Roll-to-roll, high-resolution 3D printing of shape-specific particles.

Particle fabrication has attracted recent attention owing to its diverse applications in bioengineering1,2, drug and vaccine delivery3-5, microfluidics6,7, granular systems8,9, self-assembly5,10,11, microelectronics12,13 and abrasives14. Herein we introduce a scalable, high-resolution, 3D printing technique for the fabrication of shape-specific particles based on roll-to-roll continuous liquid interface production (r2rCLIP). We demonstrate r2rCLIP using single-digit, micron-resolution optics in combination with a continuous roll of film (in lieu of a static platform), enabling the rapidly permutable fabrication and harvesting of shape-specific particles from a variety of materials and with complex geometries, including geometries not possible to achieve with advanced mould-based techniques. We demonstrate r2rCLIP production of mouldable and non-mouldable shapes with voxel sizes as small as 2.0 × 2.0 µm2 in the print plane and 1.1 ± 0.3 µm unsupported thickness, at speeds of up to 1,000,000 particles per day. Such microscopic particles with permutable, intricate designs enable direct integration within biomedical, analytical and advanced materials applications.

Growing three-dimensional objects with light.

Vat photopolymerization (VP) additive manufacturing enables fabrication of complex 3D objects by using light to selectively cure a liquid resin. Developed in the 1980s, this technique initially had few practical applications due to limitations in print speed and final part material properties. In the four decades since the inception of VP, the field has matured substantially due to simultaneous advances in light delivery, interface design, and materials chemistry. Today, VP materials are used in a variety of practical applications and are produced at industrial scale. In this perspective, we trace the developments that enabled this printing revolution by focusing on the enabling themes of light, interfaces, and materials. We focus on these fundamentals as they relate to continuous liquid interface production (CLIP), but provide context for the broader VP field. We identify the fundamental physics of the printing process and the key breakthroughs that have enabled faster and higher-resolution printing, as well as production of better materials. We show examples of how in situ print process monitoring methods such as optical coherence tomography can drastically improve our understanding of the print process. Finally, we highlight areas of recent development such as multimaterial printing and inorganic material printing that represent the next frontiers in VP methods.

Transdermal vaccination via 3D-printed microneedles induces potent humoral and cellular immunity.

Vaccination is an essential public health measure for infectious disease prevention. The exposure of the immune system to vaccine formulations with the appropriate kinetics is critical for inducing protective immunity. In this work, faceted microneedle arrays were designed and fabricated utilizing a three-dimensional (3D)-printing technique called continuous liquid interface production (CLIP). The faceted microneedle design resulted in increased surface area as compared with the smooth square pyramidal design, ultimately leading to enhanced surface coating of model vaccine components (ovalbumin and CpG). Utilizing fluorescent tags and live-animal imaging, we evaluated in vivo cargo retention and bioavailability in mice as a function of route of delivery. Compared with subcutaneous bolus injection of the soluble components, microneedle transdermal delivery not only resulted in enhanced cargo retention in the skin but also improved immune cell activation in the draining lymph nodes. Furthermore, the microneedle vaccine induced a potent humoral immune response, with higher total IgG (Immunoglobulin G) and a more balanced IgG1/IgG2a repertoire and achieved dose sparing. Furthermore, it elicited T cell responses as characterized by functional cytotoxic CD8+ T cells and CD4+ T cells secreting Th1 (T helper type 1)-cytokines. Taken together, CLIP 3D-printed microneedles coated with vaccine components provide a useful platform for a noninvasive, self-applicable vaccination.

Botfly Myiasis Masquerading as Dacryocystitis.

Cutaneous myiasis is an infection most commonly caused globally by Dermatobia hominus , the human botfly, which is endemic to Central and South America. In North America, the most common cause of cutaneous myiasis is infestation with the larvae of Cuterebra , the North American botfly. The authors describe a 44-year-old man who presented with a 1-month history of intermittent, severe, boring pain along the side of his nose that progressed to swelling and redness along his right inferior orbital rim and lacrimal sac. CT imaging showed a rim-enhancing collection at the right medial canthus with surrounding phlegmonous changes communicating with the skin. Pathologic evaluation revealed curvilinear pigmented material associated with a granulomatous and eosinophil-rich inflammatory infiltrate, consistent with botfly myiasis. This case describes the pathogenesis of the botfly infestation in humans, as well as the clinical, radiographic, and histopathologic features of this rare orbital infection, with an emphasis on its treatment.

REGIONAL POPULATION MOBILITY AND OUTPATIENT RETINA VISITS IN THE UNITED STATES DURING THE COVID-19 PANDEMIC.

PURPOSE: To characterize how community mobility patterns across the United States correlate with local changes in retina visits during the pandemic. METHODS: Outpatient retina clinic visits were linked to population mobility by combining multiple public data sets, including the Google Community Mobility Reports and data from the Centers for Disease Control and Infection. Percentage change from baseline in daily-average number of retina visits by county and mobility were measured by county. RESULTS: A total of 2,159,689 patient visits were examined across 332 counties. Daily-average retina visits decreased by 7.0%, 19.0%, 5.0%, and 4.0% from Quarter 1 to 4 of 2020. This decrease was negatively correlated with increased incident of COVID-19 deaths for Quarters 1 to 3 (r = -0.13, r = -0.16, and r = -0.15, respectively, P < 0.001) and increased incident cases for Quarters 1 and 2 (r = -0.18, r = -0.13, respectively, P < 0.001). Daily-average retina visits relative to baseline were significantly lower for metropolitan counties in Quarters 1 and 2, compared with urban and rural (P < 0.001). The decline in retina visits had greatest association with decline in workplace visits in Quarters 1 to 3 (r = 0.27, r = 0.09, r = 0.12, respectively, P < 0.001 for all). CONCLUSION: This study provides insight into how regional mobility patterns may help to explain and predict patient behaviors and retina outpatient visit responses during the COVID-19 pandemic.

Multicenter experience with valve-in-valve transcatheter aortic valve replacement compared with primary, native valve transcatheter aortic valve replacement.

BACKGROUND: Valve-in-valve (ViV) transcatheter aortic valve replacement (TAVR) offers an alternative to reoperative surgical aortic valve replacement. The short- and intermediate-term outcomes after ViV TAVR in the real world are not entirely clear. PATIENTS AND METHODS: A multicenter, retrospective analysis of a consecutive series of 121 ViV TAVR patients and 2200 patients undergoing primary native valve TAVR from 2012 to 2017 at six medical centers. The main outcome measures were in-hospital mortality, 30-day mortality, stroke, myocardial infarction, acute kidney injury, and pacemaker implantation. RESULTS: ViV patients were more likely male, younger, prior coronary artery bypass graft, "hostile chest," and urgent. 30% of the patients had Society of Thoracic Surgeons risk score <4%, 36.3% were 4%-8% and 33.8% were >8%. In both groups many patients had concomitant coronary artery disease. Median time to prosthetic failure was 9.6 years (interquartile range: 5.5-13.5 years). 82% of failed surgical valves were size 21, 23, or 25 mm. Access was 91% femoral. After ViV, 87% had none or trivial aortic regurgitation. Mean gradients were <20 mmHg in 54.6%, 20-29 mmHg in 30.6%, 30-39 mmHg in 8.3% and ≥40 mmHg in 5.87%. Median length of stay was 4 days. In-hospital mortality was 0%. 30-day mortality was 0% in ViV and 3.7% in native TAVR. There was no difference in in-hospital mortality, postprocedure myocardial infarction, stroke, or acute kidney injury. CONCLUSION: Compared to native TAVR, ViV TAVR has similar peri-procedural morbidity with relatively high postprocedure mean gradients. A multidisciplinary approach will help ensure patients receive the ideal therapy in the setting of structural bioprosthetic valve degeneration.

Outcomes of Innominate Artery Revascularization Through Endovascular, Hybrid, or Open Approach.

BACKGROUND: Atherosclerotic disease of the innominate artery (IA) is rare and can lead to cerebral, upper extremity, and vertebral steal symptoms. Nonocclusive lesions can be treated with endovascular interventions, often with a hybrid approach while performing a right carotid endarterectomy (RCEA). Calcified IA lesions have a high risk of embolization to bilateral cerebral hemispheres. Occlusive lesions may require treatment through a median sternotomy and bypass. The purpose of our study is to review our short-term and long-term outcomes of IA revascularization. METHODS: Our operative database was used to identify patients who underwent IA revascularization between January 1998 and December 2018. Patients who underwent innominate artery stenting (IAS), combined with RCEA and IAS as well as aortoinnominate bypass (AIB), were identified. Our primary end points were freedom from neurologic event, all-cause mortality, and need for reintervention. RESULTS: Thirty-three patients (18 females [55%]) who underwent IA revascularization were identified. Average age was 67 ± 8 years, and mean clinical follow-up was 51 ± 21 months. Most patients (30 [91%]) were on a statin and antiplatelet therapy. Twenty-one patients (64%) were symptomatic. Twelve patients (36%) were asymptomatic and underwent combined RCEA with retrograde IAS for critical right carotid stenosis and IA stenosis. Preoperative imaging included a carotid duplex and computed tomography angiography. Eighteen patients (55%) underwent RCEA + IAS, 11 patients (33%) underwent isolated IAS, and 4 patients (12%) underwent AIB. In our attempt to protect bilateral hemispheres during IAS for heavily calcified lesions, we used right common carotid artery (CCA) clamping although open exposure and left CCA embolic protection filter was placed through transfemoral approach. Patients who underwent AIB had chronic heavily calcified IA occlusions or occluded IA stents with failed endovascular interventions. Perioperative stroke rate was 3%, involving 1 patient who developed reperfusion syndrome after RCEA + IAS. Perioperative mortality was 0%. Long-term stroke rate was 0%, and long-term mortality was 15% (5 of 33) because of cardiac disease. Overall restenosis rate was 9%, involving 3 patients who required secondary interventions for IA in-stent restenosis. CONCLUSIONS: IA interventions through a hybrid approach or an open approach are safe, with acceptable perioperative stroke and mortality rates. Long-term patency of these interventions is acceptable. Bilateral cerebral embolic protection can be accomplished by clamping the right CCA through an open exposure and placing a filter in the left CCA through a transfemoral approach. Patients undergoing IAS appear to have a higher rate of restenosis compared with AIB, and therefore, close follow-up with noninvasive imaging is recommended.

Difference between approximate and rigorously measured transference numbers in fluorinated electrolytes.

The performance of binary electrolytes is governed by three transport properties: conductivity, salt diffusion coefficient, and transference number. Rigorous methods for measuring conductivity and the salt diffusion coefficient are well established and used routinely in the literature. The commonly used methods for measuring transference number are the steady-state current method, t+,id, and pulsed field gradient NMR, t+,NMR. These methods yield the transference number only if the electrolyte is ideal, i.e., the salt dissociates completely into non-interacting anions and cations. In this work, we present a complete set of ion transport properties for mixtures of a functionalized perfluoroether, dimethyl carbonate terminated perfluorinated tetraethylene ether, and lithium bis(fluorosulfonyl)imide (LiFSI). The equations used to determine these properties from experimental data are based on Newman's concentrated solution theory. The concentrated-solution-theory-based transference number, t, is negative across all salt concentrations, and it increases with increasing salt concentration. In contrast, the ideal transference number, t+,id, is positive across all salt concentrations and it decreases with salt concentration. The NMR-based transference number, t+,NMR, is approximately 0.5, independent of salt concentration. The disparity between the three transference numbers, which indicates the dominance of ion clustering, is resolved by the use of Newman's concentrated solution theory.

Layerless fabrication with continuous liquid interface production.

Despite the increasing popularity of 3D printing, also known as additive manufacturing (AM), the technique has not developed beyond the realm of rapid prototyping. This confinement of the field can be attributed to the inherent flaws of layer-by-layer printing and, in particular, anisotropic mechanical properties that depend on print direction, visible by the staircasing surface finish effect. Continuous liquid interface production (CLIP) is an alternative approach to AM that capitalizes on the fundamental principle of oxygen-inhibited photopolymerization to generate a continual liquid interface of uncured resin between the growing part and the exposure window. This interface eliminates the necessity of an iterative layer-by-layer process, allowing for continuous production. Herein we report the advantages of continuous production, specifically the fabrication of layerless parts. These advantages enable the fabrication of large overhangs without the use of supports, reduction of the staircasing effect without compromising fabrication time, and isotropic mechanical properties. Combined, these advantages result in multiple indicators of layerless and monolithic fabrication using CLIP technology.

Compliant glass-polymer hybrid single ion-conducting electrolytes for lithium batteries.

Despite high ionic conductivities, current inorganic solid electrolytes cannot be used in lithium batteries because of a lack of compliance and adhesion to active particles in battery electrodes as they are discharged and charged. We have successfully developed a compliant, nonflammable, hybrid single ion-conducting electrolyte comprising inorganic sulfide glass particles covalently bonded to a perfluoropolyether polymer. The hybrid with 23 wt% perfluoropolyether exhibits low shear modulus relative to neat glass electrolytes, ionic conductivity of 10(-4) S/cm at room temperature, a cation transference number close to unity, and an electrochemical stability window up to 5 V relative to Li(+)/Li. X-ray absorption spectroscopy indicates that the hybrid electrolyte limits lithium polysulfide dissolution and is, thus, ideally suited for Li-S cells. Our work opens a previously unidentified route for developing compliant solid electrolytes that will address the challenges of lithium batteries.

Iontophoretic device delivery for the localized treatment of pancreatic ductal adenocarcinoma.

Poor delivery and systemic toxicity of many cytotoxic agents, such as the recent promising combination chemotherapy regimen of folinic acid (leucovorin), fluorouracil, irinotecan, and oxaliplatin (FOLFIRINOX), restrict their full utility in the treatment of pancreatic cancer. Local delivery of chemotherapies has become possible using iontophoretic devices that are implanted directly onto pancreatic tumors. We have fabricated implantable iontophoretic devices and tested the local iontophoretic delivery of FOLFIRINOX for the treatment of pancreatic cancer in an orthotopic patient-derived xenograft model. Iontophoretic delivery of FOLFIRINOX was found to increase tumor exposure by almost an order of magnitude compared with i.v. delivery with substantially lower plasma concentrations. Mice treated for 7 wk with device FOLFIRINOX experienced significantly greater tumor growth inhibition compared with i.v. FOLFIRINOX. A marker of cell proliferation, Ki-67, was stained, showing a significant reduction in tumor cell proliferation. These data capitalize on the unique ability of an implantable iontophoretic device to deliver much higher concentrations of drug to the tumor compared with i.v. delivery. Local iontophoretic delivery of cytotoxic agents should be considered for the treatment of patients with unresectable nonmetastatic disease and for patients with the need for palliation of local symptoms, and may be considered as a neoadjuvant approach to improve resection rates and outcome in patients with localized and locally advanced pancreatic cancer.

Does Use of Bilateral Internal Mammary Artery Grafting Reduce Long-Term Risk of Repeat Coronary Revascularization? A Multicenter Analysis.

BACKGROUND: Although previous studies have demonstrated that patients receiving bilateral internal mammary artery (BIMA) conduits during coronary artery bypass grafting have better long-term survival than those receiving a single internal mammary artery (SIMA), data on risk of repeat revascularization are more limited. In this analysis, we compare the timing, frequency, and type of repeat coronary revascularization among patients receiving BIMA and SIMA. METHODS: We conducted a multicenter, retrospective analysis of 47 984 consecutive coronary artery bypass grafting surgeries performed from 1992 to 2014 among 7 medical centers reporting to a prospectively maintained clinical registry. Among the study population, 1482 coronary artery bypass grafting surgeries with BIMA were identified, and 1297 patients receiving BIMA were propensity-matched to 1297 patients receiving SIMA. The primary end point was freedom from repeat coronary revascularization. RESULTS: The median duration of follow-up was 13.2 (IQR, 7.4-17.7) years. Patients were well matched by age, body mass index, major comorbidities, and cardiac function. There was a higher freedom from repeat revascularization among patients receiving BIMA than among patients receiving SIMA (hazard ratio [HR], 0.78 [95% CI, 0.65-0.94]; P=0.009). Among the matched cohort, 19.4% (n=252) of patients receiving SIMA underwent repeat revascularization, whereas this frequency was 15.1% (n=196) among patients receiving BIMA (P=0.004). The majority of repeat revascularization procedures were percutaneous coronary interventions (94.2%), and this did not differ between groups (P=0.274). Groups also did not differ in the ratio of native versus graft vessel percutaneous coronary intervention (P=0.899), or regarding percutaneous coronary intervention target vessels; the most common targets in both groups were the right coronary (P=0.133) and circumflex arteries (P=0.093). In comparison with SIMA, BIMA grafting was associated with a reduction in all-cause mortality at 12 years of follow-up (HR, 0.79 [95% CI, 0.69-0.91]; P=0.001), and there was no difference in in-hospital morbidity. CONCLUSIONS: BIMA grafting was associated with a reduced risk of repeat revascularization and an improvement in long-term survival and should be considered more frequently during coronary artery bypass grafting.

Optimization of Surface Display of DENV2 E Protein on a Nanoparticle to Induce Virus Specific Neutralizing Antibody Responses.

The dengue virus (DENV) causes over 350 million infections, resulting in ∼25,000 deaths per year globally. An effective dengue vaccine requires generation of strong and balanced neutralizing antibodies against all four antigenically distinct serotypes of DENV. The leading live-attenuated tetravalent dengue virus vaccine platform has shown partial efficacy, with an unbalanced response across the four serotypes in clinical trials. DENV subunit vaccine platforms are being developed because they provide a strong safety profile and are expected to avoid the unbalanced immunization issues associated with live multivalent vaccines. Subunit vaccines often lack immunogenicity, requiring either a particulate or adjuvanted formulation. Particulate formulations adsorbing monomeric DENV-E antigen to the particle surface incite a strong immune response, but have no control of antigen presentation. Highly neutralizing epitopes are displayed by DENV-E quaternary structures. To control the display of DENV-E and produce quaternary structures, particulate formulations that covalently attach DENV-E to the particle surface are needed. Here we develop a surface attached DENV2-E particulate formulation, as well as analysis tools, using PEG hydrogel nanoparticles created with particle replication in nonwetting templates (PRINT) technology. We found that adding Tween-20 to the conjugation buffer controls DENV-E adsorption to the particle surface during conjugation, improving both protein stability and epitope display. Immunizations with the anionic but not the cationic DENV2-E conjugated particles were able to produce DENV-specific and virus neutralizing antibody in mice. This work optimized the display of DENV-E conjugated to the surface of a nanoparticle through EDC/NHS chemistry, establishing a platform that can be expanded upon in future work to fully control the display of DENV-E.

Formulation of High-Performance Dry Powder Aerosols for Pulmonary Protein Delivery.

PURPOSE: Pulmonary delivery of biologics is of great interest, as it can be used for the local treatment of respiratory diseases or as a route to systemic drug delivery. To reach the full potential of inhaled biologics, a formulation platform capable of producing high performance aerosols without altering protein native structure is required. METHODS: A formulation strategy using Particle Replication in Non-wetting Templates (PRINT) was developed to produce protein dry powders with precisely engineered particle morphology. Stability of the incorporated proteins was characterized and the aerosol properties of the protein dry powders was evaluated in vitro with an Andersen Cascade Impactor (ACI). RESULTS: Model proteins bovine serum albumin (BSA) and lysozyme were micromolded into 1 µm cylinders composed of more than 80% protein, by mass. Extensive characterization of the incorporated proteins found no evidence of alteration of native structures. The BSA formulation produced a mass median aerodynamic diameter (MMAD) of 1.77 µm ± 0.06 and a geometric standard deviation (GSD) of 1.51 ± 0.06 while the lysozyme formulation had an MMAD of 1.83 µm ± 0.12 and a GSD of 1.44 ± 0.03. CONCLUSION: Protein dry powders manufactured with PRINT could enable high-performance delivery of protein therapeutics to the lungs.

Controlled analysis of nanoparticle charge on mucosal and systemic antibody responses following pulmonary immunization.

Pulmonary immunization enhances local humoral and cell-mediated mucosal protection, which are critical for vaccination against lung-specific pathogens such as influenza or tuberculosis. A variety of nanoparticle (NP) formulations have been tested preclinically for pulmonary vaccine development, yet the role of NP surface charge on downstream immune responses remains poorly understood. We used the Particle Replication in Non-Wetting Templates (PRINT) process to synthesize hydrogel NPs that varied only in surface charge and otherwise maintained constant size, shape, and antigen loading. Pulmonary immunization with ovalbumin (OVA)-conjugated cationic NPs led to enhanced systemic and lung antibody titers compared with anionic NPs. Increased antibody production correlated with robust germinal center B-cell expansion and increased activated CD4(+) T-cell populations in lung draining lymph nodes. Ex vivo treatment of dendritic cells (DCs) with OVA-conjugated cationic NPs induced robust antigen-specific T-cell proliferation with ∼ 100-fold more potency than soluble OVA alone. Enhanced T-cell expansion correlated with increased expression of surface MHCII, T-cell coactivating receptors, and key cytokines/chemokine expression by DCs treated with cationic NPs, which were not observed with anionic NPs or soluble OVA. Together, these studies highlight the importance of NP surface charge when designing pulmonary vaccines, and our findings support the notion that cationic NP platforms engender potent humoral and mucosal immune responses.

Mediating Passive Tumor Accumulation through Particle Size, Tumor Type, and Location.

As the enhanced permeation and retention (EPR) effect continues to be a controversial topic in nanomedicine, we sought to examine EPR as a function of nanoparticle size, tumor model, and tumor location, while also evaluating tumors for EPR mediating factors such as microvessel density, vascular permeability, lymphatics, stromal content, and tumor-associated immune cells. Tumor accumulation was evaluated for 55 × 60, 80 × 180, and 80 × 320 nm PRINT particles in four subcutaneous flank tumor models (SKOV3 human ovarian, 344SQ murine nonsmall cell lung, A549 human nonsmall cell lung, and A431 human epidermoid cancer). Each tumor model revealed specific particle accumulation trends with evident particle size dependence. Immuno-histochemistry staining revealed differences in tumor microvessel densities that correlated with overall tumor accumulation. Immunofluorescence images displayed size-mediated tumor penetration with signal from the larger particles concentrated close to the blood vessels, while signal from the smaller particle was observed throughout the tissue. Differences were also observed for the 55 × 60 nm particle tumor penetration across flank tumor models as a function of stromal content. The 55 × 60 nm particles were further evaluated in three orthotopic, metastatic tumor models (344SQ, A549, and SKOV3), revealing preferential accumulation in primary tumors and metastases over healthy tissue. Moreover, we observed higher tumor accumulation in the orthotopic lung cancer models than in the flank lung cancer models, whereas tumor accumulation was constant for both orthotopic and flank ovarian cancer models, further demonstrating the variability in the EPR effect as a function of tumor model and location.

Docetaxel-Loaded PLGA Nanoparticles Improve Efficacy in Taxane-Resistant Triple-Negative Breast Cancer.

Novel treatment strategies, including nanomedicine, are needed for improving management of triple-negative breast cancer. Patients with triple-negative breast cancer, when considered as a group, have a worse outcome after chemotherapy than patients with breast cancers of other subtypes, a finding that reflects the intrinsically adverse prognosis associated with the disease. The aim of this study was to improve the efficacy of docetaxel by incorporation into a novel nanoparticle platform for the treatment of taxane-resistant triple-negative breast cancer. Rod-shaped nanoparticles encapsulating docetaxel were fabricated using an imprint lithography based technique referred to as Particle Replication in Nonwetting Templates (PRINT). These rod-shaped PLGA-docetaxel nanoparticles were tested in the C3(1)-T-antigen (C3Tag) genetically engineered mouse model (GEMM) of breast cancer that represents the basal-like subtype of triple-negative breast cancer and is resistant to therapeutics from the taxane family. This GEMM recapitulates the genetics of the human disease and is reflective of patient outcome and, therefore, better represents the clinical impact of new therapeutics. Pharmacokinetic analysis showed that delivery of these PLGA-docetaxel nanoparticles increased docetaxel circulation time and provided similar docetaxel exposure to tumor compared to the clinical formulation of docetaxel, Taxotere. These PLGA-docetaxel nanoparticles improved tumor growth inhibition and significantly increased median survival time. This study demonstrates the potential of nanotechnology to improve the therapeutic index of chemotherapies and rescue therapeutic efficacy to treat nonresponsive cancers.

Oral tetrahydrouridine and decitabine for non-cytotoxic epigenetic gene regulation in sickle cell disease: A randomized phase 1 study.

BACKGROUND: Sickle cell disease (SCD), a congenital hemolytic anemia that exacts terrible global morbidity and mortality, is driven by polymerization of mutated sickle hemoglobin (HbS) in red blood cells (RBCs). Fetal hemoglobin (HbF) interferes with this polymerization, but HbF is epigenetically silenced from infancy onward by DNA methyltransferase 1 (DNMT1). METHODS AND FINDINGS: To pharmacologically re-induce HbF by DNMT1 inhibition, this first-in-human clinical trial (NCT01685515) combined 2 small molecules-decitabine to deplete DNMT1 and tetrahydrouridine (THU) to inhibit cytidine deaminase (CDA), the enzyme that otherwise rapidly deaminates/inactivates decitabine, severely limiting its half-life, tissue distribution, and oral bioavailability. Oral decitabine doses, administered after oral THU 10 mg/kg, were escalated from a very low starting level (0.01, 0.02, 0.04, 0.08, or 0.16 mg/kg) to identify minimal doses active in depleting DNMT1 without cytotoxicity. Patients were SCD adults at risk of early death despite standard-of-care, randomized 3:2 to THU-decitabine versus placebo in 5 cohorts of 5 patients treated 2X/week for 8 weeks, with 4 weeks of follow-up. The primary endpoint was ≥ grade 3 non-hematologic toxicity. This endpoint was not triggered, and adverse events (AEs) were not significantly different in THU-decitabine-versus placebo-treated patients. At the decitabine 0.16 mg/kg dose, plasma concentrations peaked at approximately 50 nM (Cmax) and remained elevated for several hours. This dose decreased DNMT1 protein in peripheral blood mononuclear cells by >75% and repetitive element CpG methylation by approximately 10%, and increased HbF by 4%-9% (P < 0.001), doubling fetal hemoglobin-enriched red blood cells (F-cells) up to approximately 80% of total RBCs. Total hemoglobin increased by 1.2-1.9 g/dL (P = 0.01) as reticulocytes simultaneously decreased; that is, better quality and efficiency of HbF-enriched erythropoiesis elevated hemoglobin using fewer reticulocytes. Also indicating better RBC quality, biomarkers of hemolysis, thrombophilia, and inflammation (LDH, bilirubin, D-dimer, C-reactive protein [CRP]) improved. As expected with non-cytotoxic DNMT1-depletion, platelets increased and neutrophils concurrently decreased, but not to an extent requiring treatment holds. As an early phase study, limitations include small patient numbers at each dose level and narrow capacity to evaluate clinical benefits. CONCLUSION: Administration of oral THU-decitabine to patients with SCD was safe in this study and, by targeting DNMT1, upregulated HbF in RBCs. Further studies should investigate clinical benefits and potential harms not identified to date. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01685515.

The LSD1 inhibitor RN-1 induces fetal hemoglobin synthesis and reduces disease pathology in sickle cell mice.

Inhibition of lysine-specific demethylase 1 (LSD1) has been shown to induce fetal hemoglobin (HbF) levels in cultured human erythroid cells in vitro. Here we report the in vivo effects of LSD1 inactivation by a selective and more potent inhibitor, RN-1, in a sickle cell disease (SCD) mouse model. Compared with untreated animals, RN-1 administration leads to induced HbF synthesis and to increased frequencies of HbF-positive cells and mature erythrocytes, as well as fewer reticulocytes and sickle cells, in the peripheral blood of treated SCD mice. In keeping with these observations, histologic analyses of the liver and spleen of treated SCD mice verified that they do not exhibit the necrotic lesions that are usually associated with SCD. These data indicate that RN-1 can effectively induce HbF levels in red blood cells and reduce disease pathology in SCD mice, and may therefore offer new therapeutic possibilities for treating SCD.

Mechanism of ion transport in perfluoropolyether electrolytes with a lithium salt.

Perfluoropolyethers (PFPEs) are polymer electrolytes with fluorinated carbon backbones that have high flash points and have been shown to exhibit moderate conductivities and high cation transference numbers when mixed with lithium salts. Ion transport in four PFPE electrolytes with different endgroups was characterized by differential scanning calorimetry (DSC), ac impedance, and pulsed-field gradient NMR (PFG-NMR) as a function of salt concentration and temperature. In spite of the chemical similarity of the electrolytes, salt diffusion coefficients measured by PFG-NMR and the glass transition temperature measured by DSC appear to be uncorrelated to ionic conductivity measured by ac impedance. We calculate a non-dimensional parameter, ß, that depends on the salt diffusion coefficients and ionic conductivity. We also use the Vogel-Tammann-Fulcher relationship to fit the temperature dependence of conductivity. We present a linear relationship between the prefactor in the VTF fit and ß; both parameters vary by four orders of magnitude in our experimental window. Our analysis suggests that transport in electrolytes with low dielectric constants (low ß) is dictated by ion hopping between clusters.