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Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are common forms of adult onset muscular dystrophy. Pathogenesis in both diseases is largely driven by production of toxic-expanded repeat RNAs that sequester MBNL RNA-binding proteins, causing mis-splicing. Given this shared pathogenesis, we hypothesized that diamidines, small molecules that rescue mis-splicing in DM1 models, could also rescue mis-splicing in DM2 models. While several DM1 cell models exist, few are available for DM2 limiting research and therapeutic development. Here, we characterize DM1 and DM2 patient-derived fibroblasts for use in small molecule screens and therapeutic studies. We identify mis-splicing events unique to DM2 fibroblasts and common events shared with DM1 fibroblasts. We show that diamidines can partially rescue molecular phenotypes in both DM1 and DM2 fibroblasts. This study demonstrates the potential of fibroblasts as models for DM1 and DM2, which will help meet an important need for well-characterized DM2 cell models.

We hypothesized that ingested warm fluids could act as triggers for biomedical devices. We investigated heat dissipation throughout the upper gastrointestinal (GI) tract by administering warm (55°C) water to pigs and identified two zones in which thermal actuation could be applied: esophageal (actuation through warm water ingestion) and extra-esophageal (protected from ingestion of warm liquids and actuatable by endoscopically administered warm fluids). Inspired by a blooming flower, we developed a capsule-sized esophageal system that deploys using elastomeric elements and then recovers its original shape in response to thermal triggering of shape-memory nitinol springs by ingestion of warm water. Degradable millineedles incorporated into the system could deliver model molecules to the esophagus. For the extra-esophageal compartment, we developed a highly flexible macrostructure (mechanical metamaterial) that deforms into a cylindrical shape to safely pass through the esophagus and deploys into a fenestrated spherical shape in the stomach, capable of residing safely in the gastric cavity for weeks. The macrostructure uses thermoresponsive elements that dissociate when triggered with the endoscopic application of warm (55°C) water, allowing safe passage of the components through the GI tract. Our gastric-resident platform acts as a gram-level long-lasting drug delivery dosage form, releasing small-molecule drugs for 2 weeks. We anticipate that temperature-triggered systems could usher the development of the next generation of stents, drug delivery, and sensing systems housed in the GI tract.

Multigram drug depot systems for extended drug release could transform our capacity to effectively treat patients across a myriad of diseases. For example, tuberculosis (TB) requires multimonth courses of daily multigram doses for treatment. To address the challenge of prolonged dosing for regimens requiring multigram drug dosing, we developed a gastric resident system delivered through the nasogastric route that was capable of safely encapsulating and releasing grams of antibiotics over a period of weeks. Initial preclinical safety and drug release were demonstrated in a swine model with a panel of TB antibiotics. We anticipate multiple applications in the field of infectious diseases, as well as for other indications where multigram depots could impart meaningful benefits to patients, helping maximize adherence to their medication.

Aggressive neurosurgical resection to achieve sustained local control is essential for prolonging survival in patients with lower-grade glioma. However, progression in many of these patients is characterized by local regrowth. Most lower-grade gliomas harbor isocitrate dehydrogenase 1 (IDH1) or IDH2 mutations, which sensitize to metabolism-altering agents. To improve local control of IDH mutant gliomas while avoiding systemic toxicity associated with metabolic therapies, we developed a precision intraoperative treatment that couples a rapid multiplexed genotyping tool with a sustained release microparticle (MP) drug delivery system containing an IDH-directed nicotinamide phosphoribosyltransferase (NAMPT) inhibitor (GMX-1778). We validated our genetic diagnostic tool on clinically annotated tumor specimens. GMX-1778 MPs showed mutant IDH genotype-specific toxicity in vitro and in vivo, inducing regression of orthotopic IDH mutant glioma murine models. Our strategy enables immediate intraoperative genotyping and local application of a genotype-specific treatment in surgical scenarios where local tumor control is paramount and systemic toxicity is therapeutically limiting.

Targeted therapies that suppress B cell receptor (BCR) signaling have emerged as promising agents in autoimmune disease and B cell malignancies. Bruton's tyrosine kinase (Btk) plays a crucial role in B cell development and activation through the BCR signaling pathway and represents a new target for diseases characterized by inappropriate B cell activity. N-(3-(5-fluoro-2-(4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamide (CC-292) is a highly selective, covalent Btk inhibitor and a sensitive and quantitative assay that measures CC-292-Btk engagement has been developed. This translational pharmacodynamic assay has accompanied CC-292 through each step of drug discovery and development. These studies demonstrate the quantity of Btk bound by CC-292 correlates with the efficacy of CC-292 in vitro and in the collagen-induced arthritis model of autoimmune disease. Recently, CC-292 has entered human clinical trials with a trial design that has provided rapid insight into safety, pharmacokinetics, and pharmacodynamics. This first-in-human healthy volunteer trial has demonstrated that a single oral dose of 2 mg/kg CC-292 consistently engaged all circulating Btk protein and provides the basis for rational dose selection in future clinical trials. This targeted covalent drug design approach has enabled the discovery and early clinical development of CC-292 and has provided support for Btk as a valuable drug target for B-cell mediated disorders.

PI3Kα has been identified as an oncogene in human tumors. By use of rational drug design, a targeted covalent inhibitor 3 (CNX-1351) was created that potently and specifically inhibits PI3Kα. We demonstrate, using mass spectrometry and X-ray crystallography, that the selective inhibitor covalently modifies PI3Kα on cysteine 862 (C862), an amino acid unique to the α isoform, and that PI3Kβ, -γ, and -δ are not covalently modified. 3 is able to potently (EC(50) < 100 nM) and specifically inhibit signaling in PI3Kα-dependent cancer cell lines, and this leads to a potent antiproliferative effect (GI(50) < 100 nM). A covalent probe, 8 (CNX-1220), which selectively bonds to PI3Kα, was used to investigate the duration of occupancy of 3 with PI3Kα in vivo. This is the first report of a PI3Kα-selective inhibitor, and these data demonstrate the biological impact of selectively targeting PI3Kα.

Systems capable of residing for prolonged periods of time in the gastric cavity have transformed our ability to diagnose and treat patients. Gastric resident systems for drug delivery, ideally need to be: ingestible, be able to change shape or swell to ensure prolonged gastric residence, have the mechanical integrity to withstand the forces associated with gastrointestinal motility, be triggerable to address any side effects, and be drug loadable and release drug over a prolonged period of time. Materials that have been primarily utilized for these applications have been largely restricted to thermoplastics and thermosets. Here we describe a novel set of materials, triggerable tough hydrogels, meeting all these requirement, supported by evaluation in a large animal model and ultimately demonstrate the potential of triggerable tough hydrogels to serve as prolonged gastric resident drug depots. Triggerable tough hydrogels may be applied in myriad of applications, including bariatric interventions, drug delivery, and tissue engineering., The use of drug delivery systems for the gastrointestinal tract has been faced with a number of drawbacks related to their prolonged use. Here, the authors develop a drug-loaded hydrogel with high strength to withstand long-term gastrointestinal motility and can be triggered to dissolve on demand.

Efforts at elimination of scourges, such as malaria, are limited by the logistic challenges of reaching large rural populations and ensuring patient adherence to adequate pharmacologic treatment. We have developed an oral, ultra-long-acting capsule that dissolves in the stomach and deploys a star-shaped dosage form that releases drug while assuming a geometry that prevents passage through the pylorus yet allows passage of food, enabling prolonged gastric residence. This gastric-resident, drug delivery dosage form releases small-molecule drugs for days to weeks and potentially longer. Upon dissolution of the macrostructure, the components can safely pass through the gastrointestinal tract. Clinical, radiographic, and endoscopic evaluation of a swine largeanimal model that received these dosage forms showed no evidence of gastrointestinal obstruction or mucosal injury. We generated long-acting formulations for controlled release of ivermectin, a drug that targets malariatransmitting mosquitoes, in the gastric environment and incorporated these into our dosage form, which then delivered a sustained therapeutic dose of ivermectin for up to 14 days in our swine model. Further, by using mathematical models of malaria transmission that incorporate the lethal effect of ivermectin against malariatransmitting mosquitoes, we demonstrated that this system will boost the efficacy of mass drug administration toward malaria elimination goals. Encapsulated, gastric-resident dosage forms for ultra-long-acting drug delivery have the potential to revolutionize treatment options for malaria and other diseases that affect large populations around the globe for which treatment adherence is essential for efficacy., NIH (Grants EB-000244 and T32-5T32HL007604-29), Bill and Melinda Gates Foundation (Grants OPP1096734, OPP1139921 and OPP1068440)

Endogenous adenosine released locally during cerebral ischemia is neuroprotective, and agents which decrease adenosine inactivation may potentiate its protective effects. The effects of 5'-deoxy-5-iodotubercidin (5'd-5IT), an inhibitor of the adenosine-catabolizing enzyme, adenosine kinase, were studied in male Wistar rats subjected to 2 h of transient middle cerebral artery occlusion. 5'd-5IT or the vehicle (10% DMSO in saline) was administered i.p. 30 min before, and 2 h and 6 h after the induction of middle cerebral artery occlusion. The infarct volume was determine using 2,3,5-triphenyltetrazolium chloride staining 48 h after middle cerebral artery occlusion. The infarct volume was significantly reduced in rats treated with 1.85 mg/kg x 3 (57% reduction, P0.001) or 1.0 mg/kg x 3 (34% reduction, P0.05), but not 0.3 mg/kg x 3 5'd-5IT compared to vehicle-treated rats. The reduction of infarct volume was accompanied by a significant improvement in behavioral measures of neurological deficit. These data further support a role of adenosine in neuroprotection and suggest that adenosine kinase inhibition may be a useful approach to the treatment of focal cerebral ischemia.

The novel and efficient of several racemic β-sulfonamidopropionate esters is described. Treatment of the racemic esters with Chymotrypsin or Subtilisin Carlsberg (purified of detergent grade) provided the corresponding (S)-car☐ylic acids in 60–90% yield. These acids (75 to > 98% e.e.) are useful starting materials for the synthesis of P-3 site modified renin inhibitors.

(1992). A CONVENIENT PREPARATION OF 4-CYCLOPROPYLPHENOL. Organic Preparations and Procedures International: Vol. 24, No. 6, pp. 696-698.

The novel and efficient of several racemic β-sulfonamidopropionate esters is described. Treatment of the racemic esters with Chymotrypsin or Subtilisin Carlsberg (purified of detergent grade) provided the corresponding (S)-car☐ylic acids in 60–90% yield. These acids (75 to > 98% e.e.) are useful starting materials for the synthesis of P-3 site modified renin inhibitors.

The discovery of second generation N-hydroxyurea 5-lipoxygenase inhibitors was accomplished through the development of a broad structure−activity relationship (SAR) study. This study identified requirements for improving potency and also extending duration by limiting metabolism. Potency could be maintained by the incorporation of heterocyclic templates substituted with selected lipophilic substituents. Duration of inhibition after oral administration was optimized by identification of structural features in the proximity of the N-hydroxyurea which correlated to low in vitro glucuronidation rates. Furthermore, the rate of in vitro glucuronidation was shown to be stereoselective for certain analogs. (R)-N-[3-[5-(4-Fluorophenoxy)-2-furyl]-1-methyl-2-propynyl]-N-hydroxyurea (17c) was identified and selected for clinical development.

Inhibitors of IkappaB kinase (IKK) have long been sought as specific regulators of NF-kappaB. A screening effort of the endogenous IKK complex allowed us to identify 5-bromo-6-methoxy-beta-carboline as a nonspecific IKK inhibitor. Optimization of this beta-carboline natural product derivative resulted in a novel class of selective IKK inhibitors with IC(50)s in the nanomolar range. In addition, we show that one of these beta-carboline analogues inhibits the phosphorylation of IkappaBalpha and subsequent activation of NF-kappaB in whole cells, as well as blocking TNF-alpha release in LPS-challenged mice.

(1992). A CONVENIENT PREPARATION OF 4-CYCLOPROPYLPHENOL. Organic Preparations and Procedures International: Vol. 24, No. 6, pp. 696-698.

The discovery of second generation N-hydroxyurea 5-lipoxygenase inhibitors was accomplished through the development of a broad structure-activity relationship (SAR) study. This study identified requirements for improving potency and also extending duration by limiting metabolism. Potency could be maintained by the incorporation of heterocyclic templates substituted with selected lipophilic substituents. Duration of inhibition after oral administration was optimized by identification of structural features in the proximity of the N-hydroxyurea which correlated to low in vitro glucuronidation rates. Furthermore, the rate of in vitro glucuronidation was shown to be stereoselective for certain analogs. (R)-N-[3-[5-(4-Fluorophenoxy)-2-furyl]-1-methyl-2-propynyl]-N-hydroxyure a (17c) was identified and selected for clinical development.

Abstract 3485 Targeted therapies that suppress B cell receptor (BCR) signaling have emerged as promising agents in the treatment of several B cell malignancies. Bruton's tyrosine kinase (Btk) plays a key role in promoting B cell proliferation and survival through participation in the BCR signaling pathway and represents a promising new drug target. AVL-292 is a covalent, highly selective, orally active small molecule inhibitor of Btk currently being evaluated in a Phase 1b clinical trial in relapsed, refractory B cell malignancies including Chronic Lymphocytic Leukemia (CLL) and non-Hodgkin lymphomas. AVL-292 forms a covalent bond with Cys481 in Btk and potently inhibits Btk in biochemical (IC50 < 0.5nM) and cellular assays (EC50 1–10 nM) including anti-IgM stimulation of BCR signaling, B cell proliferation and activation. A quantitative pharmacodynamic assay to determine the level of AVL-292 bonded to Btk in vitro or in vivo was developed and this drug-target engagement by AVL-292 was shown to correlate directly with inhibition of Btk enzyme activity and substrate phosphorylation. To rationally determine the dose and dose frequency of AVL-292 most likely to benefit patients and to reduce the potential for sub-therapeutic dosing in initial oncology patient cohorts, AVL-292 was administered to healthy adult subjects in a double-blind, placebo controlled, single ascending dose study. This study assessed safety, pharmacokinetics, and quantitatively measured Btk protein levels and AVL-292-Btk engagement in freshly isolated peripheral B lymphocytes. In healthy human subjects, AVL-292 was found to be safe and well tolerated following oral administration at dose levels ranging from 0.5–7.0 mg/kg. AVL-292 plasma levels and pharmacodynamic measurement of Btk engagement was dose-proportional across cohorts. All subjects that received 1.0 mg/kg AVL-292 achieved >80% Btk engagement and mean peak plasma levels (Cmax 365 ng/mL) of AVL-292 were rapidly achieved (Tmax median 40 min). Subjects receiving 2.0 mg/kg AVL-292 had a mean peak plasma concentration of 542 ng/mL. All subjects demonstrated >84% Btk engagement at this dose, with 5 of 6 subjects achieving >98% drug-target engagement. Although AVL-292 plasma levels declined substantially by 8 hours, Btk engagement persisted throughout 24 hours, demonstrating that covalent inhibition of Btk with AVL-292 enables prolonged duration of activity without high levels of circulating drug. These results suggest that a once daily dosing schedule is sufficient for sustained Btk inhibition. Furthermore, the Btk protein level in circulating B lymphocytes from all study subjects was evaluated and the mean level was found to be 417.7 pg Btk/mg total protein. Interestingly, this finding in normal B cells correlates well with preclinical ex vivo analysis of Btk protein in primary CLL cells where comparable Btk protein levels were found. This suggests that the AVL-292 dose range and schedule identified for complete Btk engagement in this healthy volunteer trial is likely to inform appropriate dose selection in the subsequent phase 1b oncology study, allowing more rapid identification of a safe and clinically effective dose. Disclosures: Evans: Avila Therapeutics: Employment, Equity Ownership. Tester:Avila Therapeutics: Employment, Equity Ownership. Aslanian:Avila Therapeutics: Employment, Equity Ownership. Chaturvedi:Avila Therapeutics: Employment, Equity Ownership. Mazdiyasni:Avila Therapeutics: Employment, Equity Ownership. Sheets:Avila Therapeutics: Employment, Equity Ownership. Nacht:Avila Therapeutics: Employment, Equity Ownership. Stiede:Avila Therapeutics: Employment, Equity Ownership. Witowski:Avila Therapeutics: Employment, Equity Ownership. Lounsbury:Avila Therapeutics: Employment, Equity Ownership. Petter:Avila Therapeutics: Employment, Equity Ownership. Singh:Avila Therapeutics: Employment, Equity Ownership. Westlin:Avila Therapeutics: Employment, Equity Ownership.

B-cell receptor (BCR) signaling is essential for normal B cell development and proliferation. In addition, functional BCR signaling contributes to the proliferation and survival of many B cell lymphomas through constitutive low level or tonic signaling. Disruption of this tonic signaling pathway results in cell death or decreased viability in many B cell lymphoma cells. Bruton’s tyrosine kinase (Btk) is a member of the Tec family of protein tyrosine kinases and plays a crucial role in the development and activation of B cells through association with the BCR signalosome. We have developed a novel, potent, irreversible inhibitor of Btk that selectively disrupts the BCR signaling pathway as measured by inhibition of both Btk Y223 autophosphorylation and phosphorylation of the Btk substrate PLCg2. AVL101 inhibits Btk but leaves upstream BCR kinase activity intact; both Syk phosphorylation at Y525/526 and transphosphorylation of Btk on residue Y551 are unaltered. By mass spectrometry, we have shown that AVL101 covalently modifies Btk specifically at Cys-481, and in cell-based assays, this irreversible binding leads to prolonged inhibition of Btk kinase activity. After removal of the compound, inhibition of Btk activity is maintained in cultured B cell lymphoma cells for > 8 hours. This prolonged inhibition correlates with the half-life of the Btk protein, which we have found to be greater than 8 hours, irrespective of BCR activation state or modification by the inhibitor. We have demonstrated that AVL101 functionally inhibits BCR signaling in vitro and in vivo. In vitro, AVL101 inhibits BCR ligand-induced calcium flux as well as proliferation of the B cell lymphoma cell lines DOHH2 and WSU-DLCL2. In vivo, we have shown AVL101 is orally bioavailable and can inhibit Btk-dependent B cell function. Our data demonstrate that Btk is a valid therapeutic target for B cell lymphomas and other disorders dependent on BCR signaling. Furthermore, irreversible inhibition of Btk enables complete and prolonged inactivation of the target kinase, and suggests that clinically, a rapid and sustained therapeutic response may be possible using an orally available, irreversible Btk inhibitor.

Microbial reduction of various 2,2-disubstituted-1,3-cyclohexanediones with bakers' yeast provides efficient access to chiral cyclohexanoid synthetic precursors.

The hydroxamic acid functionally can be incorporated into simple molecules to produce potent inhibitors of 5-lipoxygenase. The ability of many of these hydroxamates to inhibit leukotriene synthesis in vivo has been measured directly with a rat peritoneal anaphylaxis model. Despite their potent enzyme inhibitory activity in vitro, many orally dosed hydroxamic acids only weakly inhibited leukotriene synthesis in vivo. This discrepancy is attributable at least in part to the rapid metabolism of hydroxamates to the corresponding carboxylic acids, which are inactive against the enzyme. A study of the structural features that affect this metabolism revealed that 2-arylpropionohydroxamic acids are relatively resistant to metabolic hydrolysis. Several members of this class of hydroxamates are described that are orally active inhibitors of leukotriene synthesis.

The hydroxamic acid functionality can be incorporated in a variety of simple molecules to produce potent inhibitors of 5-lipoxygenase. As an example of this, the structure-activity relationships in a series of omega-phenylalkyl and omega-naphthylalkyl hydroxamic acids are presented. Among the features described are the influence of hydrophobicity, aryl substitution, and modifications of the hydroxamate group on enzyme inhibitory potency. To assist in the selection of more potent hydroxamic acid inhibitors, a simple hypothesis about the nature of enzyme-inhibitor binding was devised. In this hypothesis, the structures of compounds were matched to a proposed geometry of arachidonic acid when bound to the enzyme. Compounds that match best without extending into disfavored regions were predicted to be the best inhibitors. Three series of hydroxamates selected according to this approach are described. Within these series are some of the most potent inhibitors of 5-lipoxygenase reported to date.

The hydroxamic acid functionality can be incorporated in a variety of simple molecules to produce potent inhibitors of 5-lipoxygenase. As an example of this, the structure-activity relationships in a series of omega-phenylalkyl and omega-naphthylalkyl hydroxamic acids are presented. Among the features described are the influence of hydrophobicity, aryl substitution, and modifications of the hydroxamate group on enzyme inhibitory potency. To assist in the selection of more potent hydroxamic acid inhibitors, a simple hypothesis about the nature of enzyme-inhibitor binding was devised. In this hypothesis, the structures of compounds were matched to a proposed geometry of arachidonic acid when bound to the enzyme. Compounds that match best without extending into disfavored regions were predicted to be the best inhibitors. Three series of hydroxamates selected according to this approach are described. Within these series are some of the most potent inhibitors of 5-lipoxygenase reported to date.