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1.
N Engl J Med ; 379(17): 1612-1620, 2018 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-30334692

RESUMO

BACKGROUND: VX-445 is a next-generation cystic fibrosis transmembrane conductance regulator (CFTR) corrector designed to restore Phe508del CFTR protein function in patients with cystic fibrosis when administered with tezacaftor and ivacaftor (VX-445-tezacaftor-ivacaftor). METHODS: We evaluated the effects of VX-445-tezacaftor-ivacaftor on Phe508del CFTR protein processing, trafficking, and chloride transport in human bronchial epithelial cells. On the basis of in vitro activity, a randomized, placebo-controlled, double-blind, dose-ranging, phase 2 trial was conducted to evaluate oral VX-445-tezacaftor-ivacaftor in patients heterozygous for the Phe508del CFTR mutation and a minimal-function mutation (Phe508del-MF) and in patients homozygous for the Phe508del CFTR mutation (Phe508del-Phe508del) after tezacaftor-ivacaftor run-in. Primary end points were safety and absolute change in percentage of predicted forced expiratory volume in 1 second (FEV1) from baseline. RESULTS: In vitro, VX-445-tezacaftor-ivacaftor significantly improved Phe508del CFTR protein processing, trafficking, and chloride transport to a greater extent than any two of these agents in dual combination. In patients with cystic fibrosis, VX-445-tezacaftor-ivacaftor had an acceptable safety and side-effect profile. Most adverse events were mild or moderate. The treatment also resulted in an increased percentage of predicted FEV1 of up to 13.8 points in the Phe508del-MF group (P<0.001). In patients in the Phe508del-Phe508del group, who were already receiving tezacaftor-ivacaftor, the addition of VX-445 resulted in an 11.0-point increase in the percentage of predicted FEV1 (P<0.001). In both groups, there was a decrease in sweat chloride concentrations and improvement in the respiratory domain score on the Cystic Fibrosis Questionnaire-Revised. CONCLUSIONS: The use of VX-445-tezacaftor-ivacaftor to target Phe508del CFTR protein resulted in increased CFTR function in vitro and translated to improvements in patients with cystic fibrosis with one or two Phe508del alleles. This approach has the potential to treat the underlying cause of cystic fibrosis in approximately 90% of patients. (Funded by Vertex Pharmaceuticals; VX16-445-001 ClinicalTrials.gov number, NCT03227471 ; and EudraCT number, 2017-000797-11 .).


Assuntos
Aminofenóis/uso terapêutico , Benzodioxóis/uso terapêutico , Agonistas dos Canais de Cloreto/uso terapêutico , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/tratamento farmacológico , Indóis/uso terapêutico , Pirazóis/uso terapêutico , Piridinas/uso terapêutico , Pirrolidinas/uso terapêutico , Quinolonas/uso terapêutico , Adolescente , Adulto , Alelos , Aminofenóis/efeitos adversos , Benzodioxóis/efeitos adversos , Agonistas dos Canais de Cloreto/efeitos adversos , Cloretos/análise , Cloretos/metabolismo , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Método Duplo-Cego , Combinação de Medicamentos , Feminino , Volume Expiratório Forçado/efeitos dos fármacos , Genótipo , Humanos , Indóis/efeitos adversos , Masculino , Mutação , Pirazóis/administração & dosagem , Pirazóis/farmacologia , Piridinas/administração & dosagem , Piridinas/farmacologia , Pirrolidinas/administração & dosagem , Pirrolidinas/farmacologia , Quinolonas/efeitos adversos , Suor/química , Adulto Jovem
2.
N Engl J Med ; 379(17): 1599-1611, 2018 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-30334693

RESUMO

BACKGROUND: The next-generation cystic fibrosis transmembrane conductance regulator (CFTR) corrector VX-659, in triple combination with tezacaftor and ivacaftor (VX-659-tezacaftor-ivacaftor), was developed to restore the function of Phe508del CFTR protein in patients with cystic fibrosis. METHODS: We evaluated the effects of VX-659-tezacaftor-ivacaftor on the processing, trafficking, and function of Phe508del CFTR protein using human bronchial epithelial cells. A range of oral VX-659-tezacaftor-ivacaftor doses in triple combination were then evaluated in randomized, controlled, double-blind, multicenter trials involving patients with cystic fibrosis who were heterozygous for the Phe508del CFTR mutation and a minimal-function CFTR mutation (Phe508del-MF genotypes) or homozygous for the Phe508del CFTR mutation (Phe508del-Phe508del genotype). The primary end points were safety and the absolute change from baseline in the percentage of predicted forced expiratory volume in 1 second (FEV1). RESULTS: VX-659-tezacaftor-ivacaftor significantly improved the processing and trafficking of Phe508del CFTR protein as well as chloride transport in vitro. In patients, VX-659-tezacaftor-ivacaftor had an acceptable safety and side-effect profile. Most adverse events were mild or moderate. VX-659-tezacaftor-ivacaftor resulted in significant mean increases in the percentage of predicted FEV1 through day 29 (P<0.001) of up to 13.3 points in patients with Phe508del-MF genotypes; in patients with the Phe508del-Phe508del genotype already receiving tezacaftor-ivacaftor, adding VX-659 resulted in a further 9.7-point increase in the percentage of predicted FEV1. The sweat chloride concentrations and scores on the respiratory domain of the Cystic Fibrosis Questionnaire-Revised improved in both patient populations. CONCLUSIONS: Robust in vitro activity of VX-659-tezacaftor-ivacaftor targeting Phe508del CFTR protein translated into improvements for patients with Phe508del-MF or Phe508del-Phe508del genotypes. VX-659 triple-combination regimens have the potential to treat the underlying cause of disease in approximately 90% of patients with cystic fibrosis. (Funded by Vertex Pharmaceuticals; VX16-659-101 and VX16-659-001 ClinicalTrials.gov numbers, NCT03224351 and NCT03029455 .).


Assuntos
Aminofenóis/uso terapêutico , Benzodioxóis/uso terapêutico , Agonistas dos Canais de Cloreto/uso terapêutico , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/tratamento farmacológico , Indóis/uso terapêutico , Pirazóis/uso terapêutico , Pirrolidinas/uso terapêutico , Quinolonas/uso terapêutico , Adolescente , Adulto , Alelos , Aminofenóis/efeitos adversos , Benzodioxóis/efeitos adversos , Células Cultivadas , Agonistas dos Canais de Cloreto/efeitos adversos , Cloretos/análise , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Método Duplo-Cego , Combinação de Medicamentos , Feminino , Volume Expiratório Forçado/efeitos dos fármacos , Genótipo , Humanos , Indóis/efeitos adversos , Masculino , Mutação , Pirazóis/efeitos adversos , Pirazóis/farmacologia , Pirrolidinas/efeitos adversos , Pirrolidinas/farmacologia , Quinolonas/efeitos adversos , Suor/química , Adulto Jovem
3.
Proc Natl Acad Sci U S A ; 108(46): 18843-8, 2011 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-21976485

RESUMO

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene that impair the function of CFTR, an epithelial chloride channel required for proper function of the lung, pancreas, and other organs. Most patients with CF carry the F508del CFTR mutation, which causes defective CFTR protein folding and processing in the endoplasmic reticulum, resulting in minimal amounts of CFTR at the cell surface. One strategy to treat these patients is to correct the processing of F508del-CFTR with small molecules. Here we describe the in vitro pharmacology of VX-809, a CFTR corrector that was advanced into clinical development for the treatment of CF. In cultured human bronchial epithelial cells isolated from patients with CF homozygous for F508del, VX-809 improved F508del-CFTR processing in the endoplasmic reticulum and enhanced chloride secretion to approximately 14% of non-CF human bronchial epithelial cells (EC(50), 81 ± 19 nM), a level associated with mild CF in patients with less disruptive CFTR mutations. F508del-CFTR corrected by VX-809 exhibited biochemical and functional characteristics similar to normal CFTR, including biochemical susceptibility to proteolysis, residence time in the plasma membrane, and single-channel open probability. VX-809 was more efficacious and selective for CFTR than previously reported CFTR correctors. VX-809 represents a class of CFTR corrector that specifically addresses the underlying processing defect in F508del-CFTR.


Assuntos
Aminopiridinas/uso terapêutico , Benzodioxóis/uso terapêutico , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/tratamento farmacológico , Mutação , Brônquios/citologia , Linhagem Celular , Células Cultivadas , Química Farmacêutica/métodos , Cloretos/química , Fibrose Cística/genética , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Células Epiteliais/citologia , Homozigoto , Humanos , Técnicas In Vitro , Pulmão/patologia , Modelos Genéticos
4.
Lancet Respir Med ; 11(6): 550-562, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36842446

RESUMO

BACKGROUND: Elexacaftor-tezacaftor-ivacaftor has been shown to be safe and efficacious in people with cystic fibrosis and at least one F508del allele. Our aim was to identify a novel cystic fibrosis transmembrane conductance regulator (CFTR) modulator combination capable of further increasing CFTR-mediated chloride transport, with the potential for once-daily dosing. METHODS: We conducted two phase 2 clinical trials to assess the safety and efficacy of a once-daily combination of vanzacaftor-tezacaftor-deutivacaftor in participants with cystic fibrosis who were aged 18 years or older. A phase 2 randomised, double-blind, active-controlled study (VX18-561-101; April 17, 2019, to Aug 20, 2020) was carried out to compare deutivacaftor monotherapy with ivacaftor monotherapy in participants with CFTR gating mutations, following a 4-week ivacaftor monotherapy run-in period. Participants were randomly assigned to receive either ivacaftor 150 mg every 12 h, deutivacaftor 25 mg once daily, deutivacaftor 50 mg once daily, deutivacaftor 150 mg once daily, or deutivacaftor 250 mg once daily in a 1:1:2:2:2 ratio. The primary endpoint was absolute change in ppFEV1 from baseline at week 12. A phase 2 randomised, double-blind, controlled, proof-of-concept study of vanzacaftor-tezacaftor-deutivacaftor (VX18-121-101; April 30, 2019, to Dec 10, 2019) was conducted in participants with cystic fibrosis and heterozygous for F508del and a minimal function mutation (F/MF genotypes) or homozygous for F508del (F/F genotype). Participants with F/MF genotypes were randomly assigned 1:2:2:1 to receive either 5 mg, 10 mg, or 20 mg of vanzacaftor in combination with tezacaftor-deutivacaftor or a triple placebo for 4 weeks, and participants with the F/F genotype were randomly assigned 2:1 to receive either vanzacaftor (20 mg)-tezacaftor-deutivacaftor or tezacaftor-ivacaftor active control for 4 weeks, following a 4-week tezacaftor-ivacaftor run-in period. Primary endpoints for part 1 and part 2 were safety and tolerability and absolute change in ppFEV1 from baseline to day 29. Secondary efficacy endpoints were absolute change from baseline at day 29 in sweat chloride concentrations and Cystic Fibrosis Questionnaire-Revised (CFQ-R) respiratory domain score. These clinical trials are registered with ClinicalTrials.gov, NCT03911713 and NCT03912233, and are complete. FINDINGS: In study VX18-561-101, participants treated with deutivacaftor 150 mg once daily (n=23) or deutivacaftor 250 mg once daily (n=24) had mean absolute changes in ppFEV1 of 3·1 percentage points (95% CI -0·8 to 7·0) and 2·7 percentage points (-1·0 to 6·5) from baseline at week 12, respectively, versus -0·8 percentage points (-6·2 to 4·7) with ivacaftor 150 mg every 12 h (n=11); the deutivacaftor safety profile was consistent with the established safety profile of ivacaftor 150 mg every 12 h. In study VX18-121-101, participants with F/MF genotypes treated with vanzacaftor (5 mg)-tezacaftor-deutivacaftor (n=9), vanzacaftor (10 mg)-tezacaftor-deutivacaftor (n=19), vanzacaftor (20 mg)-tezacaftor-deutivacaftor (n=20), and placebo (n=10) had mean changes relative to baseline at day 29 in ppFEV1 of 4·6 percentage points (-1·3 to 10·6), 14·2 percentage points (10·0 to 18·4), 9·8 percentage points (5·7 to 13·8), and 1·9 percentage points (-4·1 to 8·0), respectively, in sweat chloride concentration of -42·8 mmol/L (-51·7 to -34·0), -45·8 mmol/L (95% CI -51·9 to -39·7), -49·5 mmol/L (-55·9 to -43·1), and 2·3 mmol/L (-7·0 to 11·6), respectively, and in CFQ-R respiratory domain score of 17·6 points (3·5 to 31·6), 21·2 points (11·9 to 30·6), 29·8 points (21·0 to 38·7), and 3·3 points (-10·1 to 16·6), respectively. Participants with the F/F genotype treated with vanzacaftor (20 mg)-tezacaftor-deutivacaftor (n=18) and tezacaftor-ivacaftor (n=10) had mean changes relative to baseline (taking tezacaftor-ivacaftor) at day 29 in ppFEV1 of 15·9 percentage points (11·3 to 20·6) and -0·1 percentage points (-6·4 to 6·1), respectively, in sweat chloride concentration of -45·5 mmol/L (-49·7 to -41·3) and -2·6 mmol/L (-8·2 to 3·1), respectively, and in CFQ-R respiratory domain score of 19·4 points (95% CI 10·5 to 28·3) and -5·0 points (-16·9 to 7·0), respectively. The most common adverse events overall were cough, increased sputum, and headache. One participant in the vanzacaftor-tezacaftor-deutivacaftor group had a serious adverse event of infective pulmonary exacerbation and another participant had a serious rash event that led to treatment discontinuation. For most participants, adverse events were mild or moderate in severity. INTERPRETATION: Once-daily dosing with vanzacaftor-tezacaftor-deutivacaftor was safe and well tolerated and improved lung function, respiratory symptoms, and CFTR function. These results support the continued investigation of vanzacaftor-tezacaftor-deutivacaftor in phase 3 clinical trials compared with elexacaftor-tezacaftor-ivacaftor. FUNDING: Vertex Pharmaceuticals.


Assuntos
Fibrose Cística , Humanos , Adulto , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Cloretos , Volume Expiratório Forçado , Aminofenóis/efeitos adversos , Benzodioxóis/uso terapêutico , Mutação , Método Duplo-Cego , Agonistas dos Canais de Cloreto/uso terapêutico
5.
Proc Natl Acad Sci U S A ; 106(44): 18825-30, 2009 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-19846789

RESUMO

Cystic fibrosis (CF) is a fatal genetic disease caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR), a protein kinase A (PKA)-activated epithelial anion channel involved in salt and fluid transport in multiple organs, including the lung. Most CF mutations either reduce the number of CFTR channels at the cell surface (e.g., synthesis or processing mutations) or impair channel function (e.g., gating or conductance mutations) or both. There are currently no approved therapies that target CFTR. Here we describe the in vitro pharmacology of VX-770, an orally bioavailable CFTR potentiator in clinical development for the treatment of CF. In recombinant cells VX-770 increased CFTR channel open probability (P(o)) in both the F508del processing mutation and the G551D gating mutation. VX-770 also increased Cl(-) secretion in cultured human CF bronchial epithelia (HBE) carrying the G551D gating mutation on one allele and the F508del processing mutation on the other allele by approximately 10-fold, to approximately 50% of that observed in HBE isolated from individuals without CF. Furthermore, VX-770 reduced excessive Na(+) and fluid absorption to prevent dehydration of the apical surface and increased cilia beating in these epithelial cultures. These results support the hypothesis that pharmacological agents that restore or increase CFTR function can rescue epithelial cell function in human CF airway.


Assuntos
Aminofenóis/farmacologia , Brônquios/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/fisiopatologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Quinolinas/farmacologia , Quinolonas/farmacologia , Absorção/efeitos dos fármacos , Substituição de Aminoácidos/efeitos dos fármacos , Aminofenóis/química , Animais , Células Cultivadas , Cloretos/metabolismo , Cílios/efeitos dos fármacos , Cílios/metabolismo , Sinergismo Farmacológico , Canais Epiteliais de Sódio/metabolismo , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Camundongos , Mutação/genética , Células NIH 3T3 , Quinolinas/química , Quinolonas/química , Sódio/metabolismo
6.
Clin Transl Sci ; 14(2): 656-663, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33278322

RESUMO

In previous work, participants with a G970R mutation in cystic fibrosis transmembrane conductance regulator (CFTR) (c.2908G>C) had numerically lower sweat chloride responses during ivacaftor treatment than participants with other CFTR gating mutations. The objective of this substudy was to characterize the molecular defect of the G970R mutation in vitro and assess the benefit of ivacaftor in participants with this mutation. This substudy assessed sweat chloride, spirometry findings, and nasal potential difference on and off ivacaftor treatment in three participants with a G970R/F508del genotype. Intestinal organoids derived from rectal biopsy specimens were used to assess ivacaftor response ex vivo and conduct messenger RNA splice and protein analyses. No consistent or meaningful trends were observed between on-treatment and off-treatment clinical assessments. Organoids did not respond to ivacaftor in forskolin-induced swelling assays; no mature CFTR protein was detected in Western blots. Organoid RNA analysis demonstrated that 3 novel splice variants were created by G970R-CFTR: exon 17 truncation, exons 13-15 and 17 skipping, and intron 17 retention. Functional and molecular analyses indicated that the c.2908G>C mutation caused a cryptic splicing defect. Organoids lacked an ex vivo response with ivacaftor and supported identification of the mechanism underlying the CFTR defect caused by c.2908G>C. Analysis of CFTR mutations indicated that cryptic splicing was a rare cause of mutation misclassification in engineered cell lines. This substudy used organoids as an alternative in vitro model for mutations, such as cryptic splice mutations that cannot be fully assessed using cDNA expressed in recombinant cell systems.


Assuntos
Aminofenóis/administração & dosagem , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/tratamento farmacológico , Quinolonas/administração & dosagem , Adolescente , Adulto , Aminofenóis/efeitos adversos , Biópsia , Linhagem Celular , Células Cultivadas , Criança , Fibrose Cística/genética , Fibrose Cística/patologia , Éxons/genética , Feminino , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/patologia , Masculino , Mutação , Organoides , Medicina de Precisão/métodos , Cultura Primária de Células , Quinolonas/efeitos adversos , Splicing de RNA , Reto/citologia , Reto/patologia , Resultado do Tratamento , Adulto Jovem
7.
J Cyst Fibros ; 16(1): 41-44, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27773592

RESUMO

Ivacaftor, a CFTR potentiator that enhances chloride transport by acting directly on CFTR to increase its channel gating activity, has been evaluated in patients with different CFTR mutations. Several previous analyses have reported no statistical correlation between change from baseline in ppFEV1 and reduction in sweat chloride levels for individuals treated with ivacaftor. The objective of the post hoc analysis described here was to expand upon previous analyses and evaluate the correlation between sweat chloride levels and absolute ppFEV1 changes across multiple cohorts of patients with different CF-causing mutations who were treated with ivacaftor. The goal of the analysis was to help define the potential value of sweat chloride as a pharmacodynamic biomarker for use in CFTR modulator trials. For any given study, reductions in sweat chloride levels and improvements in absolute ppFEV1 were not correlated for individual patients. However, when the data from all studies were combined, a statistically significant correlation between sweat chloride levels and ppFEV1 changes was observed (p<0.0001). Thus, sweat chloride level changes in response to potentiation of the CFTR protein by ivacaftor appear to be a predictive pharmacodynamic biomarker of lung function changes on a population basis but are unsuitable for the prediction of treatment benefits for individuals.


Assuntos
Aminofenóis , Cloretos/análise , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Volume Expiratório Forçado/efeitos dos fármacos , Quinolonas , Suor/química , Aminofenóis/administração & dosagem , Aminofenóis/farmacocinética , Biomarcadores/análise , Agonistas dos Canais de Cloreto/administração & dosagem , Agonistas dos Canais de Cloreto/farmacocinética , Ensaios Clínicos como Assunto , Fibrose Cística/diagnóstico , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Humanos , Mutação , Valor Preditivo dos Testes , Quinolonas/administração & dosagem , Quinolonas/farmacocinética , Resultado do Tratamento
8.
Trends Endocrinol Metab ; 16(4): 152-9, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15860411

RESUMO

All secretory pituitary cells exhibit spontaneous and extracellular Ca2+-dependent electrical activity. Somatotrophs and lactotrophs fire plateau-bursting action potentials, which generate Ca2+ signals of sufficient amplitude to trigger hormone release. Gonadotrophs also fire action potentials spontaneously, but as single, high-amplitude spikes with limited ability to promote Ca2+ influx and secretion. However, Ca2+ mobilization in gonadotrophs transforms single spiking into plateau-bursting-type electrical activity and triggers secretion. Patch clamp analysis revealed that somatotrophs and lactotrophs, but not gonadotrophs, express BK (big)-type Ca2+-controlled K+ channels, activation of which is closely associated with voltage-gated Ca2+ influx. Conversely, pituitary gonadotrophs express SK (small)-type Ca2+-activated K+ channels that are colocalized with intracellular Ca2+ release sites. Activation of both channels is crucial for plateau-bursting-type rhythmic electrical activity and secretion.


Assuntos
Sinalização do Cálcio/fisiologia , Hipófise/fisiologia , Potenciais de Ação/fisiologia , Animais , Fenômenos Biofísicos , Biofísica , Canais de Cálcio/fisiologia , Humanos , Hipófise/citologia , Hipófise/metabolismo , Canais de Potássio/fisiologia , Canais de Sódio/fisiologia
9.
Endocrinology ; 144(7): 2912-21, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12810546

RESUMO

The coupling between nitric oxide (NO)-cGMP signaling pathway and prolactin (PRL) release in pituitary lactotrophs has been established previously. However, the messenger that mediates the action of this signaling pathway on hormone secretion and the secretory mechanism affected, calcium dependent or independent, have not been identified. In cultured pituitary cells, basal PRL release was controlled by spontaneous voltage-gated calcium influx and was further enhanced by depolarization of cells and stimulation with TRH. Inhibition of constitutively expressed neuronal NO synthase decreased NO and cGMP levels and increased basal PRL release. The addition of a slowly releasable NO donor increased cGMP levels and inhibited basal PRL release in a time-dependent manner. Expression of inducible NO synthase also increased NO and cGMP levels and inhibited basal, depolarization-induced, and TRH-induced PRL release, whereas inhibition of this enzyme decreased NO and cGMP production and recovered PRL release. None of these treatments affected spontaneous and stimulated voltage-gated calcium influx. At basal NO levels, the addition of permeable cGMP analogs did not inhibit PRL secretion. At elevated NO levels, inhibition of cGMP production and facilitation of its degradation did not reverse inhibited PRL secretion. These experiments indicate that NO inhibits calcium-dependent PRL secretion in a cGMP-independent manner and downstream of voltage-gated calcium influx.


Assuntos
Cálcio/metabolismo , Óxido Nítrico/metabolismo , Adeno-Hipófise/metabolismo , Prolactina/metabolismo , Animais , Canais de Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Células Cultivadas , GMP Cíclico/metabolismo , Feminino , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo I , Óxido Nítrico Sintase Tipo II , Técnicas de Patch-Clamp , Adeno-Hipófise/citologia , Ratos , Ratos Sprague-Dawley , Hormônio Liberador de Tireotropina/farmacologia
10.
J Cyst Fibros ; 13(1): 29-36, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23891399

RESUMO

BACKGROUND: Ivacaftor (KALYDECO™, VX-770) is a CFTR potentiator that increased CFTR channel activity and improved lung function in patients age 6 years and older with CF who have the G551D-CFTR gating mutation. The aim of this in vitro study was to evaluate the effect of ivacaftor on mutant CFTR protein forms with defects in protein processing and/or channel function. METHODS: The effect of ivacaftor on CFTR function was tested in electrophysiological studies using a panel of Fischer rat thyroid (FRT) cells expressing 54 missense CFTR mutations that cause defects in the amount or function of CFTR at the cell surface. RESULTS: Ivacaftor potentiated multiple mutant CFTR protein forms that produce functional CFTR at the cell surface. These included mutant CFTR forms with mild defects in CFTR processing or mild defects in CFTR channel conductance. CONCLUSIONS: These in vitro data indicated that ivacaftor is a broad acting CFTR potentiator and could be used to help stratify patients with CF who have different CFTR genotypes for studies investigating the potential clinical benefit of ivacaftor.


Assuntos
Aminofenóis/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/agonistas , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/tratamento farmacológico , Ativação do Canal Iônico/efeitos dos fármacos , Mutação de Sentido Incorreto , Quinolonas/farmacologia , Animais , Linhagem Celular , Cloretos/metabolismo , Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Técnicas In Vitro , Ativação do Canal Iônico/genética , Ratos , Ratos Endogâmicos F344 , Glândula Tireoide/citologia
11.
J Med Chem ; 57(23): 9776-95, 2014 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-25441013

RESUMO

Quinolinone-3-carboxamide 1, a novel CFTR potentiator, was discovered using high-throughput screening in NIH-3T3 cells expressing the F508del-CFTR mutation. Extensive medicinal chemistry and iterative structure-activity relationship (SAR) studies to evaluate potency, selectivity, and pharmacokinetic properties resulted in the identification of N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (VX-770, 48, ivacaftor), an investigational drug candidate approved by the FDA for the treatment of CF patients 6 years of age and older carrying the G551D mutation.


Assuntos
Aminofenóis/síntese química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/tratamento farmacológico , Quinolonas/síntese química , Aminofenóis/farmacocinética , Aminofenóis/farmacologia , Animais , Criança , Cães , Humanos , Macaca fascicularis , Masculino , Camundongos , Células NIH 3T3 , Quinolonas/farmacocinética , Quinolonas/farmacologia , Ratos Sprague-Dawley , Relação Estrutura-Atividade
12.
J Cyst Fibros ; 13(2): 139-47, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24660233

RESUMO

BACKGROUND: We examined data from a Phase 2 trial {NCT00457821} of ivacaftor, a CFTR potentiator, in cystic fibrosis (CF) patients with aG551D mutation to evaluate standardized approaches to sweat chloride measurement and to explore the use of sweat chloride and nasal potential difference (NPD) to estimate CFTR activity. METHODS: Sweat chloride and NPD were secondary endpoints in this placebo-controlled, multicenter trial. Standardization of sweat collection, processing,and analysis was employed for the first time. Sweat chloride and chloride ion transport (NPD) were integrated into a model of CFTR activity. RESULTS: Within-patient sweat chloride determinations showed sufficient precision to detect differences between dose-groups and assess ivacaftor treatment effects. Analysis of changes in sweat chloride and NPD demonstrated that patients treated with ivacaftor achieved CFTR activity equivalent to approximately 35%­40% of normal. CONCLUSIONS: Sweat chloride is useful in multicenter trials as a biomarker of CFTR activity and to test the effect of CFTR potentiators.


Assuntos
Aminofenóis/farmacologia , Cloretos , Regulador de Condutância Transmembrana em Fibrose Cística , Mucosa Nasal , Quinolonas/farmacologia , Suor , Adulto , Biomarcadores , Criança , Cloretos/análise , Cloretos/metabolismo , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Relação Dose-Resposta a Droga , Método Duplo-Cego , Monitoramento de Medicamentos/métodos , Feminino , Humanos , Masculino , Mutação , Mucosa Nasal/metabolismo , Mucosa Nasal/fisiopatologia , Reprodutibilidade dos Testes , Medicamentos para o Sistema Respiratório/farmacologia , Manejo de Espécimes/métodos , Manejo de Espécimes/normas , Suor/química , Suor/metabolismo , Resultado do Tratamento
13.
J Cyst Fibros ; 12(6): 706-13, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23628510

RESUMO

BACKGROUND: Ivacaftor, a cystic fibrosis transmembrane regulator (CFTR) potentiator, decreased sweat chloride concentrations and improved clinical measures in cystic fibrosis (CF) patients with the G551D mutation. RESULTS: Sweat chloride measurements at day 15 had an overall positive predictive value (PPV) of 86.3%, a negative predictive value (NPV) of 65.5%, sensitivity of 73.9%, and specificity of 80.9% for an FEV1 improvement of ≥5% from baseline at week 16. For ivacaftor patients the median FEV1 improvement was 16.7%; for placebo patients 0.4%. For patients aged 6-11 years who received ivacaftor and who had a sweat chloride decrease of ≥40 mmol/L from baseline at day 15, a median weight gain of 11.2% at week 16, compared to 6% for those with a smaller decrease was observed. CONCLUSIONS: Changes in sweat chloride concentration at day 15 following treatment with ivacaftor may have sufficient predictive potential to identify individuals that show improvement in pulmonary function and weight gain after 16 weeks of treatment.


Assuntos
Aminofenóis/uso terapêutico , Cloretos/análise , Fibrose Cística/genética , Quinolonas/uso terapêutico , Suor/química , Administração Oral , Adolescente , Criança , Fibrose Cística/tratamento farmacológico , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Feminino , Volume Expiratório Forçado , Humanos , Masculino , Mutação , Sensibilidade e Especificidade , Aumento de Peso
14.
Mol Biol Cell ; 24(19): 3016-24, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23924900

RESUMO

Cystic fibrosis (CF) is a fatal genetic disorder associated with defective hydration of lung airways due to the loss of chloride transport through the CF transmembrane conductance regulator protein (CFTR). CFTR contains two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a regulatory domain, and its channel assembly requires multiple interdomain contacts. The most common CF-causing mutation, F508del, occurs in NBD1 and results in misfolding and premature degradation of F508del-CFTR. VX-809 is an investigational CFTR corrector that partially restores CFTR function in people who are homozygous for F508del-CFTR. To identify the folding defect(s) in F508del-CFTR that must be repaired to treat CF, we explored the mechanism of VX-809 action. VX-809 stabilized an N-terminal domain in CFTR that contains only MSD1 and efficaciously restored function to CFTR forms that have missense mutations in MSD1. The action of VX-809 on MSD1 appears to suppress folding defects in F508del-CFTR by enhancing interactions among the NBD1, MSD1, and MSD2 domains. The ability of VX-809 to correct F508del-CFTR is enhanced when combined with mutations that improve F508del-NBD1 interaction with MSD2. These data suggest that the use of VX-809 in combination with an additional CFTR corrector that suppresses folding defects downstream of MSD1 may further enhance CFTR function in people with F508del-CFTR.


Assuntos
Aminopiridinas/administração & dosagem , Benzodioxóis/administração & dosagem , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/tratamento farmacológico , Dobramento de Proteína/efeitos dos fármacos , Fibrose Cística/genética , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/química , Humanos , Mutação de Sentido Incorreto , Conformação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína/genética , Transdução de Sinais/genética
15.
Nat Genet ; 45(10): 1160-7, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23974870

RESUMO

Allelic heterogeneity in disease-causing genes presents a substantial challenge to the translation of genomic variation into clinical practice. Few of the almost 2,000 variants in the cystic fibrosis transmembrane conductance regulator gene CFTR have empirical evidence that they cause cystic fibrosis. To address this gap, we collected both genotype and phenotype data for 39,696 individuals with cystic fibrosis in registries and clinics in North America and Europe. In these individuals, 159 CFTR variants had an allele frequency of l0.01%. These variants were evaluated for both clinical severity and functional consequence, with 127 (80%) meeting both clinical and functional criteria consistent with disease. Assessment of disease penetrance in 2,188 fathers of individuals with cystic fibrosis enabled assignment of 12 of the remaining 32 variants as neutral, whereas the other 20 variants remained of indeterminate effect. This study illustrates that sourcing data directly from well-phenotyped subjects can address the gap in our ability to interpret clinically relevant genomic variation.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Feminino , Frequência do Gene , Genótipo , Humanos , Masculino , Fenótipo
16.
J Cyst Fibros ; 11(3): 237-45, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22293084

RESUMO

BACKGROUND: The investigational CFTR potentiator ivacaftor (VX-770) increased CFTR channel activity and improved lung function in subjects with CF who have the G551D CFTR gating mutation. The aim of this in vitro study was to determine whether ivacaftor potentiates mutant CFTR with gating defects caused by other CFTR gating mutations. METHODS: The effects of ivacaftor on CFTR channel open probability and chloride transport were tested in electrophysiological studies using Fischer rat thyroid (FRT) cells expressing different CFTR gating mutations. RESULTS: Ivacaftor potentiated multiple mutant CFTR forms with defects in CFTR channel gating. These included the G551D, G178R, S549N, S549R, G551S, G970R, G1244E, S1251N, S1255P and G1349D CFTR gating mutations. CONCLUSION: These in vitro data suggest that ivacaftor has a similar effect on all CFTR forms with gating defects and support investigation of the potential clinical benefit of ivacaftor in CF patients who have CFTR gating mutations beyond G551D.


Assuntos
Aminofenóis/uso terapêutico , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , DNA/genética , Mutação/efeitos dos fármacos , Quinolonas/uso terapêutico , Animais , Fibrose Cística/tratamento farmacológico , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/efeitos dos fármacos , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Análise Mutacional de DNA , Modelos Animais de Doenças , Ativação do Canal Iônico/genética , Transporte de Íons/genética , Prognóstico , Ratos , Ratos Endogâmicos F344 , Glândula Tireoide/metabolismo , Glândula Tireoide/patologia
17.
Methods Mol Biol ; 741: 39-54, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21594777

RESUMO

The use of human bronchial epithelial (HBE) cell cultures derived from the bronchi of CF patients offers the opportunity to study the effects of CFTR correctors and potentiators on CFTR function and epithelial cell biology in the native pathological environment. Cultured HBE cells derived from CF patients exhibit many of the morphological and functional characteristics believed to be associated with CF airway disease in vivo, including abnormal ion and fluid transport leading to dehydration of the airway surface and the loss of cilia beating. In addition, they can be generated in sufficient quantities to support routine lab testing of compound potency and efficacy and retain reproducible levels of CFTR function over time. Here we describe the development and validation of the CF HBE pharmacology model and its use to characterize, optimize, and select clinical candidates. It is expected that the pre-clinical testing of CFTR potentiators and correctors using epithelial cell cultures derived from CF patients will help to increase their likelihood of clinical efficacy.


Assuntos
Brônquios/patologia , Técnicas de Cultura de Células/métodos , Separação Celular/métodos , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/patologia , Descoberta de Drogas/métodos , Células Epiteliais/patologia , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Humanos , Camundongos , Células NIH 3T3
18.
Ann N Y Acad Sci ; 1152: 174-86, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19161388

RESUMO

G(i/o) protein-coupled receptors, signaling through G protein-dependent and protein-independent pathways, have prominent effects on secretion by modulating calcium signaling and regulating the size of the releasable secretory pool, the rates of exocytosis and endocytosis, and de novo synthesis. Pituitary cells fire action potentials spontaneously, and the associated calcium influx is sufficient to maintain prolactin (PRL) release but not gonadotropin release at high and steady levels for many hours. Such secretion, termed intrinsic, spontaneous, or basal, reflects fusion of secretory vesicles triggered by the cell type-specific pattern of action potentials. In lactotrophs, activation of endothelin ET(A) and dopamine D(2) receptors causes inhibition of spontaneous electrical activity and basal adenylyl cyclase activity accompanied with inhibition of basal PRL release. Agonist-induced inhibition of cAMP production and firing of action potentials is abolished in cells with blocked pertussis toxin (PTX)-sensitive G(i/o) signaling pathway. However, agonist-induced inhibition of PRL release is only partially relieved in such treated cells, indicating that both receptors also inhibit exocytosis downstream of cAMP/calcium signaling. The PTX-insensitive step in agonist-induced inhibition of PRL release is not affected by inhibition of phosphoinositide 3-kinase and glycogen synthase kinase-3 but is partially rescued by downregulation of the G(z)alpha expression. Thus, ET(A) and D(2) receptors inhibit basal PRL release not only by blocking electrical activity but also by desensitizing calcium-secretion coupling.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Lactotrofos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Humanos , Prolactina/metabolismo , Ligação Proteica , Transdução de Sinais
19.
Am J Physiol Lung Cell Mol Physiol ; 290(6): L1117-30, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16443646

RESUMO

Cystic fibrosis (CF) is a fatal genetic disease caused by mutations in cftr, a gene encoding a PKA-regulated Cl(-) channel. The most common mutation results in a deletion of phenylalanine at position 508 (DeltaF508-CFTR) that impairs protein folding, trafficking, and channel gating in epithelial cells. In the airway, these defects alter salt and fluid transport, leading to chronic infection, inflammation, and loss of lung function. There are no drugs that specifically target mutant CFTR, and optimal treatment of CF may require repair of both the folding and gating defects. Here, we describe two classes of novel, potent small molecules identified from screening compound libraries that restore the function of DeltaF508-CFTR in both recombinant cells and cultures of human bronchial epithelia isolated from CF patients. The first class partially corrects the trafficking defect by facilitating exit from the endoplasmic reticulum and restores DeltaF508-CFTR-mediated Cl(-) transport to more than 10% of that observed in non-CF human bronchial epithelial cultures, a level expected to result in a clinical benefit in CF patients. The second class of compounds potentiates cAMP-mediated gating of DeltaF508-CFTR and achieves single-channel activity similar to wild-type CFTR. The CFTR-activating effects of the two mechanisms are additive and support the rationale of a drug discovery strategy based on rescue of the basic genetic defect responsible for CF.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/fisiopatologia , Células 3T3 , Animais , Biotinilação , Linhagem Celular , Células Cultivadas , Cloretos/metabolismo , Cresóis/metabolismo , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Humanos , Ativação do Canal Iônico , Camundongos , Pirazóis/metabolismo , Ratos , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Glândula Tireoide/fisiologia
20.
Mol Pharmacol ; 61(6): 1329-39, 2002 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12021394

RESUMO

In excitable cells, receptor-induced Ca(2+) release from intracellular stores is usually accompanied by sustained depolarization of cells and facilitated voltage-gated Ca(2+) influx (VGCI). In quiescent pituitary lactotrophs, however, endothelin-1 (ET-1) induced rapid Ca(2+) release without triggering Ca(2+) influx. Furthermore, in spontaneously firing and depolarized lactotrophs, the Ca(2+)-mobilizing action of ET-1 was followed by inhibition of spontaneous VGCI caused by prolonged cell hyperpolarization and abolition of action potential-driven Ca(2+) influx. Agonist-induced depolarization of cells and enhancement of VGCI upon Ca(2+) mobilization was established in both quiescent and firing lactotrophs treated overnight with pertussis toxin (PTX). Activation of adenylyl cyclase by forskolin and addition of cell-permeable 8-bromo-cAMP did not affect ET-1-induced sustained inhibition of VGCI, suggesting that the cAMP-protein kinase A signaling pathway does not mediate the inhibitory action of ET-1 on VGCI. Consistent with the role of PTX-sensitive K(+) channels in ET-1-induced hyperpolarization of control cells, but not PTX-treated cells, ET-1 decreased the cell input resistance and activated a 5 mM Cs(+)-sensitive K(+) current. In the presence of Cs(+), ET-1 stimulated VGCI in a manner comparable with that observed in PTX-treated cells, whereas E-4031, a specific blocker of ether-a-go-go-related gene-like K(+) channels, was ineffective. Similar effects of PTX and Cs(+) were also observed in GH(3) immortalized cells transiently expressing ET(A) receptors. These results indicate that signaling of ET(A) receptors through the G(i/o) pathway in lactotrophs and the subsequent activation of inward rectifier K(+) channels provide an effective and adenylyl cyclase-independent mechanism for a prolonged uncoupling of Ca(2+) mobilization and influx pathways.


Assuntos
Canais de Cálcio/fisiologia , Cálcio/metabolismo , Endotelina-1/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Hipófise/metabolismo , Receptores de Endotelina/metabolismo , Animais , Eletrofisiologia , Feminino , Técnicas In Vitro , Canais de Potássio/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor de Endotelina A , Transdução de Sinais
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