Absorption, distribution, metabolism and excretion of 14C-Emvododstat following a single oral dose in rats and dogs.

Emvododstat is a potent inhibitor of dihydroorotate dehydrogenase and is now in clinical development for the treatment of acute myeloid leukaemia and COVID-19.Following an oral dose administration in Long-Evans rats, 14C-emvododstat-derived radioactivity was widely distributed throughout the body, with the highest distribution in the endocrine, fatty, and secretory tissues and the lowest in central nervous system.Following a single oral dose of 14C-emvododstat in rats, 54.7% of the dose was recovered in faeces while less than 0.4% of dose was recovered in urine 7 days post-dose. Emvododstat was the dominant radioactive component in plasma and faeces.Following a single oral dose of 14C-emvododstat in dogs, 75.2% of the dose was recovered in faeces while 0.5% of dose was recovered in urine 8 days post-dose. Emvododstat was the dominant radioactive component in faeces, while emvododstat and its two metabolites (O-desmethyl emvododstat and emvododstat amide bond hydrolysis product) were the major circulating radioactivity in dog plasma.

In vitro metabolism, pharmacokinetics and drug interaction potentials of emvododstat, a DHODH inhibitor.

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.

Metabolism and Disposition of Ataluren after Oral Administration to Mice, Rats, Dogs, and Humans.

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.

PTC124 targets genetic disorders caused by nonsense mutations.

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.

Ataluren metabolism: Ataluren-O-1ß-acyl glucuronide is a stable circulating metabolite in mouse, rat, dog and human.

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.

Effectiveness of PTC124 treatment of cystic fibrosis caused by nonsense mutations: a prospective phase II trial.

BACKGROUND: In about 10% of patients worldwide and more than 50% of patients in Israel, cystic fibrosis results from nonsense mutations (premature stop codons) in the messenger RNA (mRNA) for the cystic fibrosis transmembrane conductance regulator (CFTR). PTC124 is an orally bioavailable small molecule that is designed to induce ribosomes to selectively read through premature stop codons during mRNA translation, to produce functional CFTR. METHODS: This phase II prospective trial recruited adults with cystic fibrosis who had at least one nonsense mutation in the CFTR gene. Patients were assessed in two 28-day cycles. During the first cycle, patients received PTC124 at 16 mg/kg per day in three doses every day for 14 days, followed by 14 days without treatment; in the second cycle, patients received 40 mg/kg of PTC124 in three doses every day for 14 days, followed by 14 days without treatment. The primary outcome had three components: change in CFTR-mediated total chloride transport; proportion of patients who responded to treatment; and normalisation of chloride transport, as assessed by transepithelial nasal potential difference (PD) at baseline, at the end of each 14-day treatment course, and after 14 days without treatment. The trial was registered with who.int/ictrp, and with clinicaltrials.gov, number NCT00237380. FINDINGS: Transepithelial nasal PD was evaluated in 23 patients in the first cycle and in 21 patients in the second cycle. Mean total chloride transport increased in the first treatment phase, with a change of -7.1 (SD 7.0) mV (p<0.0001), and in the second, with a change of -3.7 (SD 7.3) mV (p=0.032). We recorded a response in total chloride transport (defined as a change in nasal PD of -5 mV or more) in 16 of the 23 patients in the first cycle's treatment phase (p<0.0001) and in eight of the 21 patients in the second cycle (p<0.0001). Total chloride transport entered the normal range for 13 of 23 patients in the first cycle's treatment phase (p=0.0003) and for nine of 21 in the second cycle (p=0.02). Two patients given PTC124 had constipation without intestinal obstruction, and four had mild dysuria. No drug-related serious adverse events were recorded. INTERPRETATION: In patients with cystic fibrosis who have a premature stop codon in the CFTR gene, oral administration of PTC124 to suppress nonsense mutations reduces the epithelial electrophysiological abnormalities caused by CFTR dysfunction.

Safety, tolerability, and pharmacokinetics of PTC124, a nonaminoglycoside nonsense mutation suppressor, following single- and multiple-dose administration to healthy male and female adult volunteers.

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.

Phase 1 Study of Safety, Tolerability, and Pharmacokinetics of PTC299, an Inhibitor of Stress-Regulated Protein Translation.

PTC299 is a novel small molecule that specifically blocks the production of protein from selected mRNAs that under certain conditions use noncanonical ribosomal translational pathways. Hypoxia, oncogenic transformation, and viral infections limit normal translation and turn on these noncanonical translation pathways that are sensitive to PTC299. Vascular endothelial cell growth factor (VEGF) is an example of a transcript that is posttranscriptionally regulated. Single doses of PTC299 (0.03 to 3 mg/kg) were administered orally to healthy volunteers in a phase 1 single ascending-dose study. In a subsequent multiple ascending-dose study in healthy volunteers, multiple-dose regimens (0.3 to 1.2 mg/kg twice a day or 1.6 mg/kg 3 times a day for 7 days) were evaluated. PTC299 was well tolerated in these studies. As expected in healthy volunteers, mean plasma VEGF levels did not change. Increases in Cmax and AUC of PTC299 were dose-proportional. The target trough plasma concentration associated with preclinical efficacy was achieved within 7 days at doses of 0.6 mg/kg twice daily and above. These data demonstrate that PTC299 is orally bioavailable and well tolerated and support clinical evaluation of PTC299 in cancer, certain viral infections, or other diseases in which deregulation of translational control is a causal factor.

Phase 2a study of ataluren-mediated dystrophin production in patients with nonsense mutation Duchenne muscular dystrophy.

BACKGROUND: Approximately 13% of boys with Duchenne muscular dystrophy (DMD) have a nonsense mutation in the dystrophin gene, resulting in a premature stop codon in the corresponding mRNA and failure to generate a functional protein. Ataluren (PTC124) enables ribosomal readthrough of premature stop codons, leading to production of full-length, functional proteins. METHODS: This Phase 2a open-label, sequential dose-ranging trial recruited 38 boys with nonsense mutation DMD. The first cohort (n = 6) received ataluren three times per day at morning, midday, and evening doses of 4, 4, and 8 mg/kg; the second cohort (n = 20) was dosed at 10, 10, 20 mg/kg; and the third cohort (n = 12) was dosed at 20, 20, 40 mg/kg. Treatment duration was 28 days. Change in full-length dystrophin expression, as assessed by immunostaining in pre- and post-treatment muscle biopsy specimens, was the primary endpoint. FINDINGS: Twenty three of 38 (61%) subjects demonstrated increases in post-treatment dystrophin expression in a quantitative analysis assessing the ratio of dystrophin/spectrin. A qualitative analysis also showed positive changes in dystrophin expression. Expression was not associated with nonsense mutation type or exon location. Ataluren trough plasma concentrations active in the mdx mouse model were consistently achieved at the mid- and high- dose levels in participants. Ataluren was generally well tolerated. INTERPRETATION: Ataluren showed activity and safety in this short-term study, supporting evaluation of ataluren 10, 10, 20 mg/kg and 20, 20, 40 mg/kg in a Phase 2b, double-blind, long-term study in nonsense mutation DMD. TRIAL REGISTRATION: ClinicalTrials.gov NCT00264888.