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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 and 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 (i.c.v.) injection resulted in broader, more optimal biodistribution than did intra-cisterna magna (i.c.m.) delivery. AAV9.Syn.hCDKL5 treatment increased phosphorylation of EB2, a bona fide CDKL5 substrate, demonstrating biological activity in vivo. Our data provide proof of concept that i.c.v. 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/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.

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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.

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Familial dysautonomia (FD) is a rare neurodegenerative disease caused by a splicing mutation in elongator acetyltransferase complex subunit 1 (ELP1). This mutation leads to the skipping of exon 20 and a tissue-specific reduction of ELP1, mainly in the central and peripheral nervous systems. FD is a complex neurological disorder accompanied by severe gait ataxia and retinal degeneration. There is currently no effective treatment to restore ELP1 production in individuals with FD, and the disease is ultimately fatal. After identifying kinetin as a small molecule able to correct the ELP1 splicing defect, we worked on its optimization to generate novel splicing modulator compounds (SMCs) that can be used in individuals with FD. Here, we optimize the potency, efficacy, and bio-distribution of second-generation kinetin derivatives to develop an oral treatment for FD that can efficiently pass the blood-brain barrier and correct the ELP1 splicing defect in the nervous system. We demonstrate that the novel compound PTC258 efficiently restores correct ELP1 splicing in mouse tissues, including brain, and most importantly, prevents the progressive neuronal degeneration that is characteristic of FD. Postnatal oral administration of PTC258 to the phenotypic mouse model TgFD9;Elp1Δ20/flox increases full-length ELP1 transcript in a dose-dependent manner and leads to a 2-fold increase in functional ELP1 in the brain. Remarkably, PTC258 treatment improves survival, gait ataxia, and retinal degeneration in the phenotypic FD mice. Our findings highlight the great therapeutic potential of this novel class of small molecules as an oral treatment for FD.

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Antibiotic-resistant Neisseria gonorrhoeae (Ng) are an emerging public health threat due to increasing numbers of multidrug resistant (MDR) organisms. We identified two novel orally active inhibitors, PTC-847 and PTC-672, that exhibit a narrow spectrum of activity against Ng including MDR isolates. By selecting organisms resistant to the novel inhibitors and sequencing their genomes, we identified a new therapeutic target, the class Ia ribonucleotide reductase (RNR). Resistance mutations in Ng map to the N-terminal cone domain of the α subunit, which we show here is involved in forming an inhibited α4ß4 state in the presence of the ß subunit and allosteric effector dATP. Enzyme assays confirm that PTC-847 and PTC-672 inhibit Ng RNR and reveal that allosteric effector dATP potentiates the inhibitory effect. Oral administration of PTC-672 reduces Ng infection in a mouse model and may have therapeutic potential for treatment of Ng that is resistant to current drugs.

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A 6-month-old intact male Standard Dachshund was referred for evaluation of a soft tissue swelling above the right eye. Examination of the right eye revealed mild lateral deviation of the globe, normal vision, and a dorsonasal soft tissue swelling. Examination of the posterior segment was normal. Dual-phase computed tomography angiography was consistent with an orbital varix of the angularis oculi and right dorsal external ophthalmic veins with no evidence of arterial involvement. Treatment involved fluoroscopically guided coil embolization of the venous outflow with nine platinum microcoils, followed by sclerotherapy of the varix using 1.5 mL of 3% sodium tetradecyl sulfate foam. Moderate-to-marked swelling was noted at the treatment site in the weeks following therapy, which gradually resolved. At final reexamination 3 months post-therapy, complete sclerosis and resolution of the orbital varix were documented. To the authors' knowledge, this is the first reported case involving the use of a sclerotic agent for successful treatment of a venous malformation in a dog.

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PTC725 is a small molecule NS4B-targeting inhibitor of hepatitis C virus (HCV) genotype (gt) 1 RNA replication that lacks activity against HCV gt2. We analyzed the Los Alamos HCV sequence database to predict susceptible/resistant HCV gt's according to the prevalence of known resistance-conferring amino acids in the NS4B protein. Our analysis predicted that HCV gt3 would be highly susceptible to the activity of PTC725. Indeed, PTC725 was shown to be active against a gt3 subgenomic replicon with a 50% effective concentration of ∼5 nM. De novo resistance selection identified mutations encoding amino acid substitutions mapping to the first predicted transmembrane region of NS4B, a finding consistent with results for PTC725 and other NS4B-targeting compounds against HCV gt1. This is the first report of the activity of an NS4B targeting compound against HCV gt3. In addition, we have identified previously unreported amino acid substitutions selected by PTC725 treatment which further demonstrate that these compounds target the NS4B first transmembrane region.

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BACKGROUND: Previous studies have shown an increased risk of pneumonia with benzodiazepines (BZD) and an increased risk of any infection with non-BZD hypnotics, but no analysis has specifically investigated the risk of pneumonia with non-BZD hypnotic use. OBJECTIVE: To evaluate the risk of pneumonia associated with non-BZD hypnotic use in the elderly. METHODS: This was a retrospective case-control study of members aged 65 years and older enrolled in an integrated health care system. Cases were identified as patients aged 65 years and older with a diagnosis of pneumonia from January 2011 to December 2012. Controls were matched in a 4:1 ratio to cases based on age, gender, and active enrollment. Non-BZD hypnotic exposure was evaluated for all cases and controls 1 year before the index date. Proximity of exposure to index date and duration of use were analyzed. Conditional logistic regression adjusted for covariates was performed. RESULTS: We identified 51,029 cases with pneumonia and matched 188,391 controls without pneumonia. Of the cases with pneumonia, 5.5% (2,790) of cases had exposure to a non-BZD hypnotic, compared with 3.4% (6,345) of controls. Non-BZD hypnotic exposure was associated with an increased risk of pneumonia (OR = 1.14; 95% CI = 1.08-1.20). When exposure was stratified by proximity to index date, only current exposure was associated with an increased risk of pneumonia (OR = 1.27; 95% CI = 1.18-1.36). Short-term exposure was associated with a relatively higher risk of pneumonia (OR = 1.57; 95% CI = 1.39-1.77) compared with long-term use (OR = 1.16; 95% CI = 1.06-1.25). CONCLUSIONS: Current use of non-BZD hypnotics in older adults is associated with an increased risk of pneumonia. The findings of this study provide additional support for reducing the use of non-BZD hypnotics in older adults and for pursuing safer alternatives for treating insomnia. DISCLOSURES: No outside funding supported this study. At the time of this study, Jung was a PGY2 resident in drug information at Kaiser Permanente Drug Information Services. All authors are employed by Kaiser Permanente and report no other potential financial conflicts of interest. Study concept and design were contributed by Jung, Spence, Lee, and Gibbs. Jung, Spence, and Hui were responsible for data collection, and data interpretation was performed by Jung and Spence, with assistance from Escasa, Lee, and Hui. The manuscript was primarily written by Jung, along with Spence and Escasa, and revised by Spence, Escasa, and Lee, along with the other authors.

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A novel series of 2-(4-sulfonamidophenyl)-indole 3-carboxamides was identified and optimized for activity against the HCV genotype 1b replicon resulting in compounds with potent and selective activity. Further evaluation of this series demonstrated potent activity across HCV genotypes 1a, 2a and 3a. Compound 4z had reduced activity against HCV genotype 1b replicons containing single mutations in the NS4B coding sequence (F98C and V105M) indicating that NS4B is the target. This novel series of 2-(4-sulfonamidophenyl)-indole 3-carboxamides serves as a promising starting point for a pan-genotype HCV discovery program.

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A structure-activity relationship investigation of various 6-(azaindol-2-yl)pyridine-3-sulfonamides using the HCV replicon cell culture assay led to the identification of a potent series of 7-azaindoles that target the hepatitis C virus NS4B. Compound 2ac, identified via further optimization of the series, has excellent potency against the HCV 1b replicon with an EC50 of 2nM and a selectivity index of >5000 with respect to cellular GAPDH RNA. Compound 2ac also has excellent oral plasma exposure levels in rats, dogs and monkeys and has a favorable liver to plasma distribution profile in rats.

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OBJECTIVE: To describe successful transarterial coil embolization of a palatine artery pseudoaneurysm that extended into the caudal maxillary sinus of a gelding. STUDY DESIGN: Clinical report. ANIMAL: A 24-year-old Morgan gelding with right-sided epistaxis. METHODS: The right maxillary sinus was imaged by radiography, computed tomography, and sinoscopy. Angiography was performed to locate the source of bleeding, and transarterial coil embolization of a right palatine artery pseudoaneurysm was performed. RESULTS: There was some mucoid nasal discharge and an intermittent cough postoperatively. No epistaxis was seen after embolization. There was moderate swelling of the surgical incision over the mid-cervical common carotid artery. The horse was discharged from the hospital 4 days after surgery, and had been doing well, with no signs of bleeding, for 2 months postoperatively. The horse had acute colic secondary to a strangulating lipoma at 2 months and was euthanatized after exploratory celiotomy. Placement of embolization coils in the right palatine artery was confirmed by CT and necropsy. CONCLUSIONS: Severe epistaxis in the horse may be caused by a ruptured major palatine artery pseudoaneurysm. Occlusion of this vessel can be successfully accomplished by transarterial coil embolization.

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A novel, potent, and orally bioavailable inhibitor of hepatitis C RNA replication targeting NS4B, compound 4t (PTC725), has been identified through chemical optimization of the 6-(indol-2-yl)pyridine-3-sulfonamide 2 to improve DMPK and safety properties. The focus of the SAR investigations has been to identify the optimal combination of substituents at the indole N-1, C-5, and C-6 positions and the sulfonamide group to limit the potential for in vivo oxidative metabolism and to achieve an acceptable pharmacokinetic profile. Compound 4t has excellent potency against the HCV 1b replicon, with an EC50 = 2 nM and a selectivity index of >5000 with respect to cellular GAPDH. Compound 4t has an overall favorable pharmacokinetic profile with oral bioavailability values of 62%, 78%, and 18% in rats, dogs, and monkeys, respectively, as well as favorable tissue distribution properties with a liver to plasma exposure ratio of 25 in rats.

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While new direct-acting antiviral agents for the treatment of chronic hepatitis C virus (HCV) infection have been approved, there is a continued need for novel antiviral agents that act on new targets and can be used in combination with current therapies to enhance efficacy and to restrict the emergence of drug-resistant viral variants. To this end, we have identified a novel class of small molecules, exemplified by PTC725, that target the nonstructural protein 4B (NS4B). PTC725 inhibited HCV 1b (Con1) replicons with a 50% effective concentration (EC50) of 1.7 nM and an EC90 of 9.6 nM and demonstrated a >1,000-fold selectivity window with respect to cytotoxicity. The compounds were fully active against HCV replicon mutants that are resistant to inhibitors of NS3 protease and NS5B polymerase. Replicons selected for resistance to PTC725 harbored amino acid substitutions F98L/C and V105M in NS4B. Anti-replicon activity of PTC725 was additive to synergistic in combination with alpha interferon or with inhibitors of HCV protease and polymerase. Immunofluorescence microscopy demonstrated that neither the HCV inhibitors nor the F98C substitution altered the subcellular localization of NS4B or NS5A in replicon cells. Oral dosing of PTC725 showed a favorable pharmacokinetic profile with high liver and plasma exposure in mice and rats. Modeling of dosing regimens in humans indicates that a once-per-day or twice-per-day oral dosing regimen is feasible. Overall, the preclinical data support the development of PTC725 for use in the treatment of chronic HCV infection.

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U1 interference (U1i) is a novel method to block gene expression. U1i requires expression of a 5'-end-mutated U1 snRNA designed to base pair to the 3'-terminal exon of the target gene's pre-mRNA that leads to inhibition of polyadenylation. Here, we show U1i is robust (> or =95%) and a 10-nt target length is sufficient for good silencing. Surprisingly, longer U1 snRNAs, which could increase annealing to the target, fail to improve silencing. Extensive mutagenesis of the 10-bp U1 snRNA:target duplex shows that any single mismatch different from GU at positions 3-8, destroys silencing. However, mismatches within the other positions give partial silencing, suggesting that off-target inhibition could occur. The specificity of U1i may be enhanced, however, by the fact that silencing is impaired by RNA secondary structure or by splicing factors binding nearby, the latter mediated by Arginine-Serine (RS) domains. U1i inhibition can be reconstituted in vivo by tethering of RS domains of U1-70K and U2AF65. These results help to: (i) define good target sites for U1i; (ii) identify and understand natural cellular examples of U1i; (iii) clarify the contribution of hydrogen bonding to U1i and to U1 snRNP binding to 5' splice sites and (iv) understand the mechanism of U1i.

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Reducing or eliminating expression of a given gene is likely to require multiple methods to ensure coverage of all of the genes in a given mammalian cell. We and others [Furth, P. A., Choe, W. T., Rex, J. H., Byrne, J. C., and Baker, C. C. (1994) Mol. Cell. Biol. 14, 5278-5289] have previously shown that U1 small nuclear (sn) RNA, both natural or with 5' end mutations, can specifically inhibit reporter gene expression in mammalian cells. This inhibition occurs when the U1 snRNA 5' end base pairs near the polyadenylation signal of the reporter gene's pre-mRNA. This base pairing inhibits poly(A) tail addition, a key, nearly universal step in mRNA biosynthesis, resulting in degradation of the mRNA. Here we demonstrate that expression of endogenous mammalian genes can be efficiently inhibited by transiently or stably expressed 5' end-mutated U1 snRNA. Also, we determine the inhibitory mechanism and establish a set of rules to use this technique and to improve the efficiency of inhibition. Two U1 snRNAs base paired to a single pre-mRNA act synergistically, resulting in up to 700-fold inhibition of the expression of specific reporter genes and 25-fold inhibition of endogenous genes. Surprisingly, distance from the U1 snRNA binding site to the poly(A) signal is not critical for inhibition, instead the U1 snRNA must be targeted to the terminal exon of the pre-mRNA. This could reflect a disruption by the 5' end-mutated U1 snRNA of the definition of the terminal exon as described by the exon definition model.

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