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1.
Bioanalysis ; 12(21): 1545-1555, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33064028

RESUMO

Background: This paper describes for the first-time analytical procedures established to resolve the challenges associated with simultaneous and direct quantification of ataluren and ataluren-O-1ß-acyl glucuronide (AAG) by LC-MS/MS in human plasma and urine matrices. Methodology/results: The plasma quantification method was validated for calibration range of 12.5-12500 ng/ml for ataluren and 6.25-2500 ng/ml for AAG. The urine quantification method was validated for calibration range of 0.01-10 and 1-1000 µg/ml for ataluren and AAG, respectively. Plasma and urine samples were stabilized upon collection and through storage to prevent hydrolysis and acyl migration of AAG. Conclusion: Methods described in this paper enabled successful completion of ataluren clinical pharmacology studies for simultaneous pharmacokinetic assessment of ataluren and AAG.


Assuntos
Cromatografia Líquida/métodos , Oxidiazóis/sangue , Oxidiazóis/urina , Espectrometria de Massas em Tandem/métodos , Humanos , Oxidiazóis/farmacologia
2.
Pharmacol Res Perspect ; 8(2): e00576, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32196986

RESUMO

Ataluren promotes ribosomal readthrough of premature termination codons in mRNA which result from nonsense mutations. In vitro studies were performed to characterize the metabolism and enzyme kinetics of ataluren and its interaction potential with CYP enzymes. Incubation of [14 C]-ataluren with human liver microsomes indicated that the major metabolic pathway for ataluren is via direct glucuronidation and that the drug is not metabolized via cytochrome P450 (CYP). Glucuronidation was also observed in the incubation in human intestinal and kidney microsomes, but not in human pulmonary microsomes. UGT1A9 was found to be the major uridine diphosphate glucuronosyltransferase (UGT) responsible for ataluren glucuronidation in the liver and kidney microsomes. Enzyme kinetic analysis of the formation of ataluren acyl glucuronide, performed in human liver, kidney, and intestinal microsomes and recombinant human UGT1A9, found that increasing bovine serum albumin (BSA) levels enhanced the glucuronidation Michaelis-Menten constant (Km ) and ataluren protein binding but had a minimal effect on maximum velocity (Vmax ) of glucuronidation. Due to the decreased unbound Michaelis-Menten constant (Km,u ), the ataluren unbound intrinsic clearance (CLint,u ) increased for all experimental systems and BSA concentrations. Human kidney microsomes were about 3.7-fold more active than human liver microsomes, in terms of CLint,u /mg protein, indicating that the kidney is also a key organ for the metabolism and disposition of ataluren in humans. Ataluren showed no or little potential to inhibit or induce most of the CYP enzymes.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Glucuronosiltransferase/metabolismo , Oxidiazóis/farmacologia , Proteínas Sanguíneas/metabolismo , Indução Enzimática , Glucuronídeos/metabolismo , Glucuronosiltransferase/genética , Humanos , Intestinos , Rim , Cinética , Fígado , Microssomos/metabolismo , Fenótipo , Ligação Proteica , Proteínas Recombinantes/metabolismo
3.
Drug Metab Dispos ; 48(4): 317-325, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31980502

RESUMO

Ataluren is a unique small molecule developed for the treatment of diseases caused by nonsense mutations, which result in premature termination of ribosomal translation and lack of full-length protein production. This study investigated the in vivo metabolism and disposition of ataluren in mice, rats, dogs, and humans. After single oral administration of [14C]ataluren, the overall recovery of radioactivity was ≥93.7%, with approximately 39%, 17%-21%, 12%, and 55% in the urine and 54%, 70%-72%, 80%, and 47% in the feces from intact mice, rats, dogs, and humans, respectively. In bile duct-cannulated (BDC) rats, approximately 10%, 7%, and 82% of the dose was recovered in the urine, feces, and bile, respectively, suggesting that biliary secretion was a major route for the elimination of ataluren in the rats. Ataluren was extensively metabolized after oral administration, and the metabolic profiles of ataluren were quantitatively similar across all species. Unchanged ataluren was the dominant radioactive component in plasma. Ataluren acyl glucuronide was the most prominent metabolite in plasma of all species and the dominant metabolite in BDC rat bile and human urine, whereas the oxadiazole cleavage products were the major or prominent metabolites in the feces of all species. Overall, the results indicate that phase I metabolism is negligible and that the pathway largely involves glucuronidation. No other circulatory conjugation metabolite was detected across investigated species. SIGNIFICANCE STATEMENT: Ataluren is a novel carboxylic acid-containing small molecule drug for treating nonsense mutation Duchenne muscular dystrophy. In vivo metabolism and disposition after a single dose of the drug were investigated in mice, rats, dogs, and humans. Phase I metabolism of ataluren was negligible, and the pathway largely involves glucuronidation. No other circulatory conjugation metabolite was detected across investigated species.


Assuntos
Distrofia Muscular de Duchenne/tratamento farmacológico , Oxidiazóis/farmacocinética , Administração Oral , Adolescente , Adulto , Animais , Códon sem Sentido , Cães , Feminino , Voluntários Saudáveis , Humanos , Masculino , Taxa de Depuração Metabólica , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Distrofia Muscular de Duchenne/genética , Oxidiazóis/administração & dosagem , Terminação Traducional da Cadeia Peptídica/efeitos dos fármacos , Ratos , Distribuição Tecidual , Adulto Jovem
4.
Mol Cancer Ther ; 18(1): 3-16, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30352802

RESUMO

PTC299 was identified as an inhibitor of VEGFA mRNA translation in a phenotypic screen and evaluated in the clinic for treatment of solid tumors. To guide precision cancer treatment, we performed extensive biological characterization of the activity of PTC299 and demonstrated that inhibition of VEGF production and cell proliferation by PTC299 is linked to a decrease in uridine nucleotides by targeting dihydroorotate dehydrogenase (DHODH), a rate-limiting enzyme for de novo pyrimidine nucleotide synthesis. Unlike previously reported DHODH inhibitors that were identified using in vitro enzyme assays, PTC299 is a more potent inhibitor of DHODH in isolated mitochondria suggesting that mitochondrial membrane lipid engagement in the DHODH conformation in situ is required for its optimal activity. PTC299 has broad and potent activity against hematologic cancer cells in preclinical models, reflecting a reduced pyrimidine nucleotide salvage pathway in leukemia cells. Archived serum samples from patients treated with PTC299 demonstrated increased levels of dihydroorotate, the substrate of DHODH, indicating target engagement in patients. PTC299 has advantages over previously reported DHODH inhibitors, including greater potency, good oral bioavailability, and lack of off-target kinase inhibition and myelosuppression, and thus may be useful for the targeted treatment of hematologic malignancies.


Assuntos
Neoplasias Hematológicas/tratamento farmacológico , Imidazóis/administração & dosagem , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Tiazóis/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Di-Hidro-Orotato Desidrogenase , Neoplasias Hematológicas/sangue , Neoplasias Hematológicas/enzimologia , Humanos , Imidazóis/farmacologia , Células K562 , Camundongos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/sangue , Tiazóis/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Nature ; 447(7140): 87-91, 2007 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-17450125

RESUMO

Nonsense mutations promote premature translational termination and cause anywhere from 5-70% of the individual cases of most inherited diseases. Studies on nonsense-mediated cystic fibrosis have indicated that boosting specific protein synthesis from <1% to as little as 5% of normal levels may greatly reduce the severity or eliminate the principal manifestations of disease. To address the need for a drug capable of suppressing premature termination, we identified PTC124-a new chemical entity that selectively induces ribosomal readthrough of premature but not normal termination codons. PTC124 activity, optimized using nonsense-containing reporters, promoted dystrophin production in primary muscle cells from humans and mdx mice expressing dystrophin nonsense alleles, and rescued striated muscle function in mdx mice within 2-8 weeks of drug exposure. PTC124 was well tolerated in animals at plasma exposures substantially in excess of those required for nonsense suppression. The selectivity of PTC124 for premature termination codons, its well characterized activity profile, oral bioavailability and pharmacological properties indicate that this drug may have broad clinical potential for the treatment of a large group of genetic disorders with limited or no therapeutic options.


Assuntos
Códon sem Sentido/genética , Doenças Genéticas Inatas/tratamento farmacológico , Doenças Genéticas Inatas/genética , Oxidiazóis/farmacologia , Oxidiazóis/uso terapêutico , Biossíntese de Proteínas/efeitos dos fármacos , Alelos , Animais , Disponibilidade Biológica , Distrofina/biossíntese , Distrofina/genética , Doenças Genéticas Inatas/sangue , Humanos , Camundongos , Camundongos Endogâmicos mdx , Oxidiazóis/administração & dosagem , Oxidiazóis/farmacocinética , Fenótipo , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Especificidade por Substrato
6.
J Clin Pharmacol ; 47(4): 430-44, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17389552

RESUMO

Nonsense (premature stop codon) mutations are causative in 5% to 15% of patients with monogenetic inherited disorders. PTC124, a 284-Dalton 1,2,4-oxadiazole, promotes ribosomal readthrough of premature stop codons in mRNA and offers therapeutic potential for multiple genetic diseases. The authors conducted 2 phase I studies of PTC124 in 62 healthy adult volunteers. The initial, single-dose study evaluated doses of 3 to 200 mg/kg and assessed fed-fasting status on pharmacokinetics following a dose of 50 mg/kg. The subsequent multiple-dose study evaluated doses from 10 to 50 mg/kg/dose twice per day (bid) for up to 14 days. PTC124 administered orally as a liquid suspension was palatable and well tolerated through single doses of 100 mg/kg. At 150 and 200 mg/kg, PTC124 induced mild headache, dizziness, and gastrointestinal events. With repeated doses through 50 mg/kg/dose bid, reversible transaminase elevations <2 times the upper limit of normal were sometimes observed. Immunoblot analyses of peripheral blood mononuclear cell extracts revealed no protein elongation due to nonspecific ribosomal readthrough of normal stop codons. PTC124 plasma concentrations exceeding the 2- to 10-microg/mL values associated with activity in preclinical genetic disease models were safely achieved. No sex-related differences in pharmacokinetics were seen. No drug accumulation with repeated dosing was apparent. Diurnal variation was observed, with greater PTC124 exposures after evening doses. PTC124 excretion in the urine was <2%. PTC124 pharmacokinetics were described by a 1-compartment model. Collectively, the data support initiation of phase II studies of PTC124 in patients with nonsense mutation-mediated cystic fibrosis and Duchenne muscular dystrophy.


Assuntos
Códon sem Sentido/antagonistas & inibidores , Oxidiazóis/farmacocinética , Adolescente , Adulto , Área Sob a Curva , Ritmo Circadiano , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Interações Alimento-Droga , Meia-Vida , Humanos , Immunoblotting , Masculino , Oxidiazóis/administração & dosagem , Oxidiazóis/efeitos adversos
7.
J Biol Chem ; 278(40): 39092-103, 2003 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-12857725

RESUMO

Antiretroviral therapy to treat AIDS uses molecules that target the reverse transcriptase and protease enzymes of human immunodeficiency virus, type 1 (HIV-1). A major problem associated with these treatments, however, is the emergence of drug-resistant strains. Thus, there is a compelling need to find drugs against other viral targets. One such target is the interaction between Tat, an HIV-1 regulatory protein essential for viral replication, and trans-activation-responsive (TAR) RNA. Here we describe the design and synthesis of an encoded combinatorial library containing 39,304 unnatural small molecules. Using a rapid high through-put screening technology, we identified 59 compounds. Structure-activity relationship studies led to the synthesis of 19 compounds that bind TAR RNA with high affinities. In the presence of a representative Tat-TAR inhibitor (5 microM TR87), we observed potent and sustained suppression of HIV replication in cultured cells over 24 days. The same concentration of this inhibitor did not exhibit any toxicity in cell cultures or in mice. TR87 was also shown to specifically disrupt Tat-TAR binding in vitro and inhibit Tat-mediated transcriptional activation in vitro and in vivo, providing a strong correlation between its activities and inhibition of HIV-1 replication. These results provide a structural scaffold for further development of new drugs, alone or in combination with other drugs, for treatment of HIV-1-infected individuals. Our results also suggest a general strategy for discovering pharmacophores targeting RNA structures that are essential in progression of other infectious, inflammatory, and genetic diseases.


Assuntos
Produtos do Gene tat/química , HIV-1/fisiologia , Oligopeptídeos/farmacologia , Ativação Transcricional , Animais , Antivirais/farmacologia , Automação , Carbamatos/química , Células Cultivadas , Cromatografia Gasosa , Relação Dose-Resposta a Droga , Transferência Ressonante de Energia de Fluorescência , HIV-1/química , HIV-1/metabolismo , Células HeLa , Humanos , Ligantes , Camundongos , Modelos Biológicos , Modelos Químicos , Modelos Estatísticos , Conformação de Ácido Nucleico , Oligopeptídeos/química , Biblioteca de Peptídeos , Ligação Proteica , RNA/química , RNA/metabolismo , DNA Polimerase Dirigida por RNA/metabolismo , Relação Estrutura-Atividade , Produtos do Gene tat do Vírus da Imunodeficiência Humana
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