Molecular characterization of myotonic dystrophy fibroblast cell lines for use in small molecule screening.

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.

Temperature-responsive biometamaterials for gastrointestinal applications.

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.

A gastric resident drug delivery system for prolonged gram-level dosing of tuberculosis treatment.

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.

Scalable Gastric Resident Systems for Veterinary Application.

Gastric resident dosage forms have been used successfully in farm animals for the delivery of a variety of drugs helping address the challenge of extended dosing. Despite these advances, there remains a significant challenge across the range of species with large variation in body size. To address this, we investigate a scalable gastric resident platform capable of prolonged retention. We investigate prototypes in dimensions consistent with administration and retention in the stomachs of two species (rabbit and pig). We investigate sustained gastric retention of our scalable dosage form platform, and in pigs show the capacity to modulate drug release kinetics of a model drug in veterinary practice, meloxicam, with our dosage form. The ability to achieve gastric residence and thereby enable sustained drug levels across different species may have a significant impact in the welfare of animals in both research, agricultural, zoological, and clinical practice settings.

Genotype-targeted local therapy of glioma.

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.

Development of an oral once-weekly drug delivery system for HIV antiretroviral therapy.

The efficacy of antiretroviral therapy is significantly compromised by medication non-adherence. Long-acting enteral systems that can ease the burden of daily adherence have not yet been developed. Here we describe an oral dosage form composed of distinct drug-polymer matrices which achieved week-long systemic drug levels of the antiretrovirals dolutegravir, rilpivirine and cabotegravir in a pig. Simulations of viral dynamics and patient adherence patterns indicate that such systems would significantly reduce therapeutic failures and epidemiological modelling suggests that using such an intervention prophylactically could avert hundreds of thousands of new HIV cases. In sum, weekly administration of long-acting antiretrovirals via a novel oral dosage form is a promising intervention to help control the HIV epidemic worldwide.

Triggerable tough hydrogels for gastric resident dosage forms.

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.

Oral, ultra-long-lasting drug delivery: Application toward malaria elimination goals.

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 large-animal 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 malaria-transmitting 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 malaria-transmitting 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.

Inhibition of Btk with CC-292 provides early pharmacodynamic assessment of activity in mice and humans.

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.

Discovery of a potent and isoform-selective targeted covalent inhibitor of the lipid kinase PI3Kα.

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 PI3Kß, -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.

Novel IKK inhibitors: beta-carbolines.

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.