RESUMO
Psoriasis and atopic dermatitis are skin diseases affecting millions of patients. Here, we characterize benzoxaborole phosphodiesterase (PDE)-4 inhibitors, a new topical class that has demonstrated therapeutic benefit for psoriasis and atopic dermatitis in phase 2 or phase 3 studies. Crisaborole [AN2728, 4-((1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-yl)oxy)benzonitrile], compd2 [2-ethoxy-6-((1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-yl)oxy)nicotinonitrile], compd3 [6-((1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-yl)oxy)-2-(2-isopropoxyethoxy)nicotinonitrile], and compd4 [5-chloro-6-((1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-yl)oxy)-2-((4-oxopentyl)oxy)nicotinonitrile] are potent PDE4 inhibitors with similar affinity for PDE4 isoforms and equivalent inhibition on the catalytic domain and the full-length enzyme. These benzoxaboroles are less active on other PDE isozymes. Compd4 binds to the catalytic domain of PDE4B2 with the oxaborole group chelating the catalytic bimetal and overlapping with the phosphate in cAMP during substrate hydrolysis, and the interaction extends into the adenine pocket. In cell culture, benzoxaborole PDE4 inhibitors suppress the release of tumor necrosis factor-α, interleukin (IL)-23, IL-17, interferon-γ, IL-4, IL-5, IL-13, and IL-22, and these cytokines contribute to the pathologic changes in skin structure and barrier functions as well as immune dysregulation in atopic dermatitis and psoriasis. Treatment with compd3 or N(6),2'-O-dibutyryladenosine 3',5'-cyclic monophosphate increases cAMP response element binding protein phosphorylation in human monocytes and decreases extracellular signal-regulated kinase phosphorylation in human T cells; these changes lead to reduced cytokine production and are among the mechanisms by which compd3 blocks cytokine release. Topical compd3 penetrates the skin and suppresses phorbol myristate acetate-induced IL-13, IL-22, IL-17F, and IL-23 transcription and calcipotriol-induced thymic stromal lymphopoietin expression in mouse skin. Skin thinning is a major dose-limiting side effect of glucocorticoids. By contrast, repeated application of compd3 did not thin mouse skin. These findings show the potential benefits and safety of benzoxaborole PDE4 inhibitors for the treatment of psoriasis and atopic dermatitis.
Assuntos
Compostos de Boro/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Dermatite Atópica/tratamento farmacológico , Inibidores da Fosfodiesterase 4/farmacologia , Psoríase/tratamento farmacológico , Pele/efeitos dos fármacos , Pele/patologia , Administração Tópica , Animais , Compostos de Boro/administração & dosagem , Compostos de Boro/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Domínio Catalítico , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/química , Citocinas/metabolismo , Dermatite Atópica/metabolismo , Dermatite Atópica/patologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Modelos Moleculares , Inibidores da Fosfodiesterase 4/administração & dosagem , Inibidores da Fosfodiesterase 4/uso terapêutico , Fosforilação/efeitos dos fármacos , Psoríase/metabolismo , Psoríase/patologia , Pele/metabolismo , Linfopoietina do Estroma do TimoRESUMO
Crisaborole topical ointment, 2% (formerly known as AN2728) is a benzoxaborole, nonsteroidal, topical, anti-inflammatory phosphodiesterase 4 (PDE4) inhibitor investigational compound that recently completed phase 3 studies for the treatment of mild to moderate atopic dermatitis (AD). The unique configuration of boron within the crisaborole molecule enables selective targeting and inhibition of PDE4, an enzyme that converts the intracellular second messenger 3'5'-cyclic adenosine monophosphate (cAMP) into the active metabolite adenosine monophosphate (AMP). By inhibiting PDE4 and thus increasing levels of cAMP, crisaborole controls inflammation. The use of boron chemistry enabled synthesis of a low-molecular-weight compound (251 daltons), thereby facilitating effective penetration of crisaborole through human skin. In vitro experiments showed that crisaborole inhibits cytokine production from peripheral blood mononuclear cells in a pattern similar to other PDE4 inhibitors and distinct from corticosteroids. Crisaborole also displayed topical anti-inflammatory activity in a skin inflammation model. Once crisaborole reaches systemic circulation after topical application, it is metabolized to inactive metabolites. This limits systemic exposure to crisaborole and systemic PDE4 inhibition. In phase 1 and 2 clinical studies, crisaborole ointment, 2% was generally well tolerated and improved AD disease severity scores, pruritus, and all other AD signs and symptoms. Two large, randomized, controlled, phase 3, pivotal clinical trials assessing the efficacy and safety of crisaborole topical ointment, 2% in children, adolescents, and adults with mild to moderate AD were recently completed with positive results.
Assuntos
Anti-Inflamatórios não Esteroides/administração & dosagem , Compostos de Boro/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Dermatite Atópica/tratamento farmacológico , Inibidores da Fosfodiesterase 4/administração & dosagem , Administração Tópica , Animais , Anti-Inflamatórios não Esteroides/química , Compostos de Boro/química , Compostos Bicíclicos Heterocíclicos com Pontes/química , Citocinas/antagonistas & inibidores , Citocinas/metabolismo , Dermatite Atópica/diagnóstico , Dermatite Atópica/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Pomadas , Inibidores da Fosfodiesterase 4/química , Resultado do TratamentoRESUMO
Benzoxaboroles are a novel class of drug-like compounds that have been rich sources of novel inhibitors for various enzymes and of new drugs. While examining benzoxaborole activity in phenotypic screens, our attention was attracted by the (aminomethylphenoxy)benzoxaborole family, which potently inhibited Toll-like receptor-stimulated cytokine secretion from leukocytes. After considering their structure-activity relationships and the central role of kinases in leukocyte biology, we performed a kinome-wide screen to investigate the members of the (aminomethylphenoxy)benzoxaborole family. This technique identified Rho-activated kinase (ROCK) as a target. We showed competitive behavior, with respect to ATP, and then determined the ROCK2-drug cocrystal structure. The drug occupies the ATP site in which the oxaborole moiety provides hydrogen bond donors and acceptors to the hinge, and the aminomethyl group interacts with the magnesium/ATP-interacting aspartic acid common to protein kinases. The series exhibits excellent selectivity against most of the kinome, with greater than 15-fold selectivity against the next best member of the AGC protein kinase subfamily. Medicinal chemistry efforts with structure-based design resulted in a compound with a Ki of 170 nM. Cellular studies revealed strong enzyme inhibition rank correlation with suppression of intracellular phosphorylation of a ROCK substrate. The biochemical potencies of these compounds also translated to functional activity, causing smooth muscle relaxation in rat aorta and guinea pig trachea. The series exhibited oral availability and one member reduced rat blood pressure, consistent with ROCK's role in smooth muscle contraction. Thus, the benzoxaborole moiety represents a novel hinge-binding kinase scaffold that may have potential for therapeutic use.
Assuntos
Compostos de Boro/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismo , Animais , Aorta Torácica/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Citocinas/sangue , Humanos , Células Jurkat , Modelos Moleculares , Contração Muscular/efeitos dos fármacos , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Fosforilação , Inibidores de Proteínas Quinases/química , Proteína Fosfatase 1/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Traqueia/efeitos dos fármacos , Quinases Associadas a rho/genéticaRESUMO
Pro-inflammatory cytokines play a critical role in the development of autoimmune and inflammatory diseases. Targeting the cytokine environment has proven efficient for averting inflammation. In this study, we reported that 6-[4-(aminomethyl)-2-chlorophenoxyl]benzo[c][1,2]oxaborol-1(3H)-ol (AN3485), a benzoxaborole analog, inhibited TLR2-, TLR3-, TLR4-, and TLR5-mediated TNF-α, IL-1ß, and IL-6 release from human PBMCs and isolated monocytes with IC(50) values ranging from 18 to 580 nM, and the inhibition was mediated at the transcriptional level. Topical administration of AN3485 significantly reduced PMA-induced contact dermatitis and oxazolone-induced delayed-type hypersensitivity in mice, indicating its capability of penetrating skin and potential topical application in skin inflammation. Oral administration of AN3485 showed dose-dependent suppression of LPS-induced TNF-α and IL-6 production in mice with an ED(90) of 30 mg/kg. Oral AN3485, 35 mg/kg, twice a day, suppressed collagen-induced arthritis in mice over a 20-day period. The potent anti-inflammatory activity in in vitro and in vivo disease models makes AN3485 an attractive therapeutic lead for a variety of cutaneous and systemic inflammatory diseases.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Artrite/tratamento farmacológico , Compostos de Boro/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Dermatite Alérgica de Contato/tratamento farmacológico , Hipersensibilidade a Drogas/tratamento farmacológico , Hipersensibilidade Tardia/tratamento farmacológico , Receptores Toll-Like/antagonistas & inibidores , Administração Oral , Animais , Anti-Inflamatórios não Esteroides/administração & dosagem , Anti-Inflamatórios não Esteroides/farmacocinética , Anti-Inflamatórios não Esteroides/toxicidade , Artrite/imunologia , Artrite/metabolismo , Compostos de Boro/administração & dosagem , Compostos de Boro/farmacocinética , Compostos de Boro/toxicidade , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/farmacocinética , Compostos Bicíclicos Heterocíclicos com Pontes/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/biossíntese , Citocinas/metabolismo , Dermatite Alérgica de Contato/etiologia , Dermatite Alérgica de Contato/imunologia , Dermatite Alérgica de Contato/metabolismo , Relação Dose-Resposta a Droga , Hipersensibilidade a Drogas/etiologia , Hipersensibilidade a Drogas/imunologia , Hipersensibilidade a Drogas/metabolismo , Feminino , Humanos , Hipersensibilidade Tardia/induzido quimicamente , Hipersensibilidade Tardia/imunologia , Hipersensibilidade Tardia/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
A series of novel 6-(aminomethylphenoxy)benzoxaborole analogs was synthesized for the investigation of the structure-activity relationship of the inhibition of TNF-alpha, IL-1beta, and IL-6, from lipopolysaccharide stimulated peripheral blood mononuclear cells. Compounds 9d and 9e showed potent activity against all three cytokines with IC50 values between 33 and 83nM. Chloro substituted analog 9e (AN3485) is considered to be a promising lead for novel anti-inflammatory agent with a favorable pharmacokinetic profile.
Assuntos
Anti-Inflamatórios/química , Benzoxazóis/química , Compostos de Boro/química , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Animais , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacocinética , Compostos de Boro/metabolismo , Compostos de Boro/farmacocinética , Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/farmacocinética , Meia-Vida , Humanos , Interleucina-1beta/antagonistas & inibidores , Interleucina-1beta/metabolismo , Interleucina-6/antagonistas & inibidores , Interleucina-6/metabolismo , Cinética , Leucócitos Mononucleares/efeitos dos fármacos , Lipopolissacarídeos/toxicidade , Camundongos , Ligação Proteica , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Structure-activity relationships of 6-(benzoylamino)benzoxaborole analogs were investigated for the inhibition of TNF-α, IL-1ß, and IL-6 from lipopolysaccharide stimulated peripheral blood mononuclear cells. Compound 1q showed potent activity against all three cytokines with IC50 values between 0.19 and 0.50µM, inhibited LPS-induced TNF-α and IL-6 elevation in mice and improved collagen-induced arthritis in mice. Compound 1q (AN4161) is considered to be a promising lead for novel anti-inflammatory agent with an excellent pharmacokinetic profile.
Assuntos
Anti-Inflamatórios/química , Anti-Inflamatórios/uso terapêutico , Artrite Experimental/tratamento farmacológico , Derivados de Benzeno/química , Derivados de Benzeno/uso terapêutico , Compostos de Boro/química , Compostos de Boro/uso terapêutico , Animais , Anti-Inflamatórios/farmacocinética , Anti-Inflamatórios/farmacologia , Derivados de Benzeno/farmacocinética , Derivados de Benzeno/farmacologia , Compostos de Boro/farmacocinética , Compostos de Boro/farmacologia , Interleucina-1beta/imunologia , Interleucina-6/imunologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Lipopolissacarídeos/imunologia , Camundongos , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Monoclonal antibodies (mAbs) are one of the fastest-growing classes of drugs and have been approved to treat several diseases, including cancers and autoimmune disorders. Preclinical pharmacokinetics studies are performed to determine the therapeutically meaningful dosages and efficacy of candidate drugs. These studies are typically performed in non-human primates; however, using primates is costly and raises ethical considerations. As a result, rodent models that better mimic human-like pharmacokinetics have been generated and remain an area of active investigation. Pharmacokinetic characteristics of a candidate drug, such as half-life, are partly controlled by antibody binding to the human neonatal receptor hFCRN. Due to the abnormally high binding of human antibodies to mouse FCRN, traditional laboratory rodents do not accurately model the pharmacokinetics of human mAbs. In response, humanized rodents expressing hFCRN have been generated. However, these models generally use large inserts randomly integrated into the mouse genome. Here, we report the production and characterization of a CRISPR/Cas9-mediated hFCRN transgenic mouse termed SYNB-hFCRN. Using CRISPR/Cas9-assisted gene targeting, we prepared a strain with a simultaneous knockout of mFcrn and insertion of a hFCRN mini-gene under the control of the endogenous mouse promoter. These mice are healthy and express hFCRN in the appropriate tissues and immune cell subtypes. Pharmacokinetic evaluation of human IgG and adalimumab (Humira®) demonstrate hFCRN-mediated protection. These newly generated SYNB-hFCRN mice provide another valuable animal model for use in preclinical pharmacokinetics studies during early drug development.
Assuntos
Antígenos de Histocompatibilidade Classe I , Receptores Fc , Camundongos , Animais , Camundongos Transgênicos , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Anticorpos Monoclonais , Imunoglobulina G/metabolismoRESUMO
INTRODUCTION: There is an urgent need for new animal models of SARS CoV-2 infection to improve research and drug development. This brief commentary examines the deficits of current models and proposes several improved alternates. The existing single transgene mouse models poorly mimic the clinical features of COVID-19; those strains get a milder disease than human COVID-19 disease. Many of the current transgenic models utilize random integration of several copies of single ACE2 transgenes, resulting in unnatural gene expression and exhibit rapid lethality. We suggest preparing precision knock-in of selected human mini genes at the mouse initiation codon and knock-out of the mouse homolog as a better option. Three genes critical for infection are suggested targets, ACE2 (the viral cellular receptor), its co-infection protease TMRPSS2, and the primary antibody clearance receptor FcγRT. To offer the best platform for COVID 19 research, preparation of single, double, and triple humanized combinations offers the researcher the opportunity to better understand the contributions of these receptors, coreceptors to therapeutic efficacy. In addition, we propose to create the humanized strains in the C57BL/6J and BALB/c backgrounds. These two backgrounds are Th1 responders and Th2 responders, respectively, and allow modeling of the variability seen in human pathology including lung pathology and late sequelae of COVID-19 disease (BALB/c). We suggest the need to do a thorough characterization of both the short-term and long-term effects of SAR-CoV-2 infection at the clinical, virologic, histopathologic, hematologic, and immunologic levels. We expect the multiply humanized strains will be superior to the single-gene and multiple-gene-copy transgenic models available to date. These mouse models will represent state-of-the-art tools for investigating mechanisms of COVID-19 pathogenesis and immunity and developing vaccines and drugs.
Assuntos
Pesquisa Biomédica/tendências , Tratamento Farmacológico da COVID-19 , COVID-19/genética , Modelos Animais de Doenças , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/genética , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/imunologia , Animais , Animais Geneticamente Modificados , Antivirais/farmacologia , Antivirais/uso terapêutico , COVID-19/imunologia , Vacinas contra COVID-19/farmacologia , Vacinas contra COVID-19/uso terapêutico , Desenvolvimento de Medicamentos/tendências , Humanos , Camundongos , SARS-CoV-2/imunologiaRESUMO
We report the discovery of novel boron-containing molecules, exemplified by N-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)-2-trifluoromethylbenzamide (AN3520) and 4-fluoro-N-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)-2-trifluoromethylbenzamide (SCYX-6759), as potent compounds against Trypanosoma brucei in vitro, including the two subspecies responsible for human disease T. b. rhodesiense and T. b. gambiense. These oxaborole carboxamides cured stage 1 (hemolymphatic) trypanosomiasis infection in mice when administered orally at 2.5 to 10 mg/kg of body weight for 4 consecutive days. In stage 2 disease (central nervous system [CNS] involvement), mice infected with T. b. brucei were cured when AN3520 or SCYX-6759 were administered intraperitoneally or orally (50 mg/kg) twice daily for 7 days. Oxaborole-treated animals did not exhibit gross signs of compound-related acute or subchronic toxicity. Metabolism and pharmacokinetic studies in several species, including nonhuman primates, demonstrate that both SCYX-6759 and AN3520 are low-clearance compounds. Both compounds were well absorbed following oral dosing in multiple species and also demonstrated the ability to cross the blood-brain barrier with no evidence of interaction with the P-glycoprotein transporter. Overall, SCYX-6759 demonstrated superior pharmacokinetics, and this was reflected in better efficacy against stage 2 disease in the mouse model. On the whole, oxaboroles demonstrate potent activity against all T. brucei subspecies, excellent physicochemical profiles, in vitro metabolic stability, a low potential for CYP450 inhibition, a lack of active efflux by the P-glycoprotein transporter, and high permeability. These properties strongly suggest that these novel chemical entities are suitable leads for the development of new and effective orally administered treatments for human African trypanosomiasis.
Assuntos
Imidazóis/uso terapêutico , Tripanossomicidas/uso terapêutico , Trypanosoma brucei brucei/patogenicidade , Tripanossomíase Africana/tratamento farmacológico , Animais , Feminino , Humanos , Imidazóis/química , Macaca fascicularis , Masculino , Camundongos , Estrutura Molecular , Ratos , Ratos Sprague-Dawley , Trypanosoma brucei brucei/efeitos dos fármacosRESUMO
A series of benzimidazole-benzoxaborole hybrid molecules linked via an amide linker are described that exhibit good in vitro activity against Onchocerca volvulus, a filarial nematode responsible for the disease onchocerciasis, also known as river blindness. The lead identified in this series, 8a (AN8799), was found to have acceptable pharmacokinetic properties to enable evaluation in animal models of human filariasis. Compound 8a was effective in killing Brugia malayi, B. pahangi, and Litomosoides sigmodontis worms present in Mongolian gerbils when dosed subcutaneously as a suspension at 100 mg/kg/day for 14 days but not when dosed orally at 100 mg/kg/day for 28 days. The measurement of plasma levels of 8a at the end of the dosing period and at the time of sacrifice revealed an interesting dependence of activity on the extended exposure for both 8a and the positive control, flubendazole.
Assuntos
Benzimidazóis/uso terapêutico , Compostos de Boro/uso terapêutico , Brugia/efeitos dos fármacos , Oncocercose/tratamento farmacológico , Amidas , Animais , Benzimidazóis/farmacocinética , Compostos de Boro/farmacocinética , Feminino , Filaricidas/farmacocinética , Filaricidas/uso terapêutico , Gerbillinae , Masculino , Onchocerca volvulus/efeitos dos fármacosRESUMO
We have used a supervised classification approach to systematically mine a large microarray database derived from livers of compound-treated rats. Thirty-four distinct signatures (classifiers) for pharmacological and toxicological end points can be identified. Just 200 genes are sufficient to classify these end points. Signatures were enriched in xenobiotic and immune response genes and contain un-annotated genes, indicating that not all key genes in the liver xenobiotic responses have been characterized. Many signatures with equal classification capabilities but with no gene in common can be derived for the same phenotypic end point. The analysis of the union of all genes present in these signatures can reveal the underlying biology of that end point as illustrated here using liver fibrosis signatures. Our approach using the whole genome and a diverse set of compounds allows a comprehensive view of most pharmacological and toxicological questions and is applicable to other situations such as disease and development.
Assuntos
Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Xenobióticos/farmacologia , Animais , Bases de Dados Genéticas , Genômica , Fígado/patologia , Cirrose Hepática/genética , Ratos , Reprodutibilidade dos TestesRESUMO
OBJECTIVE: The only therapies currently available for cutaneous neurofibromas (cNF) are procedural. The goals of the Therapies Development Working Group were to (1) summarize currently available treatment options for cNF, (2) define key considerations for drug discovery and development generally, and specifically for cNF, and (3) outline recommendations for the successful development of medical therapies for cNF. METHODS: The subgroup reviewed published and unpublished data on procedural, drug/device, and medical treatment approaches utilized for cNFs via literature search. The team defined disease- and patient-specific factors to consider for therapies development in a series of consensus meetings. RESULTS: The team identified 5 approaches entailing procedural and drug/device methods currently under study. There have been 4 clinical studies exploring various interventional therapies, from which outcomes were highly variable. The team identified 4 key factors to prioritize during the development of products for the treatment for cNF: safety, anatomic distribution of cNF, numbers of tumors to be treated, and route of administration. CONCLUSIONS: The number, size, and distribution of cNF is highly variable among patients with NF1 and it is possible that different phenotypes will require different drug development paths. The nonfatal nature of the disease and relatively limited patient numbers suggest that for any product to have a higher likelihood of acceptance, it will have to (1) demonstrate an effect that is clinically meaningful, (2) have a safety profile conducive to long-term dosing, and (3) have a low manufacturing cost.
Assuntos
Desenvolvimento de Medicamentos , Neurofibroma/tratamento farmacológico , Neurofibromatose 1/complicações , Neoplasias Cutâneas/tratamento farmacológico , Descoberta de Drogas , Humanos , Neurofibroma/complicações , Neoplasias Cutâneas/complicaçõesRESUMO
OBJECTIVE: Several clinical trials targeting cutaneous neurofibromas (cNF) have been conducted; however, none has resulted in meaningful changes to care. The Clinical Trial Design and Development subgroup's goals were to (1) define key considerations in the design of clinical trials for cNF, (2) summarize existing data in relation to these considerations, and (3) provide consensus recommendations about key elements of trial design to accelerate the clinical development of therapies for cNF. METHODS: The subgroup, with experts from genetics, dermatology, neurology, oncology, and basic science, spanning academia, government research, and regulatory programs, and industry, reviewed published and unpublished data on clinical trials for cNF and other diseases in the skin. Discussions of these data resulted in formulation of a list of priority issues to address in order to develop efficient and effective clinical trials for cNF. RESULTS: The subgroup identified 2 natural history studies of cNF, 4 priority outcome measures, and 6 patient-reported outcome tools for potential use in efficacy trials of cNF. Time to initiate intervention, patient eligibility, mechanism of action, route of administration, safety monitoring, and regulatory agency interactions were identified as key factors to consider when designing clinical trials for cNF. CONCLUSIONS: Alignment on endpoints and methods for the measurement and quantification of cNF represent a priority for therapeutic development for cNF. Advances in technological methods and outcome tools utilized in other skin diseases may be applicable to cNF studies. Patient age is an important factor guiding trial design and clinical development path.
Assuntos
Ensaios Clínicos como Assunto/métodos , Neurofibroma/terapia , Projetos de Pesquisa , Neoplasias Cutâneas/terapia , Humanos , Avaliação de Resultados em Cuidados de Saúde , Medidas de Resultados Relatados pelo PacienteRESUMO
The comparability and reliability of data generated using microarray technology would be enhanced by use of a common set of standards that allow accuracy, reproducibility and dynamic range assessments on multiple formats. We designed and tested a complex biological reagent for performance measurements on three commercial oligonucleotide array formats that differ in probe design and signal measurement methodology. The reagent is a set of two mixtures with different proportions of RNA for each of four rat tissues (brain, liver, kidney and testes). The design provides four known ratio measurements of >200 reference probes, which were chosen for their tissue-selectivity, dynamic range coverage and alignment to the same exemplar transcript sequence across all three platforms. The data generated from testing three biological replicates of the reagent at eight laboratories on three array formats provides a benchmark set for both laboratory and data processing performance assessments. Close agreement with target ratios adjusted for sample complexity was achieved on all platforms and low variance was observed among platforms, replicates and sites. The mixed tissue design produces a reagent with known gene expression changes within a complex sample and can serve as a paradigm for performance standards for microarrays that target other species.
Assuntos
Perfilação da Expressão Gênica/normas , Análise de Sequência com Séries de Oligonucleotídeos/normas , RNA Mensageiro/normas , Animais , Perfilação da Expressão Gênica/métodos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Sondas de Oligonucleotídeos , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Padrões de Referência , Reprodutibilidade dos Testes , Distribuição TecidualRESUMO
BACKGROUND: Reliance on just one drug to treat the prevalent tropical disease, schistosomiasis, spurs the search for new drugs and drug targets. Inhibitors of human cyclic nucleotide phosphodiesterases (huPDEs), including PDE4, are under development as novel drugs to treat a range of chronic indications including asthma, chronic obstructive pulmonary disease and Alzheimer's disease. One class of huPDE4 inhibitors that has yielded marketed drugs is the benzoxaboroles (Anacor Pharmaceuticals). METHODOLOGY/PRINCIPAL FINDINGS: A phenotypic screen involving Schistosoma mansoni and 1,085 benzoxaboroles identified a subset of huPDE4 inhibitors that induced parasite hypermotility and degeneration. To uncover the putative schistosome PDE4 target, we characterized four PDE4 sequences (SmPDE4A-D) in the parasite's genome and transcriptome, and cloned and recombinantly expressed the catalytic domain of SmPDE4A. Among a set of benzoxaboroles and catechol inhibitors that differentially inhibit huPDE4, a relationship between the inhibition of SmPDE4A, and parasite hypermotility and degeneration, was measured. To validate SmPDE4A as the benzoxaborole molecular target, we first generated Caenorhabditis elegans lines that express a cDNA for smpde4a on a pde4(ce268) mutant (hypermotile) background: the smpde4a transgene restored mutant worm motility to that of the wild type. We then showed that benzoxaborole inhibitors of SmPDE4A that induce hypermotility in the schistosome also elicit a hypermotile response in the C. elegans lines that express the smpde4a transgene, thereby confirming SmPDE4A as the relevant target. CONCLUSIONS/SIGNIFICANCE: The orthogonal chemical, biological and genetic strategies employed identify SmPDE4A's contribution to parasite motility and degeneration, and its potential as a drug target. Transgenic C. elegans is highlighted as a potential screening tool to optimize small molecule chemistries to flatworm molecular drug targets.
Assuntos
Anti-Helmínticos/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Inibidores da Fosfodiesterase 4/farmacologia , Schistosoma mansoni/efeitos dos fármacos , Animais , Animais Geneticamente Modificados/genética , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Domínio Catalítico , Clonagem Molecular , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Locomoção/efeitos dos fármacos , Schistosoma mansoni/anatomia & histologia , Schistosoma mansoni/fisiologiaRESUMO
Successful drug discovery requires accurate decision making in order to advance the best candidates from initial lead identification to final approval. Chemogenomics, the use of genomic tools in pharmacology and toxicology, offers a promising enhancement to traditional methods of target identification/validation, lead identification, efficacy evaluation, and toxicity assessment. To realize the value of chemogenomics information, a contextual database is needed to relate the physiological outcomes induced by diverse compounds to the gene expression patterns measured in the same animals. Massively parallel gene expression characterization coupled with traditional assessments of drug candidates provides additional, important mechanistic information, and therefore a means to increase the accuracy of critical decisions. A large-scale chemogenomics database developed from in vivo treated rats provides the context and supporting data to enhance and accelerate accurate interpretation of mechanisms of toxicity and pharmacology of chemicals and drugs. To date, approximately 600 different compounds, including more than 400 FDA approved drugs, 60 drugs approved in Europe and Japan, 25 withdrawn drugs, and 100 toxicants, have been profiled in up to 7 different tissues of rats (representing over 3200 different drug-dose-time-tissue combinations). Accomplishing this task required evaluating and improving a number of in vivo and microarray protocols, including over 80 rigorous quality control steps. The utility of pairing clinical pathology assessments with gene expression data is illustrated using three anti-neoplastic drugs: carmustine, methotrexate, and thioguanine, which had similar effects on the blood compartment, but diverse effects on hepatotoxicity. We will demonstrate that gene expression events monitored in the liver can be used to predict pathological events occurring in that tissue as well as in hematopoietic tissues.
Assuntos
Biotecnologia/métodos , Desenho de Fármacos , Indústria Farmacêutica/métodos , 5-Aminolevulinato Sintetase/biossíntese , Animais , Antineoplásicos/farmacologia , Antineoplásicos/toxicidade , Automação , Ductos Biliares/patologia , Carmustina/toxicidade , Biologia Computacional , Bases de Dados como Assunto , Relação Dose-Resposta a Droga , Regulação para Baixo , Expressão Gênica , Humanos , Hiperplasia/etiologia , Fígado/efeitos dos fármacos , Masculino , Metotrexato/toxicidade , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Tamanho do Órgão , Farmacologia/métodos , RNA/química , RNA Complementar/metabolismo , Ratos , Ratos Sprague-Dawley , Reticulócitos/citologia , Reticulócitos/metabolismo , Tioguanina/toxicidade , Fatores de Tempo , Distribuição Tecidual , Toxicologia/métodosRESUMO
There is an increasing interest in in vivo metabolite identification in early drug discovery in order to (i) give a more complete picture of metabolic profile in investigational animal models, (ii) propose phase I and phase II metabolites using the same pharmacokinetic/toxicokinetic study samples, (iii) expose metabolically labile groups where chemical modifications could improve stability, and (iv) enable early safety assessment of metabolites. In the early discovery stage of our anti-inflammatory program, one novel benzoxaborole, AN6414, exhibiting both PDE4 enzyme and TNFα inhibition activities, became our primary candidate for further investigation. The traditional metabolite identifications usually require high dosed samples with long data scans and analysis. In this study, we conducted quick and more selective core-structure related precursor scans followed by daughter ion scans and identified a total of 10 major phase I and phase II metabolites using rat plasma samples from a toxicokinetic study at an oral dosing of 30 mg/kg. Plasma samples were treated with solid phase extraction (SPE) prior to LC/MS/MS. An AB SCIEX API 4000 QTRAP mass spectrometer coupled with a Shimadzu LC system was used for LC/MS/MS analysis. We found the major metabolites of AN6414 to be oxidative deboronation, protodeboronation, oxidation products and their sulfate-conjugated species. This analysis drove analoging efforts which improved the pharmacokinetic profile, namely, lowering clearance and increasing exposure relative to AN6414. Toxicity predictions by the software program DEREK suggest the identified potential metabolites to be safe.
Assuntos
Anti-Inflamatórios/farmacocinética , Compostos de Boro/farmacocinética , Cromatografia Líquida , Descoberta de Drogas/métodos , Inibidores da Fosfodiesterase 4/farmacologia , Piridinas/farmacocinética , Espectrometria de Massas em Tandem , Administração Oral , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/sangue , Anti-Inflamatórios/urina , Área Sob a Curva , Biotransformação , Compostos de Boro/administração & dosagem , Compostos de Boro/sangue , Compostos de Boro/toxicidade , Feminino , Masculino , Taxa de Depuração Metabólica , Modelos Biológicos , Oxirredução , Inibidores da Fosfodiesterase 4/administração & dosagem , Inibidores da Fosfodiesterase 4/sangue , Inibidores da Fosfodiesterase 4/toxicidade , Piridinas/administração & dosagem , Piridinas/sangue , Piridinas/toxicidade , Ratos , Ratos Sprague-Dawley , Medição de Risco , Software , Extração em Fase Sólida , Sulfatos/farmacocinéticaRESUMO
We have used boron-based molecules to create novel, competitive, reversible inhibitors of phosphodiesterase 4 (PDE4). The co-crystal structure reveals a binding configuration which is unique compared to classical catechol PDE4 inhibitors, with boron binding to the activated water in the bimetal center. These phenoxybenzoxaboroles can be optimized to generate submicromolar potency enzyme inhibitors, which inhibit TNF-α, IL-2, IFN-γ, IL-5 and IL-10 activities in vitro and show safety and efficacy for topical treatment of human psoriasis. They provide a valuable new route for creating novel potent anti-PDE4 inhibitors.
Assuntos
Compostos de Boro/química , Compostos de Boro/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/química , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Inibidores de Fosfodiesterase/química , Inibidores de Fosfodiesterase/farmacologia , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Ligação Competitiva , Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Domínio Catalítico , Cristalografia por Raios X , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Citocinas/biossíntese , Humanos , Técnicas In Vitro , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Metais/química , Modelos Moleculares , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Brain and spinal cord injuries result in cognitive and/or sensorimotor impairments that can significantly diminish the quality of life for the patient and their carers, and result in healthcare system costs totaling in the billions. The current gold-standard of acute care for spinal cord injury is to administer high doses of glucocorticoids within 8 h of injury; administration after 8 h may be without effect or detrimental to the outcome of the patient. Therefore, improved pharmacological approaches for limiting the extent of tissue damage and neurological dysfunction in the acute injury setting are urgently needed. Early intervention in CNS injury by antagonizing or controlling the post-injury inflammatory process with pharmaceutical agents is a major focus of current clinical and preclinical investigations. In this editorial overview, recent clinical trials and preclinical studies of brain and spinal cord injuries are discussed, including studies focusing on the use of broad-spectrum immunosuppressive drugs (eg, minocycline); growth factors (eg, erythropoietin); dual anti-inflammatory and anti-vasospasm drugs, such as Rho and ROCK kinase inhibitors; and broad-spectrum anti-inflammatory drugs, such as PDE4 inhibitors. These new approaches hold great promise to improve outcomes for patients with brain and spinal injuries.
RESUMO
Toxicogenomics using a reference database can provide a better understanding and prediction of toxicity, largely by creating biomarkers that tie gene expression to actual pathology events. During the course of building a toxicogenomic database, an observation was made that a number of non-steroidal anti-inflammatory compounds (NSAIDs) at supra-pharmacologic doses induced an acute phase response (APR) and displayed hepatic gene expression patterns similar to that of intravenous lipopolysaccharide (LPS). Since NSAIDs are known to cause injury along the gastrointestinal tract, it has been suggested that NSAIDs increase intestinal permeability, allowing LPS and/or bacteria into the systemic circulation and stimulating an APR detectable in the liver. A short term study was subsequently conducted examining the effects of aspirin, indomethacin, ibuprofen, and rofecoxib to rats and a variety of endpoints were examined that included serum levels of inflammatory cytokines, histologic evaluation, and hepatic gene expression. Both indomethacin and ibuprofen injured the gastrointestinal tract, induced an APR, and increased serum levels of LPS, while rofecoxib and aspirin did not affect the GI tract or induce an APR. In treatments that eventually showed a systemic inflammatory response, hepatic expression of many inflammatory genes was noted as early as 6 hours after treatment well before alterations in traditional clinical pathology markers were detected. This finding led to the creation of a hepatic gene expression biomarker of APR that was effectively shown to be an early identifier of imminent inflammatory injury. In terms of the relative gastrointestinal safety and the NSAIDs studied, an important safety distinction can be made between the presumptive efficacious dose and the APR-inducing dose for indomethacin (1-2-fold), ibuprofen (5-fold), and rofecoxib (approximately 250-fold). Our data support the notion that NSAID-induced intestinal injury results in leakage of commensural bacteria and/or LPS into the circulation, provoking a systemic inflammatory response and that hepatic gene expression-based biomarkers can be used as early and sensitive biomarkers of APR onset.