Tools to evaluate nanodiamond-mediated delivery of tiopronin for cataract prevention

”There is a growing demand for non-surgical means of cataract treatment. This dissertation presents three bodies of work that reflect the early-stage development of eye drop formulations aimed at delaying cataract progression. These formulations consist of the antioxidant 2-mercaptopropionylglycine (MPG) loaded onto nanodiamond particles. Cataractogenesis is linked to oxidative damage to lens proteins. To investigate the potential of MPG for protection against oxidative damage, A549 cells were incubated in 0.6 mM tert-butylhydroperoxide (tBHP). Cells exposed to tBHP without MPG exhibited elevated levels of reactive oxygen species, which led to the depletion of the vital antioxidant glutathione and, ultimately, apoptosis. Co-administration of 5 mM MPG protected cells from tBHP-induced damage, resulting in maintenance of cell viability. To monitor the uptake and fate of MPG in the eye, a rapid, high sensitivity HPLC method was developed for the analysis of MPG and its metabolite, 2-mercaptopropionic acid, in ocular tissues. Method validation experiments demonstrated the reliability of this method for quantifying MPG uptake and evaluating drug delivery strategies. Achieving effective drug concentrations in the lens poses a major challenge. Nanodiamond is biocompatible, and its surface chemistry can be tailored to specific applications. Thus, it is emerging as a candidate of interest for drug delivery. Nanodiamond surfaces were functionalized with carboxyl (ND-COOH), hydroxyl (ND- OH), and amino (ND-NH2) groups to investigate the effect of ND surface chemistry on adsorption and release of MPG. The ND-NH2 exhibited the highest adsorption capacity, but the ND-OH was the most effective for sustained release”--Abstract, page iv

THE FEASIBILITY AND ACCEPTABILITY OF BEHAVIORAL ACTIVATION IN AN ADOLESCENT CLINICAL SETTING

Purpose: During the COVID-19 pandemic, estimates of adolescent depression prevalence have significantly increased. Although 80% of youth with mental health problems do not access mental health (MH) services, most interact regularly with pediatricians, suggesting primary care is a promising setting for youth MH service delivery. Behavioral activation (BA) is a straightforward intervention focusing on increasing engagement in meaningful activities and has been shown to be effective in adolescent populations. This study examines the feasibility and acceptability of implementing BA delivered by nurses to adolescents with mild to moderate depressive symptoms in an ambulatory setting. Methods: We examined the feasibility and acceptability of BA to a sociodemographic-diverse adolescent population. To examine feasibility, we retrospectively analyzed the percentage of patients presenting with mild to moderate depressive symptoms on the patient health questionnaire-9 during a random four week period. We then conducted qualitative interviews with four parent-adolescent dyads, and two additional adolescents. A semi-structured interview guide was iteratively developed to assess perceived acceptability of BA and barriers and facilitators to engaging in the intervention. Interviews were recorded, transcribed, and coded by authors JDT and JF. A thematic analysis was completed to identify whether the program as designed was acceptable to the targeted population. Results: A records review found that out of 122 unique patients presenting for any clinic visit during the four week period, 44 (35%) met criteria for mild to moderate depressive symptoms. Of those who met criteria, youth were 17.45 years old (range = 12-22) with 61% identified as female. Results of the qualitative interviews suggest that participants perceived BA to be appropriate; themes emerged suggesting that both parents and youth noted nurse-delivered BA was similar to other types of therapies they had engaged with in other settings and that participants experienced nurses’ as able to protect confidentiality and someone they could be “vulnerable” with. Participants were mixed in how feasible they felt regular visits to the pediatrician’s office was, with some noting that transportation and time were major barriers for engaging in treatment. All participants noted that telehealth options (including meeting with a nurse provider over the phone or through web conferencing) were appropriate and would facilitate treatment access. Participants also noted that time, transportation and cost were significant barriers to engaging in valued activities (a central mechanism of BA), and several parents and youth noted that these barriers were exacerbated during the pandemic. Conclusion: Our data highlights the implementation potential for delivering BA by nurses in an adolescent ambulatory setting. Qualitative themes suggest this approach (especially delivered via telehealth) is feasible and acceptable for youth with mild to moderate depressive symptoms

The Neuroprotective Effects of the Novel Thiol Antioxidant, N-acetylcysteineamide (NACA), on SH-SY5Y Neuroblastoma Cells

This study investigated the antioxidant and neuroprotective efficacy of N-acetylcysteineamide (NACA) in comparison to the well-known antioxidant, N-acetylcysteine (NAC) on SH-SY5Y neuroblastoma cells which are prominent in vitro models for studies of neurodegenerative diseases such as Parkinson’s and Alzheimer’s. Cell viability testing determined that exposure to 750 μM doses of NAC and NACA for 4 hours were optimum. Oxidative stress was induced using tert-butyl hydroperoxide (tBHP), a membrane-permeant oxidant compound that enters the cells and generates tert-butoxy radicals resulting in lipid peroxidation, depletion of cellular antioxidant defenses, and cell death. The cells were divided into six groups: control, tBHP alone, pre-treatment with NAC, pre-treatment with NACA, and controls for NAC and NACA. The levels of ROS and the extent of cytotoxicity were measured via fluorescence spectroscopy. Both NAC and NACA reduced the cytotoxicity and the levels of ROS from exposure to tBHP, but NACA out-performed NAC by a noteworthy margin

Simultaneous Determination of Tiopronin and its Primary Metabolite in Plasma and Ocular Tissues by HPLC

Tiopronin, formally 2-mercaptopropionylglycine (MPG), is currently prescribed to treat cystinuria and rheumatoid arthritis, and its antioxidant properties have led to its investigation as a treatment for cataracts, a condition in which oxidative stress is strongly implicated. To study its accumulation in the eye, a reliable, isocratic HPLC method was developed for the determination of MPG and its primary metabolite 2-mercaptopropionic acid (MPA) in plasma and relevant ocular tissues. This method utilizes pre-column derivatization and fluorescence detection. The 3.5 min separation enables high-throughput analysis, and validation experiments demonstrated that this method is suitable for evaluating ocular accumulation of MPG and MPA at concentrations as low as 66 and 33 nm, respectively. Excellent linearity was achieved over the working concentration range with R2 \u3e 0.997. Extraction recovery was reproducible within each matrix and exceeded 97%. Accuracy was within 13.3% relative error, and intra- and inter-day precisions were within 6% CV and 7% CV, respectively. Sample stability was demonstrated under various storage conditions, and the use of an internal standard conferred exceptional ruggedness. This method has been successfully applied for the determination of MPG and MPA in plasma, cornea, lens and retina following intraperitoneal administration of the drug in Wistar rats

Protective Effects of Tiopronin on Oxidatively Challenged Human Lung Carcinoma Cells (A549)

Tiopronin (MPG) is a thiol antioxidant drug that has been explored as a treatment for various oxidative stress-related disorders. However, many of its antioxidant capabilities remain untested in well-validated cell models. To more thoroughly understand the action of this promising pharmaceutical compound against acute oxidative challenge, A549 human lung carcinoma cells were exposed to tert-butyl hydroperoxide (tBHP) and treated with MPG. Analyses of cell viability, intracellular glutathione (GSH) levels, and the prevalence of reactive oxygen species (ROS) and mitochondrial superoxide were used to examine the effects of MPG on tBHP-challenged cells. MPG treatment suppressed intracellular ROS and mitochondrial superoxide and prevented tBHP-induced GSH depletion and apoptosis. These results indicate that MPG is effective at preserving redox homeostasis against acute oxidative insult in A549 cells if present at sufficient concentrations during exposure to oxidants such as tBHP. The effects of treatment gleaned from this study can inform experimental design for future in vivo work on the therapeutic potential of MPG

Medicinal Thiols: Current Status and New Perspectives

The thiol (-SH) functional group is found in a number of drug compounds and confers a unique combination of useful properties. Thiol-containing drugs can reduce radicals and other toxic electrophiles, restore cellular thiol pools, and form stable complexes with heavy metals such as lead, arsenic, and copper. Thus, thiols can treat a variety of conditions by serving as radical scavengers, GSH prodrugs, or metal chelators. Many of the compounds discussed here have been in use for decades, yet continued exploration of their properties has yielded new understanding in recent years, which can be used to optimize their clinical application and provide insights into the development of new treatments. The purpose of this narrative review is to highlight the biochemistry of currently used thiol drugs within the context of developments reported in the last five years. More specifically, this review focuses on thiol drugs that represent the standard of care for their associated conditions, including N-acetylcysteine, 2,3-meso-dimercaptosuccinic acid, British anti-Lewisite, D-penicillamine, amifostine, and others. Reports of novel dosing regimens, delivery strategies, and clinical applications for these compounds were examined with an eye toward emerging approaches to address a wide range of medical conditions in the future

Effect of Nanodiamond Surface Chemistry on Adsorption and Release of Tiopronin

Tiopronin is an FDA-approved thiol drug currently used to treat cystinuria and rheumatoid arthritis. However, due to its antioxidant properties, it may be beneficial in a variety of other conditions. One primary obstacle to its wider application is its limited bioavailability, which necessitates administration of high systemic doses to achieve localized therapeutic effects. Incorporation of a drug delivery vehicle can solve this dilemma by providing a means of controlled, targeted release. Functionalized nanodiamond is a promising theranostic platform that has demonstrated great potential for biomedical applications, including drug delivery. Design of nanodiamond theranostic platforms requires comprehensive understanding of drug-platform interactions, and the necessary physical chemical investigations have only been realized for a limited number of compounds. Towards the long-term goal of developing a nanodiamond-tiopronin treatment paradigm, this study aims to shed light on the effects of nanodiamond surface chemistry on adsorption and release of tiopronin. Specifically, adsorption isotherms were measured and fit to Langmuir and Freundlich models for carboxylated, hydroxylated, and aminated nanodiamonds, and release was monitored in solutions at pH 4.0, 5.8, 7.3, and 8.1. Our results indicate that aminated nanodiamonds exhibit the highest loading capacity while hydroxylated nanodiamonds are the most effective for sustained release. Therefore, a high degree of flexibility may be afforded by the use of nanodiamonds with different surface chemistries optimized for specific applications

Genotype, Age, Genetic Background, and Sex Influence Epha2-Related Cataract Development in Mice

PURPOSE. Age-related cataract is the leading cause of blindness worldwide. Variants in the EPHA2 gene increase the disease risk, and its knockout in mice causes cataract. We investigated whether age, sex, and genetic background, risk factors for age-related cataract, and Epha2 genotype influence Epha2-related cataract development in mice. METHODS. Cataract development was monitored in Epha2+/+, Epha2+/-, and Epha2-/- mice (Epha2Gt(KST085)Byg) on C57BL/6J and FVB:C57BL/6J (50:50) backgrounds. Cellular architecture of lenses, endoplasmic reticulum (ER) stress, and redox state were determined using histological, molecular, and analytical techniques. RESULTS. Epha2-/- and Epha2+/- mice on C57BL/6J background developed severe cortical cataracts by 18 and 38 weeks of age, respectively, compared to development of similar cataract significantly later in Epha2-/- mice and no cataract in Epha2+/- mice in this strain on FVB background, which was previously reported. On FVB:C57BL/6J background, Epha2-/- mice developed severe cortical cataract by 38 weeks and Epha2+/- mice exhibited mild cortical cataract up to 64 weeks of age. Progression of cataract in Epha2-/- and Epha2+/- female mice on C57BL/6J and mixed background, respectively, was slower than in matched male mice. N-cadherin and β-catenin immunolabeling showed disorganized lens fiber cells and disruption of lens architecture in Epha2-/- and Epha2+/− lenses, coinciding with development of severe cataracts. EPHA2 immunolabeling showed intracellular accumulation of the mutant EPHA2-β-galactosidase fusion protein that induced a cytoprotective ER stress response and in Epha2+/- lenses was also accompanied by glutathione redox imbalance. CONCLUSIONS. Both, Epha2-/- and Epha2+/- mice develop age-related cortical cataract; age as a function of Epha2 genotype, sex, and genetic background influence Epha2-related cataractogenesis in mice