Ru(II) Phenanthroline-Based Oligothienyl Complexes as Phototherapy Agents.

Ru(II) polypyridyl complexes have gained widespread attention as photosensitizers for photodynamic therapy (PDT). Herein, we systematically investigate a series of the type [Ru(phen)2(IP-nT)]2+, featuring 1,10-phenanthroline (phen) coligands and imidazo[4,5-f][1,10]phenanthroline ligands tethered to n = 0-4 thiophene rings (IP-nT). The complexes were characterized and investigated for their electrochemical, spectroscopic, and (photo)biological properties. The electrochemical oxidation of the nT unit shifted by -350 mV as n = 1 → 4 (+920 mV for Ru-1T, +570 mV for Ru-4T); nT reductions were observed in complexes Ru-3T (-2530 mV) and Ru-4T (-2300 mV). Singlet oxygen quantum yields ranged from 0.53 to 0.88, with Ru-3T and Ru-4T being equally efficient (∼0.88). Time-resolved absorption spectra of Ru-0T-1T were dominated by metal-to-ligand charge-transfer (3MLCT) states (τTA = 0.40-0.85 µs), but long-lived intraligand charge-transfer (3ILCT) states were observed in Ru-2T-4T (τTA = 25-148 µs). The 3ILCT energies of Ru-3T and Ru-4T were computed to be 1.6 and 1.4 eV, respectively. The phototherapeutic efficacy against melanoma cells (SK-MEL-28) under broad-band visible light (400-700 nm) increases as n = 0 → 4: Ru-0T was inactive up to 300 µM, Ru-1T-2T were moderately active (EC50 ∼ 600 nM, PI = 200), and Ru-3T (EC50 = 57 nM, PI > 1100) and Ru-4T (EC50 = 740 pM, PI = 114,000) were the most phototoxic. The activity diminishes with longer wavelengths of light and is completely suppressed for all complexes except Ru-3T and Ru-4T in hypoxia. Ru-4T is the more potent and robust PS in 1% O2 over seven biological replicates (avg EC50 = 1.3 µM, avg PI = 985). Ru-3T exhibited hypoxic activity in five of seven replicates, underscoring the need for biological replicates in compound evaluation. Singlet oxygen sensitization is likely responsible for phototoxic effects of the compounds in normoxia, but the presence of redox-active excited states may facilitate additional photoactive pathways for complexes with three or more thienyl groups. The 3ILCT state with its extended lifetime (30-40× longer than the 3MLCT state for Ru-3T and Ru-4T) implicates its predominant role in photocytotoxicity.

Environmental Health and Safety Offers a Biosafety Risk Assessment for a Theoretical Model of a Gene Therapy Process Transfer from Research and Development to Large-Scale Manufacturing.

Introduction: This article provides a strategy by which a manufacturing process with a Biosafety Level 2 (BL2) designation can be downgraded to Biosafety Level 1 (BL1). The principles of the downgrading process are based on the robust contamination controls in clinical and commercial manufacturing, which typically are not part of Research and Development processes. These strict requirements along with the application of current Good Manufacturing Practice (cGMP) principles provide a framework by which processes can be suitably managed and controlled to mitigate biohazard risk, specifically for cell lines that may be contaminated with human pathogenic viral agents. Purpose: We demonstrate how a risk assessment guide was used to define the risk profile of a theoretical process with a human cell line intended for clinical/commercial application. Based on the risk assessment, key BL2 elements were identified as suitable for downgrading, including facility containment controls, emergency spill response plans, and storage and shipping requirements. For various reasons, some aspects of the systems were deemed unsuitable for downgrading due to the severity of the control risk and, therefore, remained at BL2. Summary and Conclusions: We have used an established risk assessment guide to show how cGMP compliments and augments biosafety containment. We provide justification for downgrading from BL2 to BL1 for clinical and commercial cell and gene therapy manufacturing with human cell lines.

An Industry Proposal for a Cell and Gene Therapy Safety Data Sheet.

Introduction: A safety data sheet (SDS) is an established hazard communication tool for chemicals, for which no comparable document exists in the biotherapeutics industry. As the cell and gene therapy (CGT) field expands, industry leaders have identified a growing need to address this gap in communication of the unique occupational health and safety risks posed by CGT materials and products. Methods: Following the sections of a traditional chemical SDS, information was modified by industry subject matter experts, relevant to CGT biological materials. This guide was developed based on assumptions of a maximum biosafety level 2, and any chemical components present in the material were excluded from the hazard classification. Results: The guide contains necessary information to conduct a workplace risk assessment and communicate the unique workplace hazards posed by potential exposures to the material. The target audience is intended to be entities handling and producing these materials, plus collaborators, contractors, or operations sites receiving and handling the CGT material. An example of a CGT SDS is provided in Table 1. Discussion: The CGT SDS provides industry with a best practice to address an existing gap in hazard communication for CGT. We expect that, as the field evolves, so may the contents. The CGT SDS can be used as a reference for other biological modalities in the field. Conclusions: This initial CGT SDS communicates workplace hazards and assesses the unique risks posed by these biological materials and can assist in creating exposure control plans specific to the workplace hazards.

Δ9-Tetrahydrocannabinol Differentially Alters Cannabidiol Efficacy in Recovery of Phonology and Syntax Following Damage to a Songbird Cortical-Like Brain Region.

Introduction: There are few vocal learning animals that are suitable for laboratory study, and so songbirds have unique utility for evaluating drug effects on behavior learned during a critical period of development. We previously found that purified botanically-derived cannabidiol (CBD, ≥98%) mitigates effects of partial ablation of zebra finch HVC, a pre-vocal motor cortical region. Here we expand prior work to determine ability of the euphorigenic cannabis constituent, Δ9-tetrahydrocannabinol (THC) to modulate CBD efficacy. Evidence suggests relative abundance of phytocannabinoids within cannabis extracts is an important determinant of activity, with CBD:THC of particular significance. As CBD-enriched extracts have become increasingly available both by prescription and over the counter, differential efficacy associated with distinct phytocannabinoid combinations and relative CBD:THC amounts is of increasing concern. Methods and Results: To evaluate THC modulation of CBD efficacy in mitigating the effects of partial ablation of zebra finch HVC, we have tested 3 mg/kg of purified botanically derived CBD (≥98%) containing 0.02, 0.08, 1, 3 and 5% THC. Results demonstrate differential efficacy on phonology and syntax, consistent with complex, hormetic dose-responses. On phonology, CBD with the lowest THC content (3% CBD + 0.02% THC) improved recovery while that with the highest THC content (3% CBD+5% THC) slowed it. In terms of syntax, all THC concentrations improved recovery time with the higher 3 mg/kg+3% THC being distinctly effective in returning behavior to pre-injury levels, and the highest 3 mg/kg CBD+5% THC for reducing the acute magnitude of syntax disruption. Differential phonology and syntax effects likely involve distinct neural circuits that control vocal learning and production. Understanding these systems-level effects will inform mechanisms underlying both phytocannabinoid action, and learning-dependent vocal recovery. Conclusions: Overall, we have found that efficacy of purified botanically derived CBD (≥98%) to influence vocal recovery varies with THC content in complex ways. This adds to evidence of differential efficacy with phytocannabinoid combinations and ratios thereof and underscores the importance of careful control over cannabis preparations used therapeutically.

Intraligand Excited States Turn a Ruthenium Oligothiophene Complex into a Light-Triggered Ubertoxin with Anticancer Effects in Extreme Hypoxia.

Ru(II) complexes that undergo photosubstitution reactions from triplet metal-centered (3MC) excited states are of interest in photochemotherapy (PCT) due to their potential to produce cytotoxic effects in hypoxia. Dual-action systems that incorporate this stoichiometric mode to complement the oxygen-dependent photosensitization pathways that define photodynamic therapy (PDT) are poised to maintain antitumor activity regardless of the oxygenation status. Herein, we examine the way in which these two pathways influence photocytotoxicity in normoxia and in hypoxia using the [Ru(dmp)2(IP-nT)]2+ series (where dmp = 2,9-dimethyl-1,10-phenanthroline and IP-nT = imidazo[4,5-f][1,10]phenanthroline tethered to n = 0-4 thiophene rings) to switch the dominant excited state from the metal-based 3MC state in the case of Ru-phen-Ru-1T to the ligand-based 3ILCT state for Ru-3T and Ru-4T. Ru-phen-Ru-1T, having dominant 3MC states and the largest photosubstitution quantum yields, are inactive in both normoxia and hypoxia. Ru-3T and Ru-4T, with dominant 3IL/3ILCT states and long triplet lifetimes (τTA = 20-25 µs), have the poorest photosubstitution quantum yields, yet are extremely active. In the best instances, Ru-4T exhibit attomolar phototoxicity toward SKMEL28 cells in normoxia and picomolar in hypoxia, with phototherapeutic index values in normoxia of 105-1012 and 103-106 in hypoxia. While maximizing excited-state deactivation through photodissociative 3MC states did not result in bonafide dual-action PDT/PCT agents, the study has produced the most potent photosensitizer we know of to date. The extraordinary photosensitizing capacity of Ru-3T and Ru-4T may stem from a combination of very efficient 1O2 production and possibly complementary type I pathways via 3ILCT excited states.

Gestational Iron Supplementation Improves Fetal Outcomes in a Rat Model of Prenatal Alcohol Exposure.

Prenatal alcohol exposure causes neurodevelopmental disability and is associated with a functional iron deficiency in the fetus and neonate, even when the mother consumes an apparently iron-adequate diet. Here, we test whether gestational administration of the clinically relevant iron supplement Fer-In-Sol mitigates alcohol's adverse impacts upon the fetus. Pregnant Long-Evans rats consumed an iron-adequate diet and received 5 g/kg alcohol by gavage for 7 days in late pregnancy. Concurrently, some mothers received 6 mg/kg oral iron. We measured maternal and fetal weights, hematology, tissue iron content, and oxidative damage on gestational day 20.5. Alcohol caused fetal anemia, decreased fetal body and brain weight, increased hepatic iron content, and modestly elevated hepatic malondialdehyde (p's < 0.05). Supplemental iron normalized this brain weight reduction in alcohol-exposed males (p = 0.154) but not female littermates (p = 0.031). Iron also reversed the alcohol-induced fetal anemia and normalized both red blood cell numbers and hematocrit (p's < 0.05). Iron had minimal adverse effects on the mother or fetus. These data show that gestational iron supplementation improves select fetal outcomes in prenatal alcohol exposure (PAE) including brain weight and hematology, suggesting that this may be a clinically feasible approach to improve prenatal iron status and fetal outcomes in alcohol-exposed pregnancies.

Fine-Feature Modifications to Strained Ruthenium Complexes Radically Alter Their Hypoxic Anticancer Activity†.

In an earlier study of π-expansive ruthenium complexes for photodynamic and photochemo-therapies, it was shown that a pair of structural isomers differing only in the connection point of a naphthalene residue exhibited vastly different biological activity. These isomers are further explored in this paper through the activity of their functionalized derivatives. In normoxia, the inactive 2-NIP isomer (5) can be made as photocytotoxic as the active 1-NIP isomer (1) by functionalizing with methyl or methoxy groups, while methoxy variants of the 1-NIP isomer became inactive. In all cases, the singlet oxygen sensitization quantum yield was below 1%. Hypoxic photocytotoxicity was attenuated, with only three of the series showing any activity, notwithstanding the photodissociative ligands. The results here are consistent with the earlier findings in that seemingly minor structural modifications on the non-strained ligand can dramatically modulate the normoxic and hypoxic activity of these strained compounds and that these changes appear to exert a greater influence on photocytotoxicity than singlet oxygen sensitization or rates of photosubstitution in cell-free conditions would suggest.

Anticancer Agent with Inexplicable Potency in Extreme Hypoxia: Characterizing a Light-Triggered Ruthenium Ubertoxin.

Tumor hypoxia renders treatments ineffective that are directly (e.g., radiotherapy and photodynamic therapy) or indirectly (e.g., chemotherapy) dependent on tumor oxygenation. This study introduces a ruthenium compound as a light-responsive anticancer agent that is water-soluble, has minimal dark cytotoxicity, is active at concentrations as low as 170 pM in ∼18.5% O2 normoxia and near 10 nM in 1% O2 hypoxia, and exhibits phototherapeutic indices as large as >500,000 in normoxia and >5,800 in 1% O2 hypoxia using broadband visible and monochromatic blue light treatments. These are the largest values reported to date for any compound class. We highlight the response in four different cell lines to improve rigor and reproducibility in the identification of promising clinical candidates.