along with behavioral and electrophysiological reversal of the effects of NMDA antagonists in rodents. This data supports the ability of PDE2A inhibitors to potentiate NMDA signaling and their further development for clinical cognition indications.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 2/antagonistas & inhibidores , Diseño de Fármacos , Inhibidores de Fosfodiesterasa/química , Inhibidores de Fosfodiesterasa/farmacología , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Cristalografía por Rayos X , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 2/química , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 2/metabolismo , Perros , Haplorrinos , Humanos , Ratones , Simulación del Acoplamiento Molecular , Inhibidores de Fosfodiesterasa/administración & dosificación , Inhibidores de Fosfodiesterasa/farmacocinética , RatasRESUMEN
Measurements of drug occupancies using positron emission tomography (PET) can be biased if the radioligand concentration exceeds "tracer" levels. Negative bias would also arise in successive PET scans if clearance of the radioligand is slow, resulting in a carryover effect. We developed a method to (1) estimate the in vivo dissociation constant Kd of a radioligand from PET studies displaying a non-tracer carryover (NTCO) effect and (2) correct the NTCO bias in occupancy studies taking into account the plasma concentration of the radioligand and its in vivo Kd. This method was applied in a study of healthy human subjects with the histamine H3 receptor radioligand [11C]GSK189254 to measure the PK-occupancy relationship of the H3 antagonist PF-03654746. From three test/retest studies, [11C]GSK189254 Kd was estimated to be 9.5 ± 5.9 pM. Oral administration of 0.1 to 4 mg of PF-03654746 resulted in occupancy estimates of 71%-97% and 30%-93% at 3 and 24 h post-drug, respectively. NTCO correction adjusted the occupancy estimates by 0%-15%. Analysis of the relationship between corrected occupancies and PF-03654746 plasma levels indicated that PF-03654746 can fully occupy H3 binding sites ( ROmax = 100%), and its IC50 was estimated to be 0.144 ± 0.010 ng/mL. The uncorrected IC50 was 26% higher.
Asunto(s)
Benzazepinas/metabolismo , Niacinamida/análogos & derivados , Tomografía de Emisión de Positrones/métodos , Receptores Histamínicos H3/análisis , Adulto , Benzazepinas/sangre , Radioisótopos de Carbono , Ciclobutanos/administración & dosificación , Ciclobutanos/sangre , Humanos , Niacinamida/sangre , Niacinamida/metabolismo , Pirrolidinas/administración & dosificación , Pirrolidinas/sangre , Ensayo de Unión Radioligante/métodos , Receptores Histamínicos H3/metabolismo , Adulto JovenRESUMEN
Phosphodiesterase 9A inhibitors have shown activity in preclinical models of cognition with potential application as novel therapies for treating Alzheimer's disease. Our clinical candidate, PF-04447943 (2), demonstrated acceptable CNS permeability in rats with modest asymmetry between central and peripheral compartments (free brain/free plasma = 0.32; CSF/free plasma = 0.19) yet had physicochemical properties outside the range associated with traditional CNS drugs. To address the potential risk of restricted CNS penetration with 2 in human clinical trials, we sought to identify a preclinical candidate with no asymmetry in rat brain penetration and that could advance into development. Merging the medicinal chemistry strategies of structure-based design with parallel chemistry, a novel series of PDE9A inhibitors was identified that showed improved selectivity over PDE1C. Optimization afforded preclinical candidate 19 that demonstrated free brain/free plasma ≥ 1 in rat and reduced microsomal clearance along with the ability to increase cyclic guanosine monophosphosphate levels in rat CSF.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Azetidinas/química , Barrera Hematoencefálica/metabolismo , Pirazoles/química , Pirazoles/síntesis química , Pirimidinas/química , Pirimidinas/síntesis química , Pirimidinonas/química , 3',5'-AMP Cíclico Fosfodiesterasas/química , Administración Oral , Animales , Azetidinas/síntesis química , Azetidinas/farmacocinética , Cristalografía por Rayos X , GMP Cíclico/líquido cefalorraquídeo , Ciclopentanos/síntesis química , Ciclopentanos/química , Ciclopentanos/farmacocinética , Bases de Datos Factuales , Perros , Diseño de Fármacos , Humanos , Modelos Moleculares , Estructura Molecular , Pirazoles/farmacocinética , Pirimidinas/farmacocinética , Pirimidinonas/síntesis química , Pirimidinonas/farmacocinética , Ratas , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
The cognitive impairments observed in Alzheimer's disease (AD) are in part a consequence of reduced acetylcholine (ACh) levels resulting from a loss of cholinergic neurons. Preclinically, serotonin 4 receptor (5-HT(4)) agonists are reported to modulate cholinergic function and therefore may provide a new mechanistic approach for treating cognitive deficits associated with AD. Herein we communicate the design and synthesis of potent, selective, and brain penetrant 5-HT(4) agonists. The overall goal of the medicinal chemistry strategy was identification of structurally diverse clinical candidates with varying intrinsic activities. The exposure-response relationships between binding affinity, intrinsic activity, receptor occupancy, drug exposure, and pharmacodynamic activity in relevant preclinical models of AD were utilized as key selection criteria for advancing compounds. On the basis of their excellent balance of pharmacokinetic attributes and safety, two lead 5-HT(4) partial agonist candidates 2d and 3 were chosen for clinical development.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Trastornos del Conocimiento/tratamiento farmacológico , Indoles/síntesis química , Piperidinas/síntesis química , Piranos/síntesis química , Agonistas del Receptor de Serotonina 5-HT4/síntesis química , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Enfermedad de Alzheimer/psicología , Animales , Células CHO , Cricetinae , Cricetulus , AMP Cíclico/biosíntesis , Perros , Agonismo Parcial de Drogas , Células HEK293 , Haplorrinos , Humanos , Técnicas In Vitro , Indoles/farmacocinética , Indoles/farmacología , Células de Riñón Canino Madin Darby , Masculino , Microsomas Hepáticos/metabolismo , Permeabilidad , Piperidinas/farmacocinética , Piperidinas/farmacología , Isoformas de Proteínas/metabolismo , Piranos/farmacocinética , Piranos/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de Serotonina 5-HT4/metabolismo , Agonistas del Receptor de Serotonina 5-HT4/farmacocinética , Agonistas del Receptor de Serotonina 5-HT4/farmacología , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Cyclic nucleotides are critical regulators of synaptic plasticity and participate in requisite signaling cascades implicated across multiple neurotransmitter systems. Phosphodiesterase 9A (PDE9A) is a high-affinity, cGMP-specific enzyme widely expressed in the rodent central nervous system. In the current study, we observed neuronal staining with antibodies raised against PDE9A protein in human cortex, cerebellum, and subiculum. We have also developed several potent, selective, and brain-penetrant PDE9A inhibitors and used them to probe the function of PDE9A in vivo. Administration of these compounds to animals led to dose-dependent accumulation of cGMP in brain tissue and cerebrospinal fluid, producing a range of biological effects that implied functional significance for PDE9A-regulated cGMP in dopaminergic, cholinergic, and serotonergic neurotransmission and were consistent with the widespread distribution of PDE9A. In vivo effects of PDE9A inhibition included reversal of the respective disruptions of working memory by ketamine, episodic and spatial memory by scopolamine, and auditory gating by amphetamine, as well as potentiation of risperidone-induced improvements in sensorimotor gating and reversal of the stereotypic scratching response to the hallucinogenic 5-hydroxytryptamine 2A agonist mescaline. The results suggested a role for PDE9A in the regulation of monoaminergic circuitry associated with sensory processing and memory. Thus, PDE9A activity regulates neuronal cGMP signaling downstream of multiple neurotransmitter systems, and inhibition of PDE9A may provide therapeutic benefits in psychiatric and neurodegenerative diseases promoted by the dysfunction of these diverse neurotransmitter systems.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , 3',5'-GMP Cíclico Fosfodiesterasas/metabolismo , Colinérgicos/farmacología , GMP Cíclico/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Inhibidores de Fosfodiesterasa/farmacología , 3',5'-AMP Cíclico Fosfodiesterasas/genética , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Animales , Reacción de Prevención/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Femenino , Humanos , Macaca fascicularis , Masculino , Memoria/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Actividad Motora/efectos de los fármacos , Neurotransmisores/farmacología , Ratas , Ratas Long-Evans , Ratas Wistar , Filtrado Sensorial/efectos de los fármacos , Conducta Estereotipada/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacosRESUMEN
The discovery of two histamine H(3) antagonist clinical candidates is disclosed. The pathway to identification of the two clinical candidates, 6 (PF-03654746) and 7 (PF-03654764) required five hypothesis driven design cycles. The key to success in identifying these clinical candidates was the development of a compound design strategy that leveraged medicinal chemistry knowledge and traditional assays in conjunction with computational and in vitro safety tools. Overall, clinical compounds 6 and 7 exceeded conservative safety margins and possessed optimal pharmacological and pharmacokinetic profiles, thus achieving our initial goal of identifying compounds with fully aligned oral drug attributes, "best-in-class" molecules.
Asunto(s)
Ciclobutanos/síntesis química , Diseño de Fármacos , Antagonistas de los Receptores Histamínicos/síntesis química , Pirrolidinas/síntesis química , Receptores Histamínicos H3/metabolismo , Animales , Proteínas Sanguíneas/metabolismo , Barrera Hematoencefálica/metabolismo , Línea Celular , Ciclobutanos/farmacología , Ciclobutanos/toxicidad , Perros , Conducta de Ingestión de Líquido/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Antagonistas de los Receptores Histamínicos/farmacología , Antagonistas de los Receptores Histamínicos/toxicidad , Humanos , Técnicas In Vitro , Riñón/metabolismo , Lipidosis/inducido químicamente , Lipidosis/metabolismo , Pulmón/metabolismo , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Estructura Molecular , Fosfolípidos/metabolismo , Unión Proteica , Pirrolidinas/farmacología , Pirrolidinas/toxicidad , Ratas , Ratas Sprague-Dawley , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
By use of parallel chemistry coupled with physicochemical property design, a series of selective κ opioid antagonists have been discovered. The parallel chemistry strategy utilized key monomer building blocks to rapidly expand the desired SAR space. The potency and selectivity of the in vitro κ antagonism were confirmed in the tail-flick analgesia model. This model was used to build an exposure-response relationship between the κ K(i) and the free brain drug levels. This strategy identified 2-methyl-N-((2'-(pyrrolidin-1-ylsulfonyl)biphenyl-4-yl)methyl)propan-1-amine, PF-4455242, which entered phase 1 clinical testing and has demonstrated target engagement in healthy volunteers.
Asunto(s)
Compuestos de Bifenilo/farmacología , Diseño de Fármacos , Descubrimiento de Drogas , Antagonistas de Narcóticos/farmacología , Receptores Opioides kappa/antagonistas & inhibidores , Sulfonamidas/farmacología , 3,4-Dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclohexil)-bencenacetamida, (trans)-Isómero/farmacología , Analgésicos/química , Analgésicos/farmacocinética , Analgésicos/farmacología , Animales , Área Bajo la Curva , Compuestos de Bifenilo/química , Compuestos de Bifenilo/farmacocinética , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Perros , Haplorrinos , Humanos , Tasa de Depuración Metabólica , Ratones , Microsomas Hepáticos/metabolismo , Modelos Químicos , Estructura Molecular , Morfina/farmacología , Antagonistas de Narcóticos/química , Antagonistas de Narcóticos/farmacocinética , Dolor/metabolismo , Dolor/prevención & control , Ratas , Ratas Sprague-Dawley , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/agonistas , Relación Estructura-Actividad , Sulfonamidas/química , Sulfonamidas/farmacocinéticaRESUMEN
A novel alpha 7 nAChR agonist, 4-(5-methyloxazolo[4,5-b]pyridin-2-yl)-1,4-diazabicyclo[3.2.2]nonane (24, CP-810,123), has been identified as a potential treatment for cognitive deficits associated with psychiatric or neurological conditions including schizophrenia and Alzheimer's disease. Compound 24 is a potent and selective compound with excellent pharmaceutical properties. In rodent, the compound displays high oral bioavailability and excellent brain penetration affording high levels of receptor occupancy and in vivo efficacy in auditory sensory gating and novel object recognition. The structural diversity of this compound and its preclinical in vitro and in vivo package support the hypothesis that alpha 7 nAChR agonists may have potential as a pharmacotherapy for the treatment of cognitive deficits in schizophrenia.
Asunto(s)
Compuestos de Azabiciclo/síntesis química , Compuestos de Azabiciclo/farmacología , Trastornos del Conocimiento/tratamiento farmacológico , Agonistas Nicotínicos/síntesis química , Agonistas Nicotínicos/farmacología , Nootrópicos/síntesis química , Nootrópicos/farmacología , Oxazoles/síntesis química , Oxazoles/farmacología , Receptores Nicotínicos/química , Esquizofrenia/tratamiento farmacológico , Animales , Compuestos de Azabiciclo/química , Disponibilidad Biológica , Células Cultivadas , Células Epiteliales/efectos de los fármacos , Femenino , Hipocampo/efectos de los fármacos , Humanos , Riñón/citología , Riñón/efectos de los fármacos , Microsomas Hepáticos/efectos de los fármacos , Agonistas Nicotínicos/química , Nootrópicos/química , Oocitos/efectos de los fármacos , Oxazoles/química , Ratas , Piel/citología , Piel/efectos de los fármacos , Relación Estructura-Actividad , Xenopus laevis/crecimiento & desarrollo , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
The design and synthesis of novel opiates are reported. Based on the message-address principle a novel class of 4,4- and 3,3-biaryl piperidines was designed and synthesized. Biological evaluation confirmed that these compounds exhibit high affinity and selectivity for the delta opioid receptor. Key structure-activity relationships that influence affinity, selectivity, functional activity and clearance are reported.
Asunto(s)
Ligandos , Piperidinas/química , Receptores Opioides delta/antagonistas & inhibidores , Animales , Diseño de Fármacos , Humanos , Microsomas Hepáticos/metabolismo , Piperidinas/síntesis química , Piperidinas/farmacología , Unión Proteica , Ratas , Receptores Opioides delta/metabolismo , Receptores Opioides kappa/antagonistas & inhibidores , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/antagonistas & inhibidores , Receptores Opioides mu/metabolismo , Relación Estructura-ActividadRESUMEN
By use of chemical enablement and prospective design, a novel series of selective, brain penetrant PDE9A inhibitors have been identified that are capable of producing in vivo elevations of brain cGMP.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Encéfalo/enzimología , 3',5'-AMP Cíclico Fosfodiesterasas/química , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Animales , Encéfalo/metabolismo , GMP Cíclico/metabolismo , Humanos , Enlace de Hidrógeno , Ratones , Modelos Moleculares , Inhibidores de Fosfodiesterasa/química , Inhibidores de Fosfodiesterasa/farmacocinética , Inhibidores de Fosfodiesterasa/farmacologíaRESUMEN
By utilizing structure-based drug design (SBDD) knowledge, a novel class of phosphodiesterase (PDE) 10A inhibitors was identified. The structure-based drug design efforts identified a unique "selectivity pocket" for PDE10A inhibitors, and interactions within this pocket allowed the design of highly selective and potent PDE10A inhibitors. Further optimization of brain penetration and drug-like properties led to the discovery of 2-[4-(1-methyl-4-pyridin-4-yl-1H-pyrazol-3-yl)-phenoxymethyl]-quinoline (PF-2545920). This PDE10A inhibitor is the first reported clinical entry for this mechanism in the treatment of schizophrenia.
Asunto(s)
Antipsicóticos/síntesis química , Modelos Moleculares , Hidrolasas Diéster Fosfóricas/metabolismo , Pirazoles/síntesis química , Quinolinas/síntesis química , Esquizofrenia/tratamiento farmacológico , Animales , Antipsicóticos/farmacocinética , Antipsicóticos/farmacología , Reacción de Prevención/efectos de los fármacos , Sitios de Unión , Encéfalo/metabolismo , Cristalografía por Rayos X , Perros , Femenino , Humanos , Enlace de Hidrógeno , Técnicas In Vitro , Macaca fascicularis , Masculino , Ratones , Ratones Noqueados , Microsomas Hepáticos/metabolismo , Estructura Molecular , Hidrolasas Diéster Fosfóricas/química , Hidrolasas Diéster Fosfóricas/genética , Unión Proteica , Pirazoles/farmacocinética , Pirazoles/farmacología , Quinolinas/farmacocinética , Quinolinas/farmacología , Ratas , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
A series of 2-aryloxy-4-alkoxy-pyridines ( 1) was identified as novel, selective, and orally active antagonists of the corticotropin-releasing factor 1 (CRF 1) receptor. Among these, compound 2 (CP-316311) is a potent and selective CRF 1 receptor antagonist with an IC 50 value of 6.8 nM in receptor binding and demonstrates oral efficacy in central nervous system (CNS) in vivo models. The regiochemistry of compounds in this series was determined by an X-ray structural analysis. A method to control regioselectivity via pyridine- N-oxides was developed. The synthesis of compounds in series 1 (Figure ) and [ (3)H]- 2 as well as the structure-activity relationship (SAR) are discussed. The in vitro, ex vivo, and in vivo properties of representative compounds are described herein. Compound 2 was advanced to phase II depression trials to test the hypothesis that CRF 1 antagonists could be used clinically as antidepressant drugs.
Asunto(s)
Antidepresivos/síntesis química , Piridinas/síntesis química , Receptores de Hormona Liberadora de Corticotropina/antagonistas & inhibidores , Potenciales de Acción/efectos de los fármacos , Administración Oral , Hormona Adrenocorticotrópica/sangre , Animales , Antidepresivos/química , Antidepresivos/farmacología , Autorradiografía , Encéfalo/fisiología , Línea Celular , Hormona Liberadora de Corticotropina/farmacología , Cristalografía por Rayos X , Humanos , Técnicas In Vitro , Isomerismo , Masculino , Estructura Molecular , Hipófisis/metabolismo , Piridinas/química , Piridinas/farmacología , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Reflejo de Sobresalto/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
An orally active clinical candidate of corticotropin-releasing factor 1 (CRF 1) antagonist 1 showed a significant positive food effect in dog and human after oral administration. Efforts to address the food effect issue led us to explore and discover compounds in series 2 as orally active CRF 1 receptor antagonists, in which some compounds showed improved physicochemical properties while retaining desired pharmacological properties. Compound 3a (CP-376395) was selected for further development, due not only to its reduced food effects but also its greater efficacy in CNS models. Compound 3a was advanced to the clinic. The synthesis of representative potential candidates and their in vitro, ex vivo, and in vivo data are described.
Asunto(s)
Aminopiridinas/síntesis química , Antidepresivos/síntesis química , Receptores de Hormona Liberadora de Corticotropina/antagonistas & inhibidores , Potenciales de Acción/efectos de los fármacos , Administración Oral , Hormona Adrenocorticotrópica/sangre , Aminopiridinas/farmacocinética , Aminopiridinas/farmacología , Animales , Antidepresivos/farmacocinética , Antidepresivos/farmacología , Corteza Cerebral/metabolismo , Hormona Liberadora de Corticotropina/farmacología , Perros , Ayuno , Humanos , Inyecciones Intravenosas , Locus Coeruleus/fisiología , Masculino , Hipófisis/metabolismo , Periodo Posprandial , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Reflejo de Sobresalto/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
Using microdialysis in rat prefrontal cortex, we found that 1 mg/kg of the stimulant methylphenidate and the non-stimulant atomoxetine, two widely used treatments for Attention Deficit/Hyperactivity Disorder (ADHD), produce robust increases in the extracellular levels of histamine, which plays a key role in attention, learning and memory. While the clinical response to ADHD drugs is typically attributed to modulation of norepinephrine and dopamine, this finding suggests enhanced histamine release may contribute to their efficacy as ADHD treatments.
Asunto(s)
Liberación de Histamina/efectos de los fármacos , Metilfenidato/farmacología , Corteza Prefrontal/efectos de los fármacos , Propilaminas/farmacología , Animales , Clorhidrato de Atomoxetina , Trastorno por Déficit de Atención con Hiperactividad/tratamiento farmacológico , Masculino , Metilfenidato/uso terapéutico , Corteza Prefrontal/metabolismo , Propilaminas/uso terapéutico , Ratas , Ratas Sprague-DawleyRESUMEN
Treatment with the atypical antipsychotics olanzapine and clozapine has been associated with an increased risk for deterioration of glucose homeostasis, leading to hyperglycemia, ketoacidosis, and diabetes, in some cases independent of weight gain. Because these events may be a consequence of their ability to directly alter insulin secretion from pancreatic beta-cells, we determined the effects of several antipsychotics on cholinergic- and glucose-stimulated insulin secretion from isolated rat islets. At concentrations encompassing therapeutically relevant levels, olanzapine and clozapine reduced insulin secretion stimulated by 10 micromol/l carbachol plus 7 mmol/l glucose. This inhibition of insulin secretion was paralleled by significant reductions in carbachol-potentiated inositol phosphate accumulation. In contrast, risperidone or ziprasidone had no adverse effect on cholinergic-induced insulin secretion or inositol phosphate accumulation. None of the compounds tested impaired the islet secretory responses to 8 mmol/l glucose alone. Finally, in vitro binding and functional data show that olanzapine and clozapine (unlike risperidone, ziprasidone, and haloperidol) are potent muscarinic M3 antagonists. These findings demonstrate that low concentrations of olanzapine and clozapine can markedly and selectively impair cholinergic-stimulated insulin secretion by blocking muscarinic M3 receptors, which could be one of the contributing factors to their higher risk for producing hyperglycemia and diabetes in humans.
Asunto(s)
Antipsicóticos/farmacología , Carbacol/farmacología , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Antagonistas Muscarínicos/farmacología , Animales , Benzodiazepinas/farmacología , Carbacol/antagonistas & inhibidores , Clozapina/farmacología , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Cinética , Masculino , Olanzapina , Ratas , Ratas Sprague-DawleyRESUMEN
Olanzapine and clozapine produce robust increases in hippocampal acetylcholine release during acetylcholinesterase inhibition, while other antipsychotics, including thioridazine, have only small effects. Since thioridazine binds with similar high affinities to muscarinic receptors as olanzapine and clozapine, muscarinic autoreceptor blockade was ruled out as a primary mechanism [Neuropsychopharmacology 26 (2002) 583]. This study compared in vitro binding affinities and functional activities of olanzapine, clozapine, thioridazine, ziprasidone, risperidone, chlorpromazine and scopolamine at muscarinic M2 receptors with their in vivo potencies to increase acetylcholine release in the rat hippocampus. We found that scopolamine, olanzapine and clozapine, but also high doses of thioridazine and chlorpromazine, markedly increase acetylcholine release. The reduced in vivo potencies of thioridazine and chlorpromazine are consistent with their significantly weaker functional antagonist activity at human muscarinic M2 receptors, while thioridazine's reduced binding affinity for rat muscarinic M2 receptors and lower brain exposure, may further contribute to its weak in vivo potency compared to olanzapine. The excellent correlation between in vitro antagonist activities of antipsychotics at muscarinic M2 receptors and their in vivo potencies to increase acetylcholine release, suggests that olanzapine, clozapine, as well as thioridazine and chlorpromazine, increase acetylcholine release via blockade of terminal muscarinic M2 autoreceptors.
Asunto(s)
Acetilcolina/metabolismo , Antipsicóticos/farmacología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Antagonistas Muscarínicos/farmacología , Receptores Muscarínicos/fisiología , Animales , Células CHO , Cricetinae , Humanos , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
A series of arylpiperazine- and 1,2,3,4-tetrahydroisoquinoline-based arylsulfonamides was synthesized and evaluated for their interactions with the constitutively active 5-HT7 receptor. Effects on basal adenylate cyclase activity were measured using HEK-293 cells expressing the rat 5-HT7. All ligands produced a decrease of adenylate cyclase activity, indicative of their inverse agonism. Additionally, computational studies with a set of 22 inverse agonists, including these novel inverse agonists and inverse agonists known from literature, resulted in a pharmacophore model and a CoMFA model (R2 = 0.97, SE = 0.18). Docking of inverse agonists at the binding site of a model of the helical parts of the 5-HT7 receptor, based on the alpha carbon template for 7-TM GPCRs, revealed interesting molecular interactions and a possible explanation for observed structure-activity relationships.
Asunto(s)
Isoquinolinas/síntesis química , Piperazinas/síntesis química , Agonistas de Receptores de Serotonina/síntesis química , Sulfonamidas/síntesis química , Inhibidores de Adenilato Ciclasa , Animales , Sitios de Unión , Línea Celular , Humanos , Isoquinolinas/química , Isoquinolinas/farmacología , Ligandos , Modelos Moleculares , Piperazinas/química , Piperazinas/farmacología , Relación Estructura-Actividad Cuantitativa , Ensayo de Unión Radioligante , Ratas , Receptores de Serotonina/química , Receptores de Serotonina/efectos de los fármacos , Agonistas de Receptores de Serotonina/química , Agonistas de Receptores de Serotonina/farmacología , Sulfonamidas/química , Sulfonamidas/farmacologíaRESUMEN
A process of producing a receptor in HEK-293 cells used for the drug discovery program at Pfizer Inc. has been successfully developed with a novel BelloCell bioreactor to replace the conventional 2-D cell culturing devices including Cell Factories and roller bottles. A single BelloCell-500 has produced >1.4 x 10(9) HEK-293 cells, which are equivalent to those produced by 12 roller bottles, with substantially easier operation, single inoculation, less inoculum, less medium consumption and better space utilization. The receptor expression levels are better than those obtained by the traditional process. 3.7 pmoles of radioligandY mg(-1) protein were attained in the bioreactor compared to 2.3 pmoles of radioligandY mg(-1) protein in roller bottles. This may be attributed to the three dimensional attachment during cell growth. A 92% cell recovery from the bioreactor has been attained using Acutase or Trypsin treatment followed by four washes. It has been proven to be a viable and efficient device to produce adherent cells and express target components of interest for drug discovery applications.
RESUMEN
On the basis of a set of 20 diverse 5-HT(7) receptor agonists, the pharmacophore for 5-HT(7) receptor agonism was determined. Additionally two CoMFA models were developed, based on different alignments of the agonists. Both models show good correlations between experimental and predictive pK(i) values and show a high degree of similarity. The CoMFA fields were subsequently used to map the agonist binding site of the model of the 5-HT(7) receptor. Important roles in ligand binding are attributed to Asp162 of TM3 (interaction with a protonated nitrogen), and Thr244 of TM5 (interaction with a substituent at an aromatic moiety). Amino acid residues of the aromatic cluster of TM6 are hypothesized to play an important role in ligand binding as pi-pi stacking moieties. Agonists missing a hydrogen-bond-accepting moiety, but possessing an aromatic substituent instead, seem to bind the receptor with high affinity as well by occupying a lipophilic pocket hosted by residues of TM5 and TM6.