Delivery of RNA to the Blood-Brain Barrier Endothelium Using Cationic Bicelles.

Blood-brain barrier (BBB) dysfunction is prevalent in Alzheimer's disease and other neurological disorders. Restoring normal BBB function through RNA therapy is a potential avenue for addressing cerebrovascular changes in these disorders that may lead to cognitive decline. Although lipid nanoparticles have been traditionally used as drug carriers for RNA, bicelles have been emerging as a better alternative because of their higher cellular uptake and superior transfection capabilities. Cationic bicelles composed of DPPC/DC7PC/DOTAP at molar ratios of 63.8/25.0/11.2 were evaluated for the delivery of RNA in polarized hCMEC/D3 monolayers, a widely used BBB cell culture model. RNA-bicelle complexes were formed at five N/P ratios (1:1 to 5:1) by a thin-film hydration method. The RNA-bicelle complexes at N/P ratios of 3:1 and 4:1 exhibited optimal particle characteristics for cellular delivery. The cellular uptake of cationic bicelles laced with 1 mol% DiI-C18 was confirmed by flow cytometry and confocal microscopy. The ability of cationic bicelles (N/P ratio 4:1) to transfect polarized hCMEC/D3 with FITC-labeled control siRNA was tested vis-a-vis commercially available Lipofectamine RNAiMAX. These studies demonstrated the higher transfection efficiency and greater potential of cationic bicelles for RNA delivery to the BBB endothelium.

Harmonizing Biopredictive Methodologies Through the Product Quality Research Institute (PQRI) Part I: Biopredictive Dissolution of Ibuprofen and Dipyridamole Tablets.

Assessing in vivo performance to inform formulation selection and development decisions is an important aspect of drug development. Biopredictive dissolution methodologies for oral dosage forms have been developed to understand in vivo performance, assist in formulation development/optimization, and forecast the outcome of bioequivalence studies by combining them with simulation tools to predict plasma profiles in humans. However, unlike compendial dissolution methodologies, the various biopredictive methodologies have not yet been harmonized or standardized. This manuscript presents the initial phases of an effort to develop best practices and move toward standardization of the biopredictive methodologies through the Product Quality Research Institute (PQRI, https://pqri.org ) entitled "The standardization of in vitro predictive dissolution methodologies and in silico bioequivalence study Working Group." This Working Group (WG) is comprised of participants from 10 pharmaceutical companies and academic institutes. The project will be accomplished in a total of five phases including assessing the performance of dissolution protocols designed by the individual WG members, and then building "best practice" protocols based on the initial dissolution profiles. After refining the "best practice" protocols to produce equivalent dissolution profiles, those will be combined with physiologically based biopharmaceutics models (PBBM) to predict plasma profiles. In this manuscript, the first two of the five phases are reported, namely generating biopredictive dissolution profiles for ibuprofen and dipyridamole and using those dissolution profiles with PBBM to match the clinical plasma profiles. Key experimental parameters are identified, and this knowledge will be applied to build the "best practice" protocol in the next phase.

RNF130 Regulates LDLR Availability and Plasma LDL Cholesterol Levels.

BACKGROUND: Removal of circulating plasma low-density lipoprotein cholesterol (LDL-C) by the liver relies on efficient endocytosis and intracellular vesicle trafficking. Increasing the availability of hepatic LDL receptors (LDLRs) remains a major clinical target for reducing LDL-C levels. Here, we describe a novel role for RNF130 (ring finger containing protein 130) in regulating plasma membrane availability of LDLR. METHODS: We performed a combination of gain-of-function and loss-of-function experiments to determine the effect of RNF130 on LDL-C and LDLR recycling. We overexpressed RNF130 and a nonfunctional mutant RNF130 in vivo and measured plasma LDL-C and hepatic LDLR protein levels. We performed in vitro ubiquitination assays and immunohistochemical staining to measure levels and cellular distribution of LDLR. We supplement these experiments with 3 separate in vivo models of RNF130 loss-of-function where we disrupted Rnf130 using either ASO (antisense oligonucleotides), germline deletion, or AAV CRISPR (adeno-associated virus clustered regularly interspaced short palindromic repeats) and measured hepatic LDLR and plasma LDL-C. RESULTS: We demonstrate that RNF130 is an E3 ubiquitin ligase that ubiquitinates LDLR resulting in redistribution of the receptor away from the plasma membrane. Overexpression of RNF130 decreases hepatic LDLR and increases plasma LDL-C levels. Further, in vitro ubiquitination assays demonstrate RNF130-dependent regulation of LDLR abundance at the plasma membrane. Finally, in vivo disruption of Rnf130 using ASO, germline deletion, or AAV CRISPR results in increased hepatic LDLR abundance and availability and decreased plasma LDL-C levels. CONCLUSIONS: Our studies identify RNF130 as a novel posttranslational regulator of LDL-C levels via modulation of LDLR availability, thus providing important insight into the complex regulation of hepatic LDLR protein levels.

Effect of Lipidic Excipients on the Particle Properties and Aerosol Performance of High Drug Load Spray Dried Particles for Inhalation.

High drug load inhalable particles were prepared by co-spray drying a hydrophobic, crystalline, small molecule drug with various lipid or phospholipid excipients at a 9:1 molar ratio to understand the primary drivers of aerosol performance. The effect of excipient structure on solid-state, surface characteristics, and aerodynamic performance of the co-spray dried particles was studied while keeping the spray drying parameters constant. Spray drying of the drug with lipids produced crystalline drug particles, whereas phospholipids produced partially amorphous drug particles. All of the co-spray dried particles were nearly spherical with a smooth surface, except for the spray dried drug particles without excipients - which showed the presence of rough crystals on the surface. All co-spray dried particles showed surface enrichment of the excipient. The surface enrichment of the phospholipids was higher compared to the lipids. Co-spray dried particles that showed higher surface enrichment of excipients showed improved aerosol performance. In comparing all the excipients studied, distearyolphosphatidylcholine (DSPC) showed maximum enrichment on the particle surface and thereby significantly improved aerosol performance. This study demonstrated that the addition of small amounts of lipid excipients during spray drying can change surface morphology, composition, and cohesion, impacting aerosol performance of drugs.

Medication and healthcare use, parent knowledge and cough in children: A cohort study.

INTRODUCTION: Cough is an important contributor to the health burden of children and their families. There are limited data describing healthcare utilization and medication use over the course of a cough illness beyond the initial presentation. Our primary objective was to describe medication and healthcare use in children with a respiratory illness with cough as a symptom over the course of the illness. METHODS: A cohort study of children aged less than 15-years presenting to three primary healthcare centers and three emergency departments with a cough illness between July 7, 2015 and October 6, 2018. Children with immunosuppression, known chronic lung diseases (except asthma) and those requiring hospitalization at screening were excluded. The primary outcomes were cough-related frequency and type of healthcare seeking and medication use up to 28 days following enrolment. RESULTS: Data for 465 children were analyzed; median age 2.2-years (interquartile range = 1.1-5.3). Cough at Day 28 persisted in 117 children (25.2%). Overall, 436 (94%) children received medications in the week before and/or 4 weeks following enrolment. Half with upper respiratory tract infections were prescribed antibiotics. Among children with no diagnosis of asthma, reactive airways disease or croup (n = 404), 16.8% were given steroids. Fifty-eight percent of children sought healthcare at least once before their baseline presentation (median = 1, range = 0-20) and 49.7% had at least one further presentation in the following 28 days. CONCLUSIONS: High healthcare utilization, inappropriate medication use, and suboptimal parent knowledge regarding cough suggests targeted education is needed to improve management and reduce cough burden.

MicroRNA-144 Silencing Protects Against Atherosclerosis in Male, but Not Female Mice.

OBJECTIVE: Atherosclerosis is a leading cause of death in developed countries. MicroRNAs act as fine-tuners of gene expression and have been shown to have important roles in the pathophysiology and progression of atherosclerosis. We, and others, previously demonstrated that microRNA-144 (miR-144) functions to post-transcriptionally regulate ABCA1 (ATP binding cassette transporter A1) and plasma HDL (high-density lipoprotein) cholesterol levels. Here, we explore how miR-144 inhibition may protect against atherosclerosis. Approach and Results: We demonstrate that miR-144 silencing reduced atherosclerosis in male, but not female low-density lipoprotein receptor null (Ldlr-/-) mice. MiR-144 antagonism increased circulating HDL cholesterol levels, remodeled the HDL particle, and enhanced reverse cholesterol transport. Notably, the effects on HDL and reverse cholesterol transport were more pronounced in male mice suggesting sex-specific differences may contribute to the effects of silencing miR-144 on atherosclerosis. As a molecular mechanism, we identify the oxysterol metabolizing enzyme CYP7B1 (cytochrome P450 enzyme 7B1) as a miR-144 regulated gene in male, but not female mice. Consistent with miR-144-dependent changes in CYP7B1 activity, we show decreased levels of 27-hydroxycholesterol, a known proatherogenic sterol and the endogenous substrate for CYP7B1 in male, but not female mice. CONCLUSIONS: Our data demonstrate silencing miR-144 has sex-specific effects and that treatment with antisense oligonucleotides to target miR-144 might result in enhancements in reverse cholesterol transport and oxysterol metabolism in patients with cardiovascular disease.

Posttranslational regulation of CYP2J2 by nitric oxide.

Nitric oxide (NO) is an essential signaling molecule in the body, regulating numerous biological processes. Beside its physiological roles, NO affects drug metabolism by modulating the activity and/or expression of cytochrome P450 enzymes. Previously, our lab showed that NO generation caused by inflammatory stimuli results in CYP2B6 degradation via the ubiquitin-proteasome pathway. In the current study, we tested the NO-mediated regulation of CYP2J2 that metabolizes arachidonic acids to bioactive epoxyeicosatrienoic acids, as well as therapeutic drugs such as astemizole and ebastine. To investigate the effects of NO on CYP2J2 expression and activity, Huh7 cells stably transduced with CYP2J2 with a C-terminal V5 tag were treated with dipropylenetriamine-NONOate (DPTA), a NO donor. The level of CYP2J2 proteins were decreased in a time- and concentration-dependent manner, and the activity was also rapidly inhibited. However, mRNA expression was not altered and the protein synthesis inhibitor cycloheximide did not attenuate DPTA-mediated downregulation of CYP2J2. Removal of DPTA from the culture media quickly restored the activity of remaining CYP2J2, and no further CYP2J2 degradation occurred. To determine the mechanism of CYP2J2 down-regulation by NO, cells were treated with DPTA in the presence or absence of protease inhibitors including proteasomal, lysosomal and calpain inhibitors. Remarkably, the down-regulation of CYP2J2 by NO was attenuated by calpeptin, a calpain inhibitor. However, other calpain inhibitors or calcium chelator show no inhibitory effects on the degradation. The proteasome inhibitor bortezomib showed small but significant restoration of CYP2J2 levels although stimulated ubiquitination of CYP2J2 was not detected. In conclusion, these data suggest that NO regulates CYP2J2 posttranslationally and NO-evoked CYP2J2 degradation undergoes ubiquitin-independent proteasomal degradation pathway unlike CYP2B6.

RNA-binding protein ZFP36L1 maintains posttranscriptional regulation of bile acid metabolism.

Bile acids function not only as detergents that facilitate lipid absorption but also as signaling molecules that activate the nuclear receptor farnesoid X receptor (FXR). FXR agonists are currently being evaluated as therapeutic agents for a number of hepatic diseases due to their lipid-lowering and antiinflammatory properties. FXR is also essential for maintaining bile acid homeostasis and prevents the accumulation of bile acids. Elevated bile acids activate FXR, which in turn switches off bile acid synthesis by reducing the mRNA levels of bile acid synthesis genes, including cholesterol 7α-hydroxylase (Cyp7a1). Here, we show that FXR activation triggers a rapid posttranscriptional mechanism to degrade Cyp7a1 mRNA. We identified the RNA-binding protein Zfp36l1 as an FXR target gene and determined that gain and loss of function of ZFP36L1 reciprocally regulate Cyp7a1 mRNA and bile acid levels in vivo. Moreover, we found that mice lacking hepatic ZFP36L1 were protected from diet-induced obesity and steatosis. The reduced adiposity and antisteatotic effects observed in ZFP36L1-deficient mice were accompanied by impaired lipid absorption that was consistent with altered bile acid metabolism. Thus, the ZFP36L1-dependent regulation of bile acid metabolism is an important metabolic contributor to obesity and hepatosteatosis.

ABCG1 regulates pulmonary surfactant metabolism in mice and men.

Idiopathic pulmonary alveolar proteinosis (PAP) is a rare lung disease characterized by accumulation of surfactant. Surfactant synthesis and secretion are restricted to epithelial type 2 (T2) pneumocytes (also called T2 cells). Clearance of surfactant is dependent upon T2 cells and macrophages. ABCG1 is highly expressed in both T2 cells and macrophages. ABCG1-deficient mice accumulate surfactant, lamellar body-loaded T2 cells, lipid-loaded macrophages, B-1 lymphocytes, and immunoglobulins, clearly demonstrating that ABCG1 has a critical role in pulmonary homeostasis. We identify a variant in the ABCG1 promoter in patients with PAP that results in impaired activation of ABCG1 by the liver X receptor α, suggesting that ABCG1 basal expression and/or induction in response to sterol/lipid loading is essential for normal lung function. We generated mice lacking ABCG1 specifically in either T2 cells or macrophages to determine the relative contribution of these cell types on surfactant lipid homeostasis. These results establish a critical role for T2 cell ABCG1 in controlling surfactant and overall lipid homeostasis in the lung and in the pathogenesis of human lung disease.

Malignant abdominal rocks: where do they come from?

For the radiologist, calcifications in an abdominal malignancy raise questions of both diagnostic and prognostic significance. Although certain cancers are well known to calcify, such as colorectal and ovarian, malignant abdominal calcifications actually arise from a wide variety of epithelial, mesenchymal, lymphoid, or germ cell neoplasms. The pathophysiology of calcification in abdominal malignancies is heterogeneous and incompletely understood. Calcifications may present primarily, in untreated tumors, or develop during treatment; the latter can occur in variable clinical settings. A basic understanding of the varied pathogenic etiology can assist the radiologist in assessing disease status. By presenting an assortment of calcified abdominal malignancies on computed tomography in varied clinical settings, we aim not only to inform the differential diagnosis, but also to clarify the prognosis of calcifications in abdominal malignancies.

Physician knowledge and appropriate utilization of computed tomographic colonography in colorectal cancer screening.

GOALS: To assess physician understanding of computed tomographic colonography (CTC) in colorectal cancer (CRC) screening guidelines in a pilot study. BACKGROUND: CTC is a sensitive and specific method of detecting colorectal polyps and cancer. However, several factors have limited its clinical availability, and CRC screening guidelines have issued conflicting recommendations. STUDY: A web-based survey was administered to physicians at two institutions with and without routine CTC availability. RESULTS: 398 of 1655 (24%) participants completed the survey, 59% was from the institution with routine CTC availability, 52% self-identified as trainees, and 15% as gastroenterologists. 78% had no personal experience with CTC. Only 12% was aware of any current CRC screening guidelines that included CTC. In a multiple regression model, gastroenterologists had greater odds of being aware of guidelines (OR 3.49, CI 1.67-7.26), as did physicians with prior CTC experience (OR 4.81, CI 2.39-9.68), controlling for institution, level of training, sex, and practice type. Based on guidelines that recommend CTC, when given a clinical scenario, 96% of physicians was unable to select the appropriate follow-up after a CTC, which was unaffected by institution. CONCLUSIONS: Most physicians have limited experience with CTC and are unaware of recent recommendations concerning CTC in CRC screening.

Associations between maternal phthalate exposure and cord sex hormones in human infants.

It has been speculated that maternal phthalate exposure may affect reproductive development in human newborns. However, the mechanism awaits further investigation. The aim is to evaluate the association between maternal phthalate exposure and cord sex steroid hormones in pregnant women and their newborns from the general population. A total of 155 maternal and infant pair were recruited and analyzed. Levels of urinary phthalate metabolites and sex steroid hormones were determined using liquid chromatography/electrospray tandem mass spectrometry (LC-ESI-MS/MS) and radioimmunoassay (RIA), respectively. No significant correlation was found between each steroid hormones and phthalate metabolites for male newborns, except MMP was marginally significantly correlated with E(2). After adjusting for maternal age, estradiol (E(2)) levels in cord serum from male newborns were not correlated with maternal urinary phthalate metabolites. In female newborns, the maternal urinary levels of mono-(2-ethylhexyl) phthalate (MEHP) and mono-(2-ethyl-5-hydroxyhexyl) phthalate (5OH-MEHP) were negatively correlated with the free testosterone (fT) and fT/E(2) levels in cord serum with Pearson correlation coefficients ranging between -0.24 and -0.29 (p<0.05). Additionally, after gestational age was adjusted, the maternal urinary level of DEHP was negatively correlated with the free testosterone (fT) and fT/E(2) levels in cord serum. We suggest that maternal exposure to phthalates may affect sex steroid hormones status in fetal and newborn stage.

Relationship among age, race, medical funding, and cervical cancer survival.

OBJECTIVE: To examine the impact of age, race, and medical funding on cervical cancer survival. MATERIALS AND METHODS: Study design was a retrospective chart review of cervical cancer patients. Charts were abstracted for demographic characteristics, Pap smear history, clinical presentation, treatment, and survival. Descriptive studies, Spearman correlation, and Cox's proportional hazards regression model were performed. RESULTS: One hundred-twenty-five cervical cancer patients were included. Mean age at diagnosis was 46.1 +/- 13.2 years, and median survival time from cervical cancer was 31 months; 11.2% of the study population was aged greater than 65 years; 63.4% were African American; and 44.6% had no medical funding. Diagnosis at age of at least 65 years was significantly correlated with suboptimal cervical cancer screening pattern (r = 0.36, p = .0003). Women aged at least 65 years old had a 3.39 time increased hazard of death compared to younger patients (p = .02; OR, 3.39; 95% CI, 1.20-9.56) after adjusting for advanced stage of disease and treatment modality. There was no significant association between medical funding or race on cervical cancer screening pattern, stage at diagnosis, or survival. CONCLUSION: Age at diagnosis (> or = 65 years), but not medical funding or race, was correlated with suboptimal cervical cancer screening pattern and poor survival.

Robot-assisted extended pelvic lymphadenectomy.

PURPOSE: To evaluate perioperative and pathologic outcomes of patients undergoing robot-assisted extended pelvic lymphadenectomy for bladder cancer. MATERIALS AND METHODS: A retrospective chart review was performed for all 27 patients who underwent robotassisted radical cystectomy (RARC) and extended pelvic lymphadenectomy at Tulane University and Mayo Clinic Arizona between March 2005 and April 2007. Baseline demographic, perioperative, and pathologic data were evaluated. The bifurcation of the aorta was the proximal border of dissection in all patients. RESULTS: There was a total of 27 patients, and all procedures were completed laparoscopically; all urinary diversions were constructed extracorporeally in RARC patients. The mean total operative time was 400 minutes, and mean blood loss was 277 mL. All patients had transitional-cell carcinoma in the bladder cancer group. The mean total lymph node count for the RARC group was 12.3 (range 7-20). There were no intraoperative complications, and 9 (33%) patients experienced postoperative complications. CONCLUSIONS: An extended pelvic lymphadenectomy can be reliably and safely performed robotically during RARC in the management of bladder cancer. The robotic system aids in performing a meticulous dissection and in adhering to sound oncologic principles.

Lung lesion doubling times: values and variability based on method of volume determination.

PURPOSE: To determine doubling times (DTs) of lung lesions based on volumetric measurements from thin-section CT imaging. METHODS: Previously untreated patients with > or = two thin-section CT scans showing a focal lung lesion were identified. Lesion volumes were derived using direct volume measurements and volume calculations based on lesion area and diameter. Growth rates (GRs) were compared by tissue diagnosis and measurement technique. RESULTS: 54 lesions were evaluated including 8 benign lesions, 10 metastases, 3 lymphomas, 15 adenocarcinomas, 11 squamous carcinomas, and 7 miscellaneous lung cancers. Using direct volume measurements, median DTs were 453, 111, 15, 181, 139 and 137 days, respectively. Lung cancer DTs ranged from 23-2239 days. There were no significant differences in GRs among the different lesion types. There was considerable variability among GRs using different volume determination methods. CONCLUSIONS: Lung cancer doubling times showed a substantial range, and different volume determination methods gave considerably different DTs.

Detection of residual subclinical ovarian carcinoma after completion of adjuvant chemotherapy.

PURPOSE: We sought to test the hypothesis that the presence of telomerase activity in peritoneal washings of patients treated for ovarian carcinoma is a sensitive and specific indicator of the presence of residual disease. We hypothesized that this test, if added to second-look procedure protocols, could help determine whether residual disease is present or not in patients who have completed their adjuvant chemotherapy for ovarian carcinoma. EXPERIMENTAL DESIGN: Peritoneal washings were obtained from 100 consecutive patients undergoing a second-look procedure after treatment for ovarian carcinoma (cases) and from 100 patients undergoing surgery for benign gynecological conditions (controls). The washings were assayed for telomerase activity using the telomerase repeat amplification protocol. The results were compared to the histological and cytological findings. RESULTS: Among our 100 cases, 82 (82%) had either positive second-look procedures or expressed telomerase in their peritoneal washings. Fifty-three (53%) had positive second-look procedures, whereas 66 (66%) tested positive for telomerase. Twenty-nine of the 47 patients (62%) with negative second-look procedures tested positive for telomerase. Of the 53 patients with positive second-look procedures, 37 (70%) tested positive for telomerase. None of the 100 controls (0%) expressed telomerase in their peritoneal washings. CONCLUSIONS: Telomerase activity in peritoneal washings of patients treated for ovarian carcinoma and undergoing a second-look procedure may provide a means of increasing the sensitivity of such procedures for the detection of residual disease while maintaining a high level of specificity.