Toronto aortic stenosis quality of life questionnaire (TASQ): validation in TAVI patients.

BACKGROUND: Aortic stenosis (AS) is a common cardiac condition whose prevalence increases with age. The symptom burden associated with severe aortic stenosis (AS) can introduce significant lifestyle disruptions and if left untreated can lead to a poor prognosis. Quality of life (QoL) is an important consideration in these patients. The TASQ is a QoL tool that was developed for aortic stenosis patients. We evaluated the psychometric properties of this specific questionnaire in patients who underwent transcatheter aortic valve implantation (TAVI), which is a therapeutic option for patients with severe aortic stenosis (AS). METHODS: The properties of the TASQ in measuring QoL were evaluated in AS patients undergoing TAVI. Patients presenting for the TAVI procedure (N = 62) were evaluated pre-TAVI, at discharge, 1-month, and 3-month follow-ups. Demographic information as well as caregiver status, and daily activities were recorded. In addition to the TASQ, they completed the KCCQ (Kansas City Cardiomyopathy Questionnaire) and the IIRS (Illness Intrusiveness Rating Scale). RESULTS: The TASQ is a 16-item self-administered questionnaire that assesses AS-specific QoL across five domains: physical symptoms; physical limitations; emotional impact; social limitations, and health expectations. TASQ subscales are internally consistent (α = 0.74-0.96) and showed significant improvements from baseline across assessments (p < 0.001). Construct validity evidence was demonstrated by correlations consistent with theoretically derived hypotheses across time points. CONCLUSIONS: The TASQ is a brief measure of AS-specific QoL that is sensitive to change in patients undergoing TAVI. Items on the TASQ capture important QoL concerns reported by AS patients, suggesting this is a measure of relevant and meaningful outcomes for this patient population. Detection of early improvements in QoL by the TASQ is promising, with important implications for the evaluation of procedural outcomes in this population.

Water-soluble benzodiazepine prodrug/enzyme combinations for intranasal rescue therapies.

Benzodiazepines (BZDs), including diazepam (DZP) and midazolam (MDZ), are drugs of choice for rapid treatment of seizure emergencies. Current approved use of these drugs involves administration via either intravenous or rectal routes. The former requires trained medical personnel, while the latter is socially unacceptable for many patients and caregivers. In recent years, efforts have been made to formulate BZDs for nasal administration. Because of the low solubility of these molecules, organic vehicles have been used to solubilize the drugs in the nasal products under development. However, organic solvents are irritating, potentially resulting in injury to nasal tissue. Here we report preliminary studies supporting a strategy in which water-soluble BZD prodrugs and a suitable converting enzyme are coadministered in an aqueous vehicle. Diazepam and midazolam prodrugs were synthesized and were readily converted to their active forms by a protease from Aspergillus oryzae. Using a permeation assay based on monolayers of Madin-Darby canine kidney II-wild type cells, we found that enzymatically produced BZDs could be maintained at high degrees of supersaturation, enabling faster transport across the membrane than can be achieved using saturated solutions. This strategy not only obviates the need for organic solvents, but it also suggests more rapid absorption and earlier peak concentrations than can be otherwise achieved. This article is part of a Special Issue entitled "Status Epilepticus".

Physicochemical characterization of anionic lipid-based ternary siRNA complexes.

Physicochemical characterization is a useful tool in understanding lipoplex assemblies and their correlation to biological activity. Anionic lipid-based ternary siRNA complexes composed of anionic liposomes (DOPG/DOPE), calcium ions and siRNA, have recently been shown to be safe and efficient in a breast cancer cell culture model. In the present work, the effects of various formulation parameters such as liposome composition (DOPG/DOPE ratio) and anionic lipid/Ca2+/siRNA molar charge ratio, on the physicochemical attributes (particle size, surface charge, siRNA loading efficiency and serum stability) of these ternary anionic lipoplexes were evaluated. Particle size, siRNA loading efficiency and serum stability correlated with the in vitro silencing efficiency of these lipoplexes. For example, large lipoplex particles (5/2.5/1 anionic lipid/Ca2+/siRNA molar charge ratio) showed less efficient silencing while absolute serum stability and high siRNA loading (1.3/2.5/1 anionic lipid/Ca2+/siRNA molar charge ratio), exhibited maximum silencing in breast cancer cells. The physicochemical properties also indicated that the siRNA exists in the complexed and/or encapsulated form within the lipoplexes, depending on the anionic lipid/siRNA charge ratio. Based on these studies a model representing lipid-siRNA association within the anionic lipoplexes prepared under various formulation conditions is proposed. Physicochemical attributes can be utilized to estimate in vitro activity of lipid-siRNA complexes and understand their morphology.

Prodrug/Enzyme based acceleration of absorption of hydrophobic drugs: an in vitro study.

Poor water solubility of APIs is a key challenge in drug discovery and development as it results in low drug bioavailability upon local or systemic administration. The prodrug approach is commonly utilized to enhance solubility of hydrophobic drugs. However, for accelerated drug absorption, supersaturated solutions need to be employed. In this work, a novel prodrug/enzyme based system was developed wherein prodrug and enzyme are coadministered at the point of absorption (e.g., nasal cavity) to form in situ supersaturated drug solutions for enhanced bioavailability. A combination of fosphenytoin/alkaline phosphatase was used as a model system. Prodrug conversion kinetics were evaluated with various prodrug/enzyme ratios at pH 7.4 and 32 °C. Phenytoin permeation rates were determined at various degrees of supersaturation (S = 0.8-6.1), across confluent Madin Darby canine kidney II-wild type monolayers (a nasal epithelium model), with prodrug and enzyme spiked into the apical chamber. Membrane intactness was confirmed by measuring transepithelial electrical resistance and inulin permeability. Fosphenytoin and phenytoin concentrations were analyzed using HPLC. Results indicated that a supersaturated solution could be formed using such prodrug/enzyme systems. Drug absorption increased proportionately with increasing degrees of supersaturation; this flux was 1.5-6 fold greater than that for the saturated phenytoin solution. The experimental data fitted reasonably well to a two compartment pharmacokinetic (PK) model with first order conversion of prodrug to drug. This prodrug/enzyme system markedly enhances drug transport across the model membrane. Applied in vivo, this strategy could be used to facilitate drug absorption through mucosal membranes when absorption is limited by solubility.

Cellular uptake mechanisms of novel anionic siRNA lipoplexes.

PURPOSE: To investigate cellular uptake pathways of novel anionic siRNA-lipoplexes as a function of formulation composition. METHODS: Anionic formulations with anionic lipid/Ca(2+)/siRNA ratio of 1.3/2.5/1 (AF1) and 1.3/0.3/1 (AF2) were utilized. Uptake mechanisms were investigated using uptake inhibition and co-localization approaches in breast cancer cells. Actin-mediated uptake was investigated using actin polymerization and rearrangement assays. Silencing efficiency and endosomal escaping capability of lipoplexes were evaluated. The cationic formulation Lipofectamine-2000 was used as a control. RESULTS: Anionic lipoplexes entered the breast cancer cells via endocytosis specifically via macropinocytosis or via both macropinocytosis and HSPG (heparin sulfate proteoglycans) pathways, depending on the Ca(2+)/siRNA ratio. Additionally, uptake of these lipoplexes was both microtubule and actin dependent. The control cationic lipid-siRNA complexes (Lipofectamine-2000) were internalized via both endocytic (phagocytosis, HSPG) and non-endocytic (membrane fusion) pathways. Their uptake was microtubule independent but actin dependent. Silencing efficiency of the AF2 formulation was negligible mainly due to poor endosomal release (rate-limiting step). CONCLUSIONS: Formulation composition significantly influences the internalization mechanism of anionic lipoplexes. Uptake mechanism together with formulation bioactivity helped in identification of the rate-limiting steps to efficient siRNA delivery. Such studies are extremely useful for formulation optimization to achieve enhanced intracellular delivery of nucleic acids.

A comparative study of Sumudu HPM and Elzaki HPM for coupled Burgers' equation.

The two hybrid algorithms Sumudu HPM and Elzaki HPM are used in the current study to tackle coupled Burgers' equations and produce accurate results. To demonstrate the validity of the given approaches, three instances are used. Applying Sumudu HPM and Elzaki HPM yields the same approximate and exact answers in all of the examples taken into consideration, which is proved with the help of the accompanying figures. It attests to the entire acceptance and accuracy of the solutions produced by these methods. The proposed regimes also have error and convergence analyses available. The current analytical regimes offer a more effective method of handling partial differential equations than the intricate numerical systems. It is also asserted that exact and approximation solutions are compatible. Also announced is the planned regime's numerical convergence.

Covalent attachment of proteins to functionalized polypyrrole-coated metallic surfaces for improved biocompatibility.

Stainless steel has been widely used as an implant material for various biomedical applications, but its biocompatibility is still a major issue. Though polymer coating is one of the solutions, biomolecule-attached polymer coating is a better alternative. In this paper, we have synthesized a biomolecule (bovine serum albumin, BSA)-derivatized polymer coating on a stainless steel (316L) surface and evaluated it for biocompatibility. The monomer used for coating was obtained by hydrolyzing 1-(2-cyanoethyl) pyrrole to 1-(2-carboxyethyl) pyrrole followed by activation with N-hydroxysuccinimide to N-succinimidyl ester pyrrole. This monomer was electrocoated onto steel plate to provide a smooth and adherent coating of polypyrrole-N-succinimidyl ester (PPyNSE) which was characterized in terms of surface morphology and chemical composition by scanning electron microscopy and infrared spectroscopy, respectively. Further, BSA was covalently attached to PPyNSE to obtain a biomolecule-derivatized polymer coating. This coating was evaluated for biocompatibility in terms of thrombus formation, platelet adhesion and hemolysis, and was found to be more biocompatible on these parameters than the bare metal and polypyrrole-coated surfaces. Stability studies on these coated plates were also performed.

Liposomal Drug Product Development and Quality: Current US Experience and Perspective.

Research in the area of liposomes has grown substantially in the past few decades. Liposomes are lipid bilayer structures that can incorporate drug substances to modify the drug's pharmacokinetic profile thereby improving drug delivery. The agency has received over 400 liposomal drug product submissions (excluding combination therapies), and there are currently eight approved liposomal drug products on the US market. In order to identify the pain points in development and manufacturing of liposomal drug products, a retrospective analysis was performed from a quality perspective on submissions for new and generic liposomal drug products. General analysis on liposomal drug product submissions was also performed. Results indicated that 96% of the submissions were Investigational New Drug (IND) applications, 3% were New Drug Applications (NDAs), and the remaining 1% was Abbreviated New Drug Applications (ANDAs). Doxorubicin hydrochloride was the most commonly used drug substance incorporated into the liposomes (31%). The majority of the liposomal products were administered via intravenous route (84%) with cancer (various types) being the most common indication (63%). From a quality perspective, major challenges during the development of liposomal drug products included identification and (appropriate) characterization of critical quality attributes of liposomal drug products and suitable control strategies during product development. By focusing on these areas, a faster and more efficient development of liposomal drug products may be achieved. Additionally, in this way, the drug review process for such products can be streamlined.

The evolving landscape of drug products containing nanomaterials in the United States.

The Center for Drug Evaluation and Research (CDER) within the US Food and Drug Administration (FDA) is tracking the use of nanotechnology in drug products by building and interrogating a technical profile of products containing nanomaterials submitted to CDER. In this Analysis, data from more than 350 products show an increase in the submissions of drug products containing nanomaterials over the last two decades. Of these, 65% are investigational new drugs, 17% are new drug applications and 18% are abbreviated new drug applications, with the largest class of products being liposomal formulations intended for cancer treatments. Approximately 80% of products have average particle sizes of 300 nm or lower. This analysis identifies several trends in the development of drug products containing nanomaterials, including the relative rate of approvals of these products, and provides a comprehensive overview on the landscape of nanotechnology application in medicine.

A review of intranasal formulations for the treatment of seizure emergencies.

Epileptic seizure emergencies are life-threatening conditions, which in their most severe form, status epilepticus, have a high mortality rate if not quickly terminated. Treatment requires rapid delivery of anti-epileptics such as benzodiazepines to the brain. The nasal route is attractive due to its non-invasiveness, potential for direct nose to brain delivery, high vascularity, relatively large absorptive surface area, and avoidance of intestinal/liver metabolism. However, the limited volume of the nasal cavity and poor water solubility of anti-epileptics restrict absorption, leading to insufficient therapeutic brain levels. This review covers various formulation approaches adopted to improve nasal delivery of drugs, especially benzodiazepines, used to treat seizure emergencies. Other general topics such as nasal anatomy, challenges to nasal delivery, and drug/formulation considerations for nose to brain delivery are also discussed.

Chirally Pure Prodrugs and Their Converting Enzymes Lead to High Supersaturation and Rapid Transcellular Permeation of Benzodiazepines.

Water-soluble prodrugs can be rapidly converted by enzymes to hydrophobic drugs, whose aqueous thermodynamic solubilities are low, but are maintained in aqueous solution at supersaturated concentrations due to slow precipitation kinetics. Recently, we investigated avizafone (AVF) in combination with Aspergillus oryzae protease as a prodrug/enzyme system intended to produce supersaturated diazepam (DZP). Several fold enhancement of permeation of supersaturated DZP across Madin-Darby canine kidney II-wild type (MDCKII-wt) monolayers was observed, compared to saturated DZP solutions. However, prodrug conversion was incomplete, putatively due to partial racemization of AVF and stereoselectivity of A oryzae protease. Here we report synthesis of chirally pure AVF, and demonstrate complete conversion to supersaturated DZP followed by complete DZP permeation at enhanced rates across MDCKII-wt cell monolayers. We also synthesized, for the first time, a chirally pure prodrug of midazolam (MDZ-pro) and carried out the same sequence of studies. A oryzae protease was identified as a benign and efficient activating enzyme for MDZ-pro. The MDZ-pro/A oryzae protease system showed greater than 25-fold increase in absorption rate of MDZ across MDCKII-wt monolayers, compared to saturated MDZ. Such chirally pure prodrug/enzyme systems are promising candidates for efficient intranasal delivery of benzodiazepine drugs used in the treatment of seizure emergencies.

Rapid delivery of diazepam from supersaturated solutions prepared using prodrug/enzyme mixtures: toward intranasal treatment of seizure emergencies.

Current treatments for seizure emergencies, such as status epilepticus, include intravenous or rectal administration of benzodiazepines. While intranasal delivery of these drugs is desirable, the small volume of the nasal cavity and low drug solubility pose significant difficulties. Here, we prepared supersaturated diazepam solutions under physiological conditions and without precipitation, using a prodrug/enzyme system. Avizafone, a peptide prodrug of diazepam, was delivered with--Aspergillus oryzae (A.O.) protease, an enzyme identified from a pool of hydrolytic enzymes in assay buffer, pH 7.4 at 32°C. This enzyme converted avizafone to diazepam at supersaturated concentrations. In vitro permeability studies were performed at various prodrug/enzyme ratios using Madin-Darby canine kidney II-wild type (MDCKII-wt) monolayers, a representative model of the nasal epithelium. Monolayer integrity was examined using TEER measurement and the lucifer yellow permeability assay. Prodrug/drug concentrations were measured using HPLC. Enzyme kinetics with avizafone-protease mixtures revealed K(M) = 1,501 ± 232 µM and V(max) = 1,369 ± 94 µM/s. Prodrug-protease mixtures, when co-delivered apically onto MDCKII-wt monolayers, showed 2-17.6-fold greater diazepam flux (S = 1.3-15.3) compared to near-saturated diazepam (S = 0.7). Data for prodrug conversion upstream (apical side) and drug permeability downstream (basolateral side) fitted reasonably well to a previously developed in vitro two compartment pharmacokinetic model. Avizafone-protease mixtures resulted in supersaturated diazepam in less than 5 min, with the rate and extent of supersaturation determined by the prodrug/enzyme ratio. Together, these results suggest that an intranasal avizafone-protease system may provide a rapid and alternative means of diazepam delivery.

Efficient and safe delivery of siRNA using anionic lipids: Formulation optimization studies.

Novel formulations based on physiologically occurring anionic lipids have been designed to achieve safe and efficient siRNA delivery. Anionic liposomes (DOPG/DOPE) were complexed with siRNA using calcium ion bridges to prepare anionic lipoplexes. Various formulation parameters (liposome composition, lipid and calcium concentration) were evaluated and optimized to achieve efficient silencing and high cell viability in breast cancer cells. The optimal anionic lipoplexes composed of 1µg/mL lipid (40:60 (DOPG/DOPE m/m)), 2.4mM calcium and 10nM siRNA, showed maximum silencing (∼70% knockdown) without being cytotoxic. These lipoplexes also showed stability and high efficiency in the presence of serum. Additionally, optimal anionic lipoplexes showed efficient intracellular uptake and endosomal escape. Characterization studies indicated the optimal anionic formulations were 324.2±19.6nm with a surface charge of (-22.9±0.1)mV and 98.5±1.4% encapsulation efficiency. Control cationic lipoplexes (Lipofectamine 2000) showed silencing comparable to the anionic lipoplexes but were highly cytotoxic as indicated by IC50 values (cationic - 22.9µg/mL, compared to anionic - greater than 10(7)µg/mL). Calcium-siRNA complexes (without liposomes) showed low efficiency (∼50% silencing), and highly variable results. The optimized anionic formulations may offer a safer alternative to conventional cationic based systems for efficient in vitro as well as in vivo delivery of therapeutic siRNAs.

Physicochemical characterization techniques for lipid based delivery systems for siRNA.

siRNA based therapeutics is an emerging class of molecules with a high potential for fulfilling the promise of gene medicine. The high selectivity of siRNAs for their targets and subsequent gene ablation has been effectively demonstrated in a wide range of pre-clinical models. siRNA delivery in vivo has been most successfully achieved using lipid-based drug delivery systems. These lipid based formulations are designed to entrap siRNA molecules, ensure stability in in vitro and in vivo milieu, facilitate uptake, enhance cellular targeting, and facilitate delivery in the desired intracellular compartment. As more siRNA-based therapeutics enters the clinic with the associated regulatory scrutiny, there is a clear need to develop well-characterized systems that ensure consistent quality and thus reliable performance. Early clinical trials can be conducted using formulations with limited short-term stability manufactured on a small scale. However, a thorough understanding of the factors that influence the structure and stability of these particulate formulations is required to prevent any issues with optimization of large-scale industrial manufacturing, scale-up, and long-term shelf-life required to support large clinical trials and eventual market use. As newer targets for siRNA are identified and novel lipids are synthesized to optimize their in vivo efficiency, concomitant development of bio-physical methodologies that can improve understanding of the assembly and stability of these complex systems is critical. Along with bio-physical characterization, these assays are also required to reliably design, screen, develop and optimize formulations. Physicochemical characterization thus forms the basis of developing an effective analytical control strategy for siRNA delivery systems. In this review, analytical techniques used to characterize lipid-based siRNA delivery systems are discussed in detail. The importance of these physicochemical characterization techniques and analytical assays is explained. Case studies illustrating their use in siRNA formulation development and optimization are presented.