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.

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.

Isoprostanes are prostaglandin isomers produced from arachidonic acid by a free radical-catalyzed mechanism. Urinary excretion of 8-iso-prostaglandin F2alpha, an isomer of the PGG/H synthase (cyclooxygenase or COX) enzyme product, prostaglandin F2alpha (PGF2alpha), has exhibited promise as an index of oxidant stress in vivo. We have developed a quantitative method to measure isoprostane F2alpha-I, (IPF2alpha-I) a class I isomer (8-iso-PGF2alpha is class IV), using gas chromatography/mass spectrometry. IPF2alpha-I is severalfold as abundant in human urine as 8-iso-PGF2alpha, with mean values of 737 +/- 20.6 pg/mg creatinine. Both isoprostanes are formed in a free radical-dependent manner in low density lipoprotein oxidized by copper in vitro. However, IPF2alpha-I, unlike 8-iso-PGF2alpha, is not formed in a COX-dependent manner by platelets activated by thrombin or collagen in vitro. Similarly, COX inhibition in vivo has no effect on IPF2alpha-I. Neither serum IPF2alpha-I, an index of cellular capacity to generate the isoprostane, nor urinary excretion of IPF2alpha-I, an index of actual generation in vivo, is depressed by aspirin or indomethacin. In contrast, both serum thromboxane B2 and urinary excretion of its 11-dehydro metabolite are depressed by the COX inhibitors. Although serum 8-iso-PGF2alpha formation is substantially depressed by COX inhibitors, urinary excretion of the compound is unaffected. Urinary IPF2alpha-I is elevated in cigarette smokers compared with controls (1525 +/- 180 versus 740 +/- 40 pg/mg creatinine; P < 0.01) and is highly correlated with urinary 8-iso-PGF2alpha (r = 0.9; P < 0.001). Urinary IPF2alpha-I is a novel index of lipid peroxidation in vivo, which can be measured with precision and sensitivity. It is an abundant F2-isoprostane formed in a free radical- but not COX-dependent manner. Although 8-iso-PGF2alpha may be formed as a minor product of COX, this pathway contributes trivially, if at all, to levels in urine. Urinary excretion of both isoprostanes is elevated in cigarette smokers.

Isoprostanes (iPs) are free radical catalyzed prostaglandin isomers. Analysis of individual isomers of PGF 2␣-F2-iPs-in urine has reflected lipid peroxidation in humans. However, up to 64 F 2-iPs may be formed, and it is unknown whether coordinate generation, disposition, and excretion of F 2-iPs occurs in humans. To address this issue, we developed methods to measure individual members of the four structural classes of F 2-iPs, using liquid chromatography͞tandem mass spectrometry (LC͞MS͞MS), in which sample preparation is minimized. Authentic standards of F 2-iPs of classes III, IV, V, and VI were used to identify class-specific ions for multiple reaction monitoring. Using iPF 2␣-VI as a model compound, we demonstrated the reproducibility of the assay in human urine. Urinary levels of all F 2-iPs measured were elevated in patients with familial hypercholesterolemia. However, only three of eight F 2-iPs were elevated in patients with congestive heart failure, compared with controls. Paired analyses by GC͞MS and LC͞MS͞MS of iPF 2␣-VI in hypercholesterolemia and of 8,12-iso-iPF 2␣-VI in congestive heart failure were highly correlated. This approach will permit high throughput analysis of multiple iPs in human disease.