RESUMO
Despite recent approvals of anti-obesity drugs there is still a high therapeutic need for alternative options with higher efficacy in humans. As part of our MCH-R1 antagonist program for the treatment of obesity, a series of biphenylacetamide HTS hits was evaluated. Several issues of the initial lead structures had to be resolved, such as potency, selectivity over related GPCRs and P-gp efflux limiting brain exposure in this series. We could demonstrate that all parameters can be significantly improved by structural modifications resulting in BI 414 as a potent and orally available MCH-R1 antagonist tool compound with acceptable in vivo efficacy in an animal model of obesity.
Assuntos
Alcinos/síntese química , Alcinos/farmacologia , Fármacos Antiobesidade/síntese química , Fármacos Antiobesidade/farmacologia , Piridinas/síntese química , Piridinas/farmacologia , Receptores de Somatostatina/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Citocromo P-450 CYP2D6/metabolismo , Inibidores das Enzimas do Citocromo P-450/síntese química , Inibidores das Enzimas do Citocromo P-450/farmacologia , Desenho de Fármacos , Ingestão de Alimentos/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Obesidade/tratamento farmacológico , Ratos , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Receptores Acoplados a Proteínas G/metabolismo , Relação Estrutura-AtividadeRESUMO
Although overweight and obesity are highly prevalent conditions, options to treat them are still very limited. As part of our search for safe and effective MCH-R1 antagonists for the treatment of obesity, two series of pyridones and pyridazinones were evaluated. Optimization was aimed at improving DMPK properties by increasing metabolic stability and improving the safety profile by reducing inhibition of the hERG channel and reducing the potential to induce phospholipidosis. Steric shielding of a labile keto moiety with an ortho-methyl group and fine-tuning of the polarity in several parts of the molecule resulted in BI 186908 (11 g), a potent and selective MCH-R1 antagonist with favorable DMPK and CMC properties. Chronic administration of BI 186908 resulted in significant body weight reduction comparable to sibutramine in a 4 week diet-induced obesity model in rats. Based on its favorable safety profile, BI 186908 was advanced to pre-clinical development.
Assuntos
Fármacos Antiobesidade/síntese química , Fármacos Antiobesidade/farmacologia , Piridazinas/síntese química , Piridazinas/farmacologia , Piridinas/síntese química , Piridinas/farmacologia , Receptores de Somatostatina/antagonistas & inibidores , Animais , Depressores do Apetite/farmacologia , Peso Corporal/efeitos dos fármacos , Ciclobutanos/farmacologia , Descoberta de Drogas , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Hepatócitos/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Técnicas In Vitro , Lipidoses/tratamento farmacológico , Microssomos Hepáticos/metabolismo , Fosfolipídeos/metabolismo , Bloqueadores dos Canais de Potássio/síntese química , Bloqueadores dos Canais de Potássio/farmacologia , Pirazinas/síntese química , Pirazinas/farmacologia , Ratos , Relação Estrutura-AtividadeRESUMO
Despite recent success there remains a high therapeutic need for the development of drugs targeting diseases associated with the metabolic syndrome. As part of our search for safe and effective MCH-R1 antagonists for the treatment of obesity, a series of 3,6-disubstituted pyridazines was evaluated. During optimization several issues of the initial lead structures had to be resolved, such as selectivity over related GPCRs, inhibition of the hERG channel as well as the potential to induce phospholipidosis. Utilizing property-based design, we could demonstrate that all parameters can significantly be improved by consequently increasing the polarity of the compounds. By this strategy, we succeeded in identifying potent and orally available MCH-R1 antagonists with good selectivity over M1 and 5-HT2A and an improved safety profile with respect to hERG inhibition and phospholipidosis.
Assuntos
Fármacos Antiobesidade/síntese química , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Lipidoses/tratamento farmacológico , Fosfolipídeos/metabolismo , Bloqueadores dos Canais de Potássio/síntese química , Bloqueadores dos Canais de Potássio/farmacologia , Piridazinas/síntese química , Piridazinas/farmacologia , Receptores de Somatostatina/antagonistas & inibidores , Animais , Fármacos Antiobesidade/farmacologia , Bloqueadores dos Canais de Potássio/farmacocinética , Piridazinas/farmacocinética , Ratos , Ratos Wistar , Receptor 5-HT2A de Serotonina/efeitos dos fármacos , Antagonistas da Serotonina/síntese química , Antagonistas da Serotonina/farmacologia , Relação Estrutura-AtividadeRESUMO
The aim of the present study was to compare the effects of genetic mPGES-1 loss and COX-2 inhibition on myocardial damage after coronary occlusion. mPGES-1(-/-) mice and their wild-type littermates were injected with vehicle or COX-2 inhibitor (celecoxib), and 30min later the left coronary artery was surgically occluded. At 24h, myocardial infarct (MI) volume was measured histologically. Post-MI survival was reduced in WT mice receiving celecoxib (12/20) compared with vehicle-treated controls (12/12) or the loss of mPGES-1 (13/13) together with increased phosphokinase (CPK) and cardiac troponin-I release. Endogenous mPGES-1 expression was unchanged by ischemia in WT mice and absent in mPGES-1(-/-) hearts. COX-2 expression was markedly increased at 24h after MI in WT hearts; this upregulation was largely attenuated in mPGES-1(-/-) mice. We conclude that loss of mPGES-1 prevents the upregulation of COX-2 after myocardial infarct, and in contrast to inhibition of COX-2, does not increase ischemic myocardial damage.
Assuntos
Inibidores de Ciclo-Oxigenase 2/farmacologia , Deleção de Genes , Oxirredutases Intramoleculares/deficiência , Oxirredutases Intramoleculares/genética , Microssomos/enzimologia , Isquemia Miocárdica/enzimologia , Isquemia Miocárdica/genética , Animais , Oclusão Coronária/complicações , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Regulação Enzimológica da Expressão Gênica , Camundongos , Infarto do Miocárdio/complicações , Infarto do Miocárdio/patologia , Isquemia Miocárdica/complicações , Isquemia Miocárdica/patologia , Prostaglandina-E Sintases , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Migraine is a complex, debilitating neurovascular disorder. Although knowledge on the main molecular players is still incomplete, recent preclinical and clinical findings indicate that there is a clear correlation between migraine-associated headache and the release of the neuropeptide calcitonin gene-related peptide (CGRP). BIBN4096 was the first CGRP antagonist to be tested in clinical trials for the treatment of migraine. The proven efficacy of this agent, and also the CGRP antagonist MK-0974, to alleviate acute migraine headache provided significant support for the hypothesis that CGRP has an important role in migraine pathophysiology. Moreover, the recently published results from Phase II trials are encouraging and suggest that this new type of drug might offer advantages over existing therapies for patients suffering from migraine and related headaches.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/antagonistas & inibidores , Transtornos de Enxaqueca/tratamento farmacológico , Animais , Azepinas/química , Azepinas/farmacologia , Azepinas/uso terapêutico , Peptídeo Relacionado com Gene de Calcitonina/fisiologia , Ensaios Clínicos como Assunto , Dipeptídeos/química , Dipeptídeos/farmacologia , Dipeptídeos/uso terapêutico , Humanos , Imidazóis/química , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Transtornos de Enxaqueca/fisiopatologia , Modelos Biológicos , Estrutura Molecular , Quinazolinas/química , Quinazolinas/farmacologia , Quinazolinas/uso terapêuticoRESUMO
BACKGROUND: Functional 3D organ models such as precision-cut lung slices (PCLS) have recently captured the attention of biomedical research. To enable wider implementation in research and development, these new biologically relevant organ models are being constantly refined. A very important issue is to improve the preparation of high-quality RNA (ribonucleic acid) from PCLS for drug discovery and development of new therapies. Gene expression analysis at different levels is used as an important experimental readout. Genome-wide analysis using microarrays is mostly applied for biomarker selection in disease models or in comprehensive toxicological studies. Specific biomarker testing by reverse transcriptase quantitative polymerase chain reaction (RTqPCR) is often used in efficacy studies. Both applications require high-quality RNA as starting material for the generation of reliable data. Additionally, a small number of slices should be sufficient for satisfactory RNA isolation to allow as many experimental conditions as possible to be covered with a given tissue sample. Unfortunately, the vast amount of agarose in PCLS impedes RNA extraction according to the standard procedures. RESULTS: We established an optimized protocol for RNA isolation from PCLS from humans, rats, mice, marmosets, and rhesus macaques based on the separation of lysis and precipitation steps and a magnetic-bead cleanup procedure. The resulting RNA is of high purity and possesses a high degree of integrity. There are no contaminations affecting RTqPCR efficiency or any enzymatic step in sample preparation for microarray analysis. CONCLUSIONS: In summary, we isolated RNA from PCLS from different species that is well suited for RTqPCR and for microarray analysis as downstream applications.
Assuntos
Pulmão/química , Microtomia/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA/isolamento & purificação , Transcriptoma , Idoso , Animais , Callithrix , Feminino , Humanos , Pulmão/cirurgia , Macaca mulatta , Imãs , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Análise em Microsséries , Microtomia/instrumentação , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos/estatística & dados numéricos , RNA/genética , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Neuropathic pain affects many patients, and treatment today is far from being perfect. Nav1.8 Na(+) channels, which are expressed by small fibre sensory neurons, are promising targets for novel analgesics. Na(+) channel blockers used today, however, show only limited selectivity for this channel subtype, and can cause dose-limiting side effects. Recently, the secretolytic ambroxol was found to preferentially inhibit Nav1.8 channels. We used this compound as a tool to investigate whether a Nav1.8-preferring blocker can suppress symptoms of chronic, neuropathic and inflammatory pain in animal models. The drug was tested in the formalin paw model, two models of mononeuropathy, and a model of monoarthritis in rats. Ambroxol's effects were compared with those of gabapentin. Ambroxol at a dose of 1g/kg had to be administered to rats to achieve the plasma levels that are reached in clinical use (for the treatment of infant and acute respiratory distress syndrome). Ambroxol (1g/kg) was only weakly effective in models for acute pain, but effectively reduced pain symptoms in all other models; in some cases it completely reversed pain behaviour. In most cases the effects were more pronounced than those of gabapentin (at 100mg/kg). These data show that a Nav1.8-preferring Na(+) channel blocker can effectively suppress pain symptoms in a variety of models for chronic, neuropathic and inflammatory pain at plasma levels, which can be achieved in the clinic.
Assuntos
Ambroxol/farmacologia , Analgésicos , Inflamação/complicações , Proteínas do Tecido Nervoso/efeitos dos fármacos , Dor/tratamento farmacológico , Dor/etiologia , Neuropatia Ciática/complicações , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/efeitos dos fármacos , Aminas/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Constrição Patológica/prevenção & controle , Constrição Patológica/psicologia , Ácidos Cicloexanocarboxílicos/farmacologia , Relação Dose-Resposta a Droga , Antagonistas de Aminoácidos Excitatórios/farmacologia , Formaldeído , Gabapentina , Inflamação/psicologia , Ligadura , Masculino , Canal de Sódio Disparado por Voltagem NAV1.8 , Medição da Dor/efeitos dos fármacos , Ratos , Ratos Wistar , Neuropatia Ciática/psicologia , Ácido gama-Aminobutírico/farmacologiaRESUMO
The release of CGRP in humans is associated with the occurrence of migraine headaches. The vasoactive neuropeptide is released by afferent neurones originating in the peripherally located trigeminal ganglion supplying the dura mater. The role of CGRP in migraine is further supported by recently released data showing that the CGRP-antagonist BIBN4096BS is clinically effective for the treatment of migraine headaches. Yet, the trigger for CGRP release during migraine attacks is not identified. It is suggested that the peripheral CGRP release during a migraine attack might be either triggered by direct activation of afferent dural neurones, or, by indirect activation via the central nervous system. Recently, we were able to show that the CGRP-antagonist BIBN4096BS is able to inhibit vasodilation induced by trigeminal ganglion stimulation. Now, we extend our studies to the investigation of facial blood flow changes induced by electrical stimulation of the brainstem trigeminal nucleus caudalis (TNC). Here, we show that stimulation of the TNC leads to a pronounced increase of facial blood flow. The nicotinic antagonist Hexamethonium reduced the evoked flow by approximately 50% (30 mg/kg), while the muscarinic antagonist Atropin did not influence the stimulation evoked blood flow. Application of BIBN4096BS (0.3 mg/kg, i.v.) diminished the evoked flow almost completely. Therefore, we conclude that CGRP represents the key player in TNC-induced facial vasodilation, while activation of nicotinic receptors modulates centrally induced peripheral neurogenic vasodilation.
Assuntos
Velocidade do Fluxo Sanguíneo/fisiologia , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Estimulação Encefálica Profunda/métodos , Face/inervação , Face/fisiologia , Receptores Nicotínicos/metabolismo , Núcleos do Trigêmeo/fisiologia , Vasodilatação/fisiologia , Animais , Modelos Animais de Doenças , Face/irrigação sanguínea , Masculino , Transtornos de Enxaqueca/fisiopatologia , Ratos , Ratos WistarRESUMO
Calcitonin gene-related peptide (CGRP) is known to play a major role in the pathogenesis of pain syndromes, in particular migraine pain. Here we focus on its implication in a rat pain model of inflammation, induced by injection of complete Freund adjuvant (CFA). The nonpeptide CGRP receptor antagonist BIBN4096BS reduces migraine pain and trigeminal neuronal activity. Here we demonstrate that the compound reduces inflammatory pain and spinal neuronal activity. Behavioural experiments reveal a reversal of the CFA-induced mechanical hypersensitivity and monoiodoacetate (MIA)-induced weight-bearing deficit in rats after systemic drug administration. To further investigate the mechanism of action of the CGRP antagonist in inflammatory pain, in vivo electrophysiological studies were performed in CFA-injected rats. Recordings from wide dynamic range neurons in deep dorsal horn layers of the lumbar spinal cord confirmed a reduction of neuronal activity after systemic drug application. The same amount of reduction occurred after topical administration onto the paw, with resulting systemic plasma concentrations in the low nanomolar range. However, spinal administration of BIBN4096BS did not modify the neuronal activity in the CFA model. Peripheral blockade of CGRP receptors by BIBN4096BS significantly alleviates inflammatory pain.
Assuntos
Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina , Peptídeo Relacionado com Gene de Calcitonina/análogos & derivados , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Inflamação/tratamento farmacológico , Dor/tratamento farmacológico , Piperazinas/uso terapêutico , Quinazolinas/uso terapêutico , Administração Tópica , Analgésicos Opioides/administração & dosagem , Analgésicos Opioides/uso terapêutico , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Comportamento Animal , Adjuvante de Freund , Inflamação/complicações , Cetorolaco/uso terapêutico , Masculino , Morfina/administração & dosagem , Morfina/uso terapêutico , Neurônios/efeitos dos fármacos , Osteoartrite/induzido quimicamente , Osteoartrite/complicações , Dor/etiologia , Medição da Dor/efeitos dos fármacos , Piperazinas/administração & dosagem , Piperazinas/sangue , Quinazolinas/administração & dosagem , Quinazolinas/sangue , Ratos , Ratos Wistar , Medula Espinal/fisiopatologiaRESUMO
The goal of the present study was to assess how genetic loss of microsomal prostaglandin E(2) synthase-1 (mPGES-1) affects acute cardiac ischemic damage after coronary occlusion in mice. Wild type (WT), heterozygous (mPGES-1(+/-)), and homozygous (mPGES-1(-/-)) knockout mice were subjected to left coronary artery occlusion. At 24h, myocardial infarct (MI) volume was measured histologically. Post-MI survival, plasma levels of creatine phosphokinase (CPK) and cardiac troponin-I, together with MI size, were similar in WT, mPGES-1(+/-) and mPGES-1(-/-) mice. In contrast, post-MI survival was reduced in mPGES-1(-/-) mice pretreated with I prostanoid receptor (IP) antagonist (12/16) compared with vehicle-treated controls (13/13 mPGES-1(-/-)) together with increased CPK and cardiac troponin-I release. The deletion of mPGES-1 in mice results in increased prostacyclin I(2) (PGI(2)) formation and marginal effects on the circulatory prostaglandin E(2) (PGE(2)) level. We conclude that loss of mPGES-1 results in increased PGI(2) formation, and in contrast to inhibition of PGI(2), without worsening acute cardiac ischemic injury.
Assuntos
Oxirredutases Intramoleculares/deficiência , Infarto do Miocárdio/sangue , Animais , Compostos de Benzil/farmacologia , Creatina Quinase/sangue , Epoprostenol/biossíntese , Imidazóis/farmacologia , Camundongos , Camundongos Knockout , Infarto do Miocárdio/fisiopatologia , Isquemia Miocárdica/fisiopatologia , Prostaglandina-E Sintases , Troponina I/sangueRESUMO
Anxiety has been described as an important comorbidity in patients suffering from chronic pain. However, in animals the connection between persistent pain and anxiety has hardly been investigated. Therefore, in the current study it was assessed whether chronic pain also causes anxiety-like behaviour in animals and if it can be reversed by analgesic or anxiolytic drugs. Neuropathic pain was induced in rats by partial sciatic nerve ligation (PNL) and chronic constriction injury (CCI). Mechanical hypersensitivity was assessed by the "electronic algometer", while anxiety-like behaviour was measured by using the elevated plus maze. In both neuropathic pain models, rats exhibited mechanical hypersensitivity, whereas a significant increase in anxiety-like behaviour was observed only in CCI rats (time spent in open arms decreased significantly from 99+/-15.8s in sham animals to 33.4+/-7.5s in CCI animals). Furthermore, midazolam (0.5mg/kg; i.p.) significantly reduced anxiety-like behaviour in both sham- and CCI-operated animals without influencing mechanical hypersensitivity. Morphine (3mg/kg; i.p.) and gabapentin (30 mg/kg; i.p.) significantly attenuated anxiety-like behaviour in the CCI lesioned rats: morphine increased entries into open arms from 3.0+/-0.4 to 7.7+/-1.4 (P=0.01), gabapentin elevated this value from 4.7+/-1 to 7.5+/-0.9 (P=0.02). These data suggest that rats subjected to neuropathic pain models develop anxiety-like behaviour which can be reversed by appropriate analgesic treatment. Morphine and gabapentin had no anxiolytic-like effect in sham treated animals, thus their effect on anxiety-like behaviour in the neuropathic pain model is likely indirect via their anti-nociceptive properties.
Assuntos
Aminas/administração & dosagem , Ansiedade/prevenção & controle , Ansiedade/fisiopatologia , Ácidos Cicloexanocarboxílicos/administração & dosagem , Mononeuropatias/tratamento farmacológico , Mononeuropatias/fisiopatologia , Morfina/administração & dosagem , Neuralgia/tratamento farmacológico , Neuralgia/fisiopatologia , Ácido gama-Aminobutírico/administração & dosagem , Analgésicos/administração & dosagem , Animais , Modelos Animais de Doenças , Gabapentina , Humanos , Masculino , Medição da Dor/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
Three tandemly arrayed protocadherin gene clusters (Pcdh-alpha, -beta, -gamma) comprising more than 50 genes are found in human and mouse. Here, we have investigated the expression and distribution of individual gamma-protocadherins (Pcdhs-gamma) in the developing mouse brain. We find that transfection of Pcdh-gamma genes promotes calcium-dependent cell adhesion in HEK 293 cells. Furthermore, Pcdh-gamma can be recruited to synapses of transfected primary hippocampal neurons. Several individual members of the in total 22 Pcdhs-gamma were chosen to examine the expression of the three subfamilies, Pcdh-gammaA, -gammaB, and -gammaC. These Pcdh-gamma transcripts are expressed all over the brain, with minor regional and cell-type specific differences. Interestingly, a distinct, later onset of expression is observed for Pcdh-gammaC5, a gene located at the end of the Pcdh-gamma cluster. Largely overlapping expression patterns of individual Pcdh-gamma proteins are detected with anti-peptide antibodies. Small differences are observed in the staining of dendritic processes and synapse-rich layers. Our results support the idea that Pcdhs-gamma participate in neuronal differentiation and may be implicated in the fine-tuning of neuronal morphology and synaptogenesis. Cell autonomous regulation of transcription might generate the widespread distribution of individual Pcdhs-gamma in the brain, which is strikingly different from the restricted expression patterns observed for classical cadherins. Thus, a defined set of Pcdhs-gamma may engage in neuronal adhesion and signaling on the cellular level.