RESUMO
Duchenne muscular dystrophy (DMD) is a progressive disabling X-linked recessive disorder that causes gradual and irreversible loss of muscle, resulting in early death. The corticosteroids prednisone/prednisolone and deflazacort are used to treat DMD as the standard of care; however, only deflazacort is FDA approved for DMD. The novel atypical corticosteroid vamorolone is being investigated for treatment of DMD. We compared the pharmaceutical properties as well as the efficacy and safety of the three corticosteroids across multiple doses in the B10-mdx DMD mouse model. Pharmacokinetic studies in the mouse and evaluation of p-glycoprotein (P-gP) efflux in a cellular system demonstrated that vamorolone is not a strong P-gp substrate resulting in measurable central nervous system (CNS) exposure in the mouse. In contrast, deflazacort and prednisolone are strong P-gp substrates. All three corticosteroids showed efficacy, but also side effects at efficacious doses. After dosing mdx mice for two weeks, all three corticosteroids induced changes in gene expression in the liver and the muscle, but prednisolone and vamorolone induced more changes in the brain than did deflazacort. Both prednisolone and vamorolone induced depression-like behavior. All three corticosteroids reduced endogenous corticosterone levels, increased glucose levels, and reduced osteocalcin levels. Using micro-computed tomography, femur bone density was decreased, reaching significance with prednisolone. The results of these studies indicate that efficacious doses of vamorolone, are associated with similar side effects as seen with other corticosteroids. Further, because vamorolone is not a strong P-gp substrate, vamorolone distributes into the CNS increasing the potential CNS side-effects.
Assuntos
Distrofia Muscular de Duchenne , Prednisolona , Pregnadienodiois , Pregnenodionas , Animais , Camundongos , Prednisolona/uso terapêutico , Microtomografia por Raio-X , Camundongos Endogâmicos mdx , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/genética , Corticosterona/uso terapêutico , Preparações FarmacêuticasRESUMO
Cyclin dependent kinase-like 5 (CDKL5) Deficiency Disorder (CDD) is a rare neurodevelopmental disorder caused by a mutation in the X-linked CDKL5 gene. CDKL5 is a serine/threonine kinase that is critical for axon outgrowth, dendritic morphogenesis, as well as synapse formation, maturation, and maintenance. This disorder is characterized by early-onset epilepsy, hypotonia, and failure to reach cognitive and motor developmental milestones. Because the disease is monogenic, delivery of the CDKL5 gene to the brain of patients should provide clinical benefit. To this end, we designed a gene therapy vector, adeno-associated virus (AAV)9.Syn.hCDKL5, in which human CDKL5 gene expression is driven by the synapsin promoter. In biodistribution studies conducted in mice, intracerebroventricular (ICV) injection resulted in broader, more optimal biodistribution than did intracisterna magna (ICM) delivery. AAV9.Syn.hCDKL5 treatment increased phosphorylation of EB2, a bona fide CDKL5 substrate, demonstrating biological activity in vivo. Our data provides proof-of-concept that ICV delivery of AAV9.Syn.hCDKL5 to neonatal male Cdkl5 knockout mice reduces pathology and reduces aberrant behavior. Functional improvements were seen at doses of 3e11 to 5e11 vector genomes (vg)/g brain, which resulted in transfection of ≥50% of the neurons. Functional improvements were not seen at lower doses suggesting a requirement for broad distribution for efficacy.
RESUMO
Spinal muscular atrophy (SMA) is caused by the loss of the survival motor neuron 1 (SMN1) gene function. The related SMN2 gene partially compensates but produces insufficient levels of SMN protein due to alternative splicing of exon 7. Evrysdi™ (risdiplam), recently approved for the treatment of SMA, and related compounds promote exon 7 inclusion to generate full-length SMN2 mRNA and increase SMN protein levels. SMNΔ7 type I SMA mice survive without treatment for ~17 days. SMN2 mRNA splicing modulators increase survival of SMN∆7 mice with treatment initiated at postnatal day 3 (PND3). To define SMN requirements for adult mice, SMNΔ7 mice were dosed with an SMN2 mRNA splicing modifier from PND3 to PND40, then dosing was stopped. Mice not treated after PND40 showed progressive weight loss, necrosis, and muscle atrophy after ~20 days. Male mice presented a more severe phenotype than female mice. Mice dosed continuously did not show disease symptoms. The estimated half-life of SMN protein is 2 days indicating that the SMA phenotype reappeared after SMN protein levels returned to baseline. Although SMN protein levels decreased with age in mice and SMN protein levels were higher in brain than in muscle, our studies suggest that SMN protein is required throughout the life of the mouse and is especially essential in adult peripheral tissues including muscle. These studies indicate that drugs such as risdiplam will be optimally therapeutic when given as early as possible after diagnosis and potentially will be required for the life of an SMA patient.
Assuntos
Atrofia Muscular Espinal , Processamento Alternativo , Animais , Modelos Animais de Doenças , Progressão da Doença , Éxons , Feminino , Humanos , Masculino , Camundongos , Atrofia Muscular Espinal/metabolismo , Splicing de RNA , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Proteína 2 de Sobrevivência do Neurônio MotorRESUMO
Paired box protein Pax-6 (oculothrombin) is a transcription factor that plays an important regulatory role in ocular, brain, and pancreatic development. Mutations of the PAX6 gene cause aniridia and Peters anomaly. Reduction in Pax-6 protein is also associated with ocular diseases such as dry eye. An electrochemiluminescence immunoassay method using the Meso Scale Discovery platform was developed to measure Pax-6 protein levels in corneal epithelial cells obtained by impression cytology. Impression cytology involves harvesting ocular epithelial cells by applying a polyethersulfone membrane patch briefly to the ocular surface using a commercially available EYEPRIM™ device. The epithelial cells that adhere to the membrane patch of the EYEPRIM™ device provide a biological sample which can be assayed for Pax-6 protein levels. Assay development identified an antibody pair capable of detecting purified recombinant Pax-6 protein produced in mammalian cells. The optimized assay has a dynamic range of 24 pg mL-1 to 100,000 pg mL-1 and a lower limit of quantification of 24 pg mL-1. Assay selectivity was demonstrated using either HeLa or HEK293 cells transfected with inhibitory RNA. Finally, the method was validated by measuring Pax-6 protein levels in impression cytology acquired samples obtained using the EYEPRIM™ device from rabbit cornea.
Assuntos
Proteínas de Homeodomínio , Fatores de Transcrição Box Pareados , Animais , Proteínas do Olho/genética , Células HEK293 , Proteínas de Homeodomínio/genética , Humanos , Imunoensaio , Mamíferos/genética , Mamíferos/metabolismo , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/genética , RNA , Coelhos , Proteínas Repressoras/genéticaRESUMO
Using small molecules to induce readthrough of premature termination codons is a promising therapeutic approach to treating genetic diseases and cancers caused by nonsense mutations, as evidenced by the widespread use of ataluren to treat nonsense mutation Duchene muscular dystrophy. Herein we describe a series of novel guanidino quinazoline and pyrimidine scaffolds that induce readthrough in both HDQ-P1 mammary carcinoma cells and mdx myotubes. Linkage of basic, tertiary amines with aliphatic, hydrophobic substituents to the terminal guanidine nitrogen of these scaffolds led to significant potency increases. Further potency gains were achieved by flanking the pyrimidine ring with hydrophobic substituents, inducing readthrough at concentrations as low as 120 nM and demonstrating the potential of these compounds to be used either in combination with ataluren or as stand-alone therapeutics.
Assuntos
Códon sem Sentido , Quinazolinas , Quinazolinas/farmacologia , Pirimidinas/farmacologia , Guanidinas , Nitrogênio , AminasRESUMO
Emvododstat was identified as a potent inhibitor of dihydroorotate dehydrogenase and is now in clinical development for the treatment of acute myeloid leukaemia and COVID-19. The objective of this paper is to evaluate the metabolism, pharmacokinetics, and drug interaction potentials of emvododstat.Emvododstat showed high binding to plasma protein with minimal distribution into blood cells in mouse, rat, dog, monkey, and human whole blood.O-Demethylation followed by glucuronidation appeared to be the major metabolic pathway in rat, dog, monkey, and human hepatocytes. CYP2C8, 2C19, 2D6, and 3A4 were involved in O-desmethyl emvododstat metabolite formation. Both emvododstat and O-desmethyl emvododstat inhibited CYP2D6 activity and induced CYP expression to different extents in vitro.Emvododstat and O-desmethyl emvododstat inhibited BCRP transporter activity but did not inhibit bile salt transporters and other efflux or uptake transporters. Neither emvododstat nor O-desmethyl emvododstat was a substrate for common efflux or uptake transporters investigated.Emvododstat is bioavailable in mice, rats, dogs, and monkeys following a single oral dose. The absorption was generally slow with the mean plasma Tmax ranging from 2 to 5 h; plasma exposure of O-desmethyl emvododstat was lower in rodents, but relatively higher in dogs and monkeys.
Assuntos
COVID-19 , Microssomos Hepáticos , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Carbamatos , Carbazóis , Di-Hidro-Orotato Desidrogenase , Cães , Interações Medicamentosas , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Microssomos Hepáticos/metabolismo , Proteínas de Neoplasias/metabolismo , RatosRESUMO
Duchenne muscular dystrophy (DMD) is caused by mutations in the gene encoding dystrophin. Prior work has shown that DMD progression can vary, depending on the genetic makeup of the patient. Several modifier alleles have been identified including LTBP4 and SPP1. We previously showed that Spp1 exacerbates the DMD phenotype in the mdx mouse model by promoting fibrosis and by skewing macrophage polarization. Here, we studied the mechanisms involved in Spp1's promotion of fibrosis by using both isolated fibroblasts and genetically modified mice. We found that Spp1 upregulates collagen expression in mdx fibroblasts by enhancing TGFß signaling. Spp1's effects on TGFß signaling are through induction of MMP9 expression. MMP9 is a protease that can release active TGFß ligand from its latent complex. In support for activation of this pathway in our model, we showed that treatment of mdx fibroblasts with MMP9 inhibitor led to accumulation of the TGFß latent complex, decreased levels of active TGFß and reduced collagen expression. Correspondingly, we found reduced active TGFß in Spp1-/-mdxB10 and Mmp9-/-mdxB10 muscles in vivo. Taken together with previous observations of reduced fibrosis in both models, these data suggest that Spp1 acts upstream of TGFß to promote fibrosis in mdx muscles. We found that in the context of constitutively upregulated TGFß signaling (such as in the mdxD2 model), ablation of Spp1 has very little effect on fibrosis. Finally, we performed proof-of-concept studies showing that postnatal pharmacological inhibition of Spp1 reduces fibrosis and improves muscle function in mdx mice.
Assuntos
Fibrose/genética , Distrofia Muscular de Duchenne/metabolismo , Osteopontina/genética , Fator de Crescimento Transformador beta/metabolismo , Animais , Colágeno Tipo I/biossíntese , Modelos Animais de Doenças , Distrofina/genética , Distrofina/metabolismo , Matriz Extracelular/metabolismo , Feminino , Fibroblastos/metabolismo , Fibrose/metabolismo , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos mdx , Camundongos Knockout , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Osteopontina/metabolismo , Cultura Primária de Células , Regeneração/genética , Transdução de Sinais , Fator de Crescimento Transformador beta/genéticaRESUMO
Nonsense mutations resulting in a premature stop codon in an open reading frame occur in critical tumor suppressor genes in a large number of the most common forms of cancers and are known to cause or contribute to the progression of disease. Low molecular weight compounds that induce readthrough of nonsense mutations offer a new means of treating patients with genetic disorders or cancers resulting from nonsense mutations. We have identified the nucleoside analog clitocine as a potent and efficacious suppressor of nonsense mutations. We determined that incorporation of clitocine into RNA during transcription is a prerequisite for its readthrough activity; the presence of clitocine in the third position of a premature stop codon directly induces readthrough. We demonstrate that clitocine can induce the production of p53 protein in cells harboring p53 nonsense-mutated alleles. In these cells, clitocine restored production of full-length and functional p53 as evidenced by induced transcriptional activation of downstream p53 target genes, progression of cells into apoptosis, and impeded growth of nonsense-containing human ovarian cancer tumors in xenograft tumor models. Thus, clitocine induces readthrough of nonsense mutations by a previously undescribed mechanism and represents a novel therapeutic modality to treat cancers and genetic diseases caused by nonsense mutations.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Materiais Biomiméticos/farmacologia , Códon sem Sentido/efeitos dos fármacos , Furanos/farmacologia , Nucleosídeos/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Nucleosídeos de Pirimidina/farmacologia , Proteína Supressora de Tumor p53/agonistas , Animais , Antimetabólitos Antineoplásicos/síntese química , Antimetabólitos Antineoplásicos/metabolismo , Apoptose/efeitos dos fármacos , Materiais Biomiméticos/síntese química , Materiais Biomiméticos/metabolismo , Linhagem Celular Tumoral , Feminino , Furanos/síntese química , Furanos/metabolismo , Genes Reporter , Humanos , Luciferases/genética , Luciferases/metabolismo , Camundongos , Camundongos Nus , Nucleosídeos/síntese química , Nucleosídeos/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Biossíntese de Proteínas , Nucleosídeos de Pirimidina/síntese química , Nucleosídeos de Pirimidina/metabolismo , Transdução de Sinais , Ativação Transcricional , Carga Tumoral/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A premature termination codon (PTC) in the ORF of an mRNA generally leads to production of a truncated polypeptide, accelerated degradation of the mRNA, and depression of overall mRNA expression. Accordingly, nonsense mutations cause some of the most severe forms of inherited disorders. The small-molecule drug ataluren promotes therapeutic nonsense suppression and has been thought to mediate the insertion of near-cognate tRNAs at PTCs. However, direct evidence for this activity has been lacking. Here, we expressed multiple nonsense mutation reporters in human cells and yeast and identified the amino acids inserted when a PTC occupies the ribosomal A site in control, ataluren-treated, and aminoglycoside-treated cells. We find that ataluren's likely target is the ribosome and that it produces full-length protein by promoting insertion of near-cognate tRNAs at the site of the nonsense codon without apparent effects on transcription, mRNA processing, mRNA stability, or protein stability. The resulting readthrough proteins retain function and contain amino acid replacements similar to those derived from endogenous readthrough, namely Gln, Lys, or Tyr at UAA or UAG PTCs and Trp, Arg, or Cys at UGA PTCs. These insertion biases arise primarily from mRNA:tRNA mispairing at codon positions 1 and 3 and reflect, in part, the preferred use of certain nonstandard base pairs, e.g., U-G. Ataluren's retention of similar specificity of near-cognate tRNA insertion as occurs endogenously has important implications for its general use in therapeutic nonsense suppression.
Assuntos
Códon sem Sentido/genética , Oxidiazóis/farmacologia , RNA de Transferência/genética , Ribossomos/efeitos dos fármacos , Células HEK293 , Humanos , Biossíntese de Proteínas/efeitos dos fármacos , Estabilidade de RNA/efeitos dos fármacos , RNA de Transferência/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Choroideremia (CHM) is an X-linked chorioretinal dystrophy that is caused by mutations within a single gene, CHM Currently no effective treatment exists for these patients. Since over 30% of patients harbour nonsense mutations in CHM, nonsense suppression therapy using translational readthrough inducing drugs may provide functional rescue of REP1, thus attenuating progressive sight loss. Here, we employed two CHM model systems to systematically test the efficacy and safety of ataluren (PTC124) and its novel analog PTC-414: (1) the chmru848 zebrafish, the only nonsense mutation animal model of CHM harbouring a TAA nonsense mutation, and (2) a primary human fibroblast cell line from a CHM patient harbouring a TAG nonsense mutation. PTC124 or PTC-414 treatment of chmru848 embryos led to a â¼2.0-fold increase in survival, prevented the onset of retinal degeneration with reduced oxidative stress and apoptosis, increased rep1 protein by 23.1% (PTC124) and 17.2% (PTC-414) and restored biochemical function as confirmed through in vitro prenylation assays (98 ± 2% [PTC124] and 68 ± 5% [PTC-414]). In CHMY42X/y fibroblasts, there was a recovery of prenylation activity following treatment with either PTC124 (42 ± 5%) or PTC-414 (36 ± 11%), although an increase in REP1 protein was not detected in these cells, in contrast to the zebrafish model. This comprehensive study on the use of PTC124 and PTC-414 as successful nonsense suppression agents for the treatment of CHM highlights the translational potential of these drugs for inherited retinal disease.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Coroideremia/tratamento farmacológico , Degeneração Retiniana/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Coroideremia/genética , Coroideremia/patologia , Códon sem Sentido , Modelos Animais de Doenças , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Humanos , Oxidiazóis/administração & dosagem , Estresse Oxidativo/efeitos dos fármacos , Retina/efeitos dos fármacos , Retina/patologia , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , Peixe-Zebra , Proteínas de Peixe-ZebraRESUMO
Spinal muscular atrophy (SMA) is a genetic disease characterized by atrophy of muscle and loss of spinal motor neurons. SMA is caused by deletion or mutation of the survival motor neuron 1 (SMN1) gene, and the nearly identical SMN2 gene fails to generate adequate levels of functional SMN protein due to a splicing defect. Currently, several therapeutics targeted to increase SMN protein are in clinical trials. An outstanding issue in the field is whether initiating treatment in symptomatic older patients would confer a therapeutic benefit, an important consideration as the majority of patients with milder forms of SMA are diagnosed at an older age. An SMA mouse model that recapitulates the disease phenotype observed in adolescent and adult SMA patients is needed to address this important question. We demonstrate here that Δ7 mice, a model of severe SMA, treated with a suboptimal dose of an SMN2 splicing modifier show increased SMN protein, survive into adulthood and display SMA disease-relevant pathologies. Increasing the dose of the splicing modifier after the disease symptoms are apparent further mitigates SMA histopathological features in suboptimally dosed adult Δ7 mice. In addition, inhibiting myostatin using intramuscular injection of AAV1-follistatin ameliorates muscle atrophy in suboptimally dosed Δ7 mice. Taken together, we have developed a new murine model of symptomatic SMA in adolescents and adult mice that is induced pharmacologically from a more severe model and demonstrated efficacy of both SMN2 splicing modifiers and a myostatin inhibitor in mice at later disease stages.
Assuntos
Folistatina/farmacologia , Fatores Imunológicos/farmacologia , Atrofia Muscular Espinal/tratamento farmacológico , Splicing de RNA/efeitos dos fármacos , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/agonistas , Adolescente , Adulto , Idade de Início , Animais , Dependovirus/genética , Dependovirus/metabolismo , Modelos Animais de Doenças , Deleção de Genes , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Camundongos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/patologia , Miostatina/antagonistas & inibidores , Miostatina/genética , Miostatina/metabolismo , Fenótipo , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismoRESUMO
Spinal muscular atrophy (SMA) is caused by the loss or mutation of both copies of the survival motor neuron 1 (SMN1) gene. The related SMN2 gene is retained, but due to alternative splicing of exon 7, produces insufficient levels of the SMN protein. Here, we systematically characterize the pharmacokinetic and pharmacodynamics properties of the SMN splicing modifier SMN-C1. SMN-C1 is a low-molecular weight compound that promotes the inclusion of exon 7 and increases production of SMN protein in human cells and in two transgenic mouse models of SMA. Furthermore, increases in SMN protein levels in peripheral blood mononuclear cells and skin correlate with those in the central nervous system (CNS), indicating that a change of these levels in blood or skin can be used as a non-invasive surrogate to monitor increases of SMN protein levels in the CNS. Consistent with restored SMN function, SMN-C1 treatment increases the levels of spliceosomal and U7 small-nuclear RNAs and corrects RNA processing defects induced by SMN deficiency in the spinal cord of SMNΔ7 SMA mice. A 100% or greater increase in SMN protein in the CNS of SMNΔ7 SMA mice robustly improves the phenotype. Importantly, a â¼50% increase in SMN leads to long-term survival, but the SMA phenotype is only partially corrected, indicating that certain SMA disease manifestations may respond to treatment at lower doses. Overall, we provide important insights for the translation of pre-clinical data to the clinic and further therapeutic development of this series of molecules for SMA treatment.
Assuntos
Isocumarinas/administração & dosagem , Atrofia Muscular Espinal/tratamento farmacológico , Atrofia Muscular Espinal/genética , Piperazinas/administração & dosagem , Bibliotecas de Moléculas Pequenas/farmacocinética , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Processamento Alternativo/efeitos dos fármacos , Processamento Alternativo/genética , Animais , Sistema Nervoso Central/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Éxons/genética , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Atrofia Muscular Espinal/sangue , Atrofia Muscular Espinal/patologia , Splicing de RNA/efeitos dos fármacos , Splicing de RNA/genética , Pele/metabolismo , Bibliotecas de Moléculas Pequenas/administração & dosagem , Proteína 2 de Sobrevivência do Neurônio Motor/sangueRESUMO
The interplay of translation and mRNA turnover has helped unveil how the regulation of gene expression is a continuum in which events that occur during the birth of a transcript in the nucleus can have profound effects on subsequent steps in the cytoplasm. Exemplifying this continuum is nonsense-mediated mRNA decay (NMD), the process wherein a premature stop codon affects both translation and mRNA decay. Studies of NMD helped lead us to the therapeutic concept of treating a subset of patients suffering from multiple genetic disorders due to nonsense mutations with a single small-molecule drug that modulates the translation termination process at a premature nonsense codon. Here we review both translation termination and NMD, and our subsequent efforts over the past 15 years that led to the identification, characterization, and clinical testing of ataluren, a new therapeutic with the potential to treat a broad range of genetic disorders due to nonsense mutations.
Assuntos
Códon sem Sentido , Doença/genética , Degradação do RNAm Mediada por Códon sem Sentido , Oxidiazóis/farmacologia , Estabilidade de RNA/genética , Animais , Humanos , Transcrição Gênica/genéticaRESUMO
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 SubstratoRESUMO
Nonsense mutations inactivate gene function and are the underlying cause of a large percentage of the individual cases of many genetic disorders. PTC124 is an orally bioavailable compound that promotes readthrough of premature translation termination codons, suggesting that it may have the potential to treat genetic diseases caused by nonsense mutations. Using a mouse model for cystic fibrosis (CF), we show that s.c. injection or oral administration of PTC124 to Cftr-/- mice expressing a human CFTR-G542X transgene suppressed the G542X nonsense mutation and restored a significant amount of human (h)CFTR protein and function. Translational readthrough of the premature stop codon was demonstrated in this mouse model in two ways. First, immunofluorescence staining showed that PTC124 treatment resulted in the appearance of hCFTR protein at the apical surface of intestinal glands in Cftr-/- hCFTR-G542X mice. In addition, functional assays demonstrated that PTC124 treatment restored 24-29% of the average cAMP-stimulated transepithelial chloride currents observed in wild-type mice. These results indicate that PTC124 can effectively suppress the hCFTR-G542X nonsense mutation in vivo. In light of its oral bioavailability, safety toxicology profile in animal studies, and efficacy with other nonsense alleles, PTC124 has the potential to be an important therapeutic agent for the treatment of inherited diseases caused by nonsense mutations.
Assuntos
Alelos , Códon sem Sentido , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Modelos Animais de Doenças , Expressão Gênica/efeitos dos fármacos , Oxidiazóis/farmacologia , Administração Oral , Animais , Sequência de Bases , Disponibilidade Biológica , Canais de Cloreto/efeitos dos fármacos , Canais de Cloreto/metabolismo , AMP Cíclico/farmacologia , Primers do DNA , Imunofluorescência , Humanos , Injeções Subcutâneas , Camundongos , Camundongos Transgênicos , Oxidiazóis/administração & dosagem , Oxidiazóis/farmacocinética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Ataluren is an aromatic acid derivative with a 1,2,4-oxodiazole moiety. Ataluren-O-1ß-acyl glucuronide is a prominent circulatory metabolite in mice, rats, dogs, and humans following oral administration of ataluren. The objective of this paper was to evaluate the stability in vitro and in vivo of ataluren-O-1ß-acyl glucuronide metabolite. Ultrahigh performance liquid chromatography-mass spectrometry methods were developed to separate and monitor ataluren-O-1ß-acyl glucuronide and its possible migration isomers. In vitro stability was assessed in phosphate buffered saline as well as in control rat and human plasma. The disappearance of ataluren-O-1ß-acyl glucuronide and the formation of migration isomers were monitored by the ultrahigh performance liquid chromatography-mass spectrometry methods. In vitro, ataluren-O-1ß-acyl glucuronide underwent isomerization with an estimated half-life of approximately 1 h. However, ataluren-O-1ß-acyl glucuronide was stable and was the only detectable acyl glucuronide following oral administration of ataluren in mice, rats, dogs, and humans using the same analytical methods. Ataluren acyl glucuronide in mouse, rat, dog, and human plasma could be hydrolyzed by ß-glucuronidase, further confirming the structure of O-1ß-acyl glucuronide. These results demonstrated that ataluren-O-1ß-acyl glucuronide did not undergo migration in vivo. No clinical safety concern related to ataluren-O-1ß-acyl glucuronide migration has been detected.
Assuntos
Glucuronídeos/metabolismo , Oxidiazóis/metabolismo , Animais , Cães , Humanos , Isomerismo , Masculino , Espectrometria de Massas , Camundongos , Camundongos Transgênicos , Ratos , Ratos Sprague-DawleyRESUMO
The coronavirus disease 2019 (COVID-19) pandemic has created an urgent need for therapeutics that inhibit the SARS-COV-2 virus and suppress the fulminant inflammation characteristic of advanced illness. Here, we describe the anti-COVID-19 potential of PTC299, an orally bioavailable compound that is a potent inhibitor of dihydroorotate dehydrogenase (DHODH), the rate-limiting enzyme of the de novo pyrimidine nucleotide biosynthesis pathway. In tissue culture, PTC299 manifests robust, dose-dependent, and DHODH-dependent inhibition of SARS-COV-2 replication (EC50 range, 2.0-31.6 nM) with a selectivity index >3,800. PTC299 also blocked replication of other RNA viruses, including Ebola virus. Consistent with known DHODH requirements for immunomodulatory cytokine production, PTC299 inhibited the production of interleukin (IL)-6, IL-17A (also called IL-17), IL-17 F, and vascular endothelial growth factor (VEGF) in tissue culture models. The combination of anti-SARS-CoV-2 activity, cytokine inhibitory activity, and previously established favorable pharmacokinetic and human safety profiles render PTC299 a promising therapeutic for COVID-19.
Assuntos
Antivirais/farmacologia , Carbamatos/farmacologia , Carbazóis/farmacologia , Citocinas/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , SARS-CoV-2/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Chlorocebus aethiops , Síndrome da Liberação de Citocina/tratamento farmacológico , Citocinas/imunologia , Di-Hidro-Orotato Desidrogenase , Células HeLa , Humanos , Inflamação/tratamento farmacológico , Inflamação/virologia , Células Vero , Tratamento Farmacológico da COVID-19RESUMO
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/metabolismoRESUMO
G418 is currently the most potent and active aminoglycoside to promote readthrough of eukaryotic nonsense mutations. However, owing to its toxicity G418 cannot be used in vivo to study readthrough activity A robust and scalable method for selective derivatization of G418 was developed to study the biological activity and toxicity of a series of analogs. Despite our synthetic efforts, an improvement in readthrough potency was not achieved. We discovered several analogs that demonstrated reduced zebra fish hair cell toxicity (a surrogate for ototoxicity), but this reduction in cellular toxicity did not translate to reduced in vivo toxicity in rats.