Developing drug molecules for therapy with carbon monoxide

Carbon monoxide (CO) is a gaseous signaling molecule produced in humans via the breakdown of heme in an O2-dependent reaction catalyzed by heme oxygenase enzymes. A long-lived species relative to other signaling molecules (e.g., NO, H2S), CO exerts its physiological effects via binding to low-valent transition metal centers in proteins and enzymes. Studies involving the administration of low doses of CO have shown its potential as a therapeutic agent to produce vasodilation, anti-inflammatory, antiapoptotic, and anticancer effects. In pursuit of developing tools to define better the role and therapeutic potential of CO, carbon monoxide releasing molecules (CORMs) were developed. To date, the vast majority of reported CORMs have been metal carbonyl complexes, with the most well-known being Ru2Cl4(CO)6 (CORM-2), Ru(CO)3Cl(glycinate) (CORM-3), and Mn(CO)4(S2CNMe(CH2CO2H)) (CORM-401). These complexes have been used to probe the effects of CO in hundreds of cell- and animal-based experiments. However, through recent investigations, it has become evident that these reagents exhibit complicated reactivity in biological environments. The interpretation of the effects produced by some of these complexes is obscured by protein binding, such that their formulation is not clear, and by CO leakage and potential redox activity. An additional weakness with regard to CORM-2 and CORM-3 is that these compounds cannot be tracked via fluorescence. Therefore, it is unclear where or when CO release occurs, which confounds the interpretation of experiments using these molecules. To address these weaknesses, our research team has pioneered the development of metal-free CORMs based on structurally tunable extended flavonol or quinolone scaffolds. In addition to being highly controlled, with CO release only occurring upon triggering with visible light (photoCORMs), these CO donors are trackable via fluorescence prior to CO release in cellular environments and can be targeted to specific cellular locations.In the Account, we highlight the development and application of a series of structurally related flavonol photoCORMs that (1) sense characteristics of cellular environments prior to CO release; (2) enable evaluation of the influence of cytosolic versus mitochondrial-localized CO release on cellular bioenergetics; (3) probe the cytotoxicity and anti-inflammatory effects of intracellular versus extracellular CO delivery; and (4) demonstrate that albumin delivery of a photoCORM enables potent anticancer and anti-inflammatory effects. A key advantage of using triggered CO release compounds in these investigations is the ability to examine the effects of the molecular delivery vehicle in the absence and presence of localized CO release, thus providing insight into the independent contributions of CO. Overall, flavonol-based CO delivery molecules offer opportunities for triggerable, trackable, and targetable CO delivery that are unprecedented in terms of previously reported CORMs and, thus, offer significant potential for applications in biological systems.

Event Abstract Back to Event Impact of carbon monoxide releasing molecule loading and electrospun scaffold composition on engineered vascular constructs Aatish V. Patel1, Eden K. Michael1, Kenyatta S. Washington1, Nawodi Abeyrathna2, Yi Liao2 and Chris Bashur1 1 Florida Institute of Technology, Department of Biomedical Engineering, United States 2 Florida Institute of Technology, Department of Chemistry, United States Introduction: The incorporation of carbon monoxide (CO) has the potential to improve patency of tissue engineered vascular grafts since CO can enhance endothelialization in appropriate doses[1]. Further, endogenous levels of CO have recently been shown to be essential in cell signaling. For therapeutic applications, controlling the CO dose is important both for a current clinical trial to treat pulmonary fibrosis and for potential vascular applications. Our goal is to provide controlled, local delivery of CO by incorporating visible light activated carbon monoxide releasing molecules (CORM) within electrospun scaffolds that can provide a hydrophobic barrier needed to allow CORM activation. The goals of this study were to determine CORM loading concentrations and scaffold compositions that enable extended CO releases profiles in cell culture conditions as well as vascular cell attachment and function. Materials and Methods: PCL and 75% PCL/collagen solutions were electrospun with incorporated CORMs (0, 2, and 4% w/w). Fiber diameters and collagen content were characterized with SEM and EDS, respectively. CORMs / Scaffolds were incubated and activated in cell culture conditions (DMEM, 10% FBS) using 470 nm light. CO release was confirmed directly with a myoglobin assay and indirectly with fluorescence at 350/450 nm[2]. For release profiles, the fluorescent intensity was determined for different activation times (up to 60 min) and incubation times (up to 3 days). Rat smooth muscle cells (SMC) and primary rat aortic endothelial cells were seeded on the scaffolds to test cell viability (DNA assay) and phenotype. Result and Discussion: Solution concentrations were adjusted to obtain similar average fiber diameters for PCL scaffolds with 0 and 2% CORM loading (1.3±0.4 and 1.5±0.1, respectively). Fluorescence intensity, indicating CO release, was greatest after 30 min of activation in cell culture conditions. CO release from PCL meshes occurred after up to 60 min of pre-incubation in cell culture conditions. The myoglobin assay confirmed a high CO yield for scaffolds (e.g., 92% for PCL with 2% w/w CORM). Neither the CORM itself nor the released CO were toxic SMCs at 2% loading (n=9, 1-way ANOVA, Tukey). However, the cellular response to activation was limited. Thus, we are including higher CORM loadings (i.e., 4%) as well as collagen to modulate the endothelial cell response. In preliminary work, we demonstrated that CORM in collagen/PCL meshes can still be activated after 60 min of pre-incubation. Conclusion: The results show that the release of CO from CORM-loaded electrospun meshes can be controlled by varying irradiation time. While the CORM was not toxic at 2% loading, the early results suggest that a higher dose of CO is required to impact vascular cell function. The addition of collagen within the electrospun mesh provided cell-binding sequences and still allowed for activation after similar pre-incubation times demonstrated with pure PCL. Support from The National Science Foundation

Carbon monoxide (CO) has recently gained recognition as a therapeutic agent. A controlled and localized delivery of CO can be achieved through carbon monoxide-releasing molecules (CORMs). In this work, we report a new manganese-based CORM, Py-CO, which incorporates a pyrene fluorophore. Pyrene integration not only enables green light (525 nm)-triggered CO release but also imparts aggregation-induced emission (AIE) behavior, making Py-CO a dual-functional theranostic platform. The aggregation behavior of Py-CO was validated by scanning electron microscopy (SEM), fluorescence lifetime measurements, dynamic light scattering (DLS), and computational studies. Kinetic studies revealed a CO release rate constant of 0.1579 min-1 (half-life: 3.94 min) in the nonaggregated state, which increased to 0.3279 min-1 (half-life: 2.11 min) upon aggregation, underscoring the enhanced CO release dynamics in the aggregated state. The therapeutic significance of Py-CO was assessed in vitro using the human neuroblastoma SH-SY5Y cell line. Cytotoxicity assays confirmed its ability to induce cell death, demonstrating potential anticancer efficacy. Moreover, cellular uptake studies and bioimaging experiments revealed the feasibility of simultaneous imaging and therapy. Overall, Py-CO represents a novel class of light-activated, AIE-active CORMs that uniquely combine efficient CO release with intrinsic fluorescence, thereby enabling simultaneous therapeutic delivery and real-time imaging for potential cancer theranostics.

Carbon monoxide (CO) is a bioactive signalling molecule that is produced endogenously via the breakdown of heme. Beneficial health effects associated with the delivery of CO gas have spurred the development of CO-releasing molecules (CORMs) that can be used to provide specific amounts of the gas. In addition to their potential use as therapeutics, CORMs are needed to provide insight into the biological targets of CO. In this regard, light-activated CO-releasing molecules (photoCORMs), are valuable for examining the effects of localized CO release. Herein we examine luminescent CORMs and photoCORMs that have been reported for tracking CO delivery in cells. A variety of motifs are available that exhibit differing luminescence properties and cover a wide range of wavelengths. Trackable CO donors have been successfully applied to targeting CO delivery to mitochondria, thus demonstrating the feasibility of using such molecules in detailed investigations of the biological roles of CO.

Controlled therapeutic delivery of CO from carbon monoxide-releasing molecules (CORMs).

As a by-product of heme catabolism by the heme oxygenase system, carbon monoxide (CO) has been neglected for many years, and only recently has its role as an essential signaling molecule been appreciated. In the past decade, the use of CO gas in pre-clinical experimental models of disease has produced some remarkable data indicating that its therapeutic delivery to mammals could alleviate inflammatory processes and cardiovascular disorders. However, the inherent toxic nature of CO cannot be ignored, knowing that inhalation of uncontrolled amounts of this gas can ultimately lead to serious systemic complications and neuronal derangements. From a clinical perspective, a key question is whether a safe and therapeutically effective threshold of CO can be reached locally in organs and tissues without delivering potentially toxic amounts through the lung. The advent of CO-releasing molecules (CO-RMs), a group of compounds capable of carrying and liberating controlled quantities of CO in cellular systems, appears a plausible alternative in the attempt to overcome the limitations of CO gas. Although in its infancy and far from being used for clinical applications, the CO-RMs technology is supported by very encouraging biological results and reflected by the chemical versatility of these compounds and their endless potential to be transformed into CO-based pharmaceuticals.

H2O2 responsive CO gas release platform with CA IX-Targeting for inhibiting tumor growth and metastasis

The cytoprotective properties of carbon monoxide (CO) gas and CO-releasing molecules (CORMs) are well established. Despite promising pre-clinical results, little attention has been paid to the toxicological profile of CORMs. The effects of CORM-2 and its CO-depleted molecule (iCORM-2) (20-400 μM) were compared in primary rat cardiomyocytes and two cell lines [human embryonic kidney (HeK) and Madine-Darby canine kidney Cells (MDCK)]. Cells were assessed for cell viability, apoptosis, necrosis, cytology, mitochondrial energetics, oxidative stress and cell cycle arrest markers. In separate experiments, the anti-apoptotic effects of CORM-2 and i-CORM-2 treatment were compared against CO gas treatment in HeK and MDCK lines. H(2)O(2) -induced cellular damage, measured by lactate dehydrogenase (LDH) release from primary cardiomyocytes, was reduced by 20 μM CORM-2; LDH activity, however, was directly inhibited by 400 μM CORM-2. Both CORM-2/iCORM-2 and CO gas decreased cisplatin-induced caspase-3 activity in MDCK and HeK cells suggesting an anti-apoptotic effect. Conversely, both CORM-2 and iCORM-2 induced significant cellular toxicity in the form of decreased cell viability, abnormal cell cytology, increased apoptosis and necrosis, cell cycle arrest and reduced mitochondrial enzyme activity. Comparison of these markers after CO gas administration to MDCK cells found significantly less cellular toxicity than in 100 μM CORM-2/iCORM-2-treated cells. CO gas did not have an adverse effect on mitochondrial energetics and integrity. Release of CO by low concentrations of intact CORM-2 molecules provides cytoprotective effects. These results show, however, that the ruthenium-based CORM by-product, iCORM-2, is cytotoxic and suggest that the accumulation of iCORM-2 would seriously limit any clinical application of the ruthenium-based CORMs.

Fabrication of CO-releasing surface to enhance the blood compatibility and endothelialization of TiO2 nanotubes on titanium surface

Carbon monoxide (CO) therapy has emerged as a hot topic under exploration in the field of gas therapy as it shows the promise of treating various diseases. Due to the gaseous property and the high affinity for human hemoglobin, the main challenges of administrating medicinal CO are the lack of target selectivity as well as the toxic profile at relatively high concentrations. Although abundant CO releasing molecules (CORMs) with the capacity to deliver CO in biological systems have been developed, several disadvantages related to CORMs, including random diffusion, poor solubility, potential toxicity, and lack of on-demand CO release in deep tissue, still confine their practical use. Recently, the advent of versatile nanomedicine has provided a promising chance for improving the properties of naked CORMs and simultaneously realizing the therapeutic applications of CO. This review presents a brief summarization of the emerging delivery strategies of CO based on nanomaterials for therapeutic application. First, an introduction covering the therapeutic roles of CO and several frequently used CORMs is provided. Then, recent advancements in the synthesis and application of versatile CO releasing nanomaterials are elaborated. Finally, the current challenges and future directions of these important delivery strategies are proposed.

Carbon monoxide (CO) is attracting increasing attention because of its role as a gasotransmitter with cytoprotective and homeostatic properties. Carbon monoxide releasing molecules (CORMs) are spatially and temporally controlled CO releasers that exhibit superior and more effective pharmaceutical traits than gaseous CO because of their chemistry and structure. Experimental and preclinical research in animal models has shown the therapeutic potential of inhaled CO and CORMs, and the biological effects of CO and CORMs have also been observed in preclinical trials via the genetic modulation of heme oxygenase-1 (HO-1). In this review, we describe the pharmaceutical use of CO and CORMs, methods of detecting CO release, and developments in CORM design and synthesis. Many valuable clinical CORMs formulated using macromolecules and nanomaterials are also described.

The pathophysiological roles of the endogenous signaling molecule, carbon monoxide (CO), have been extensively studied and validated in cell culture and animal models. Further, evidence supporting the therapeutic effects of CO in various human diseases has been mounting over the last two decades. Along this line, there has been intensive interest in developing various delivery forms including CO gas, CO in solution, metal–carbonyl complexes widely known as CO-releasing molecules (CO-RMs), and organic CO prodrugs. Among them, two ruthenium-based carbonyl complexes, CORM-2 and -3, occupy a very special place because they have been used in over 500 published studies. One of the mechanisms for CO's actions is known to be through attenuation of oxidative stress and regulation of production of reactive oxygen species (ROS). For this reason, it is important that CO delivery forms do not have intrinsic chemical redox properties. Herein, we describe our findings of catalase-like activities of CORM-2 and -3 in a CO-independent fashion, leading to the rapid degradation of hydrogen peroxide (H2O2) in PBS buffer (pH = 7.4) and in cell culture media. Further, we have found that CORM-2 and CORM-3 possess potent radical scavenging abilities. We have also studied two other widely used CO donors: CORM-401 and CORM-A1. Both showed chemical reactivity with ROS, but to a lesser degree than CORM-2 and -3. Because of the central role of ROS in some of the proposed mechanisms of actions for CO biology, the discovery of intrinsic chemical redox properties for these CO-RMs means that additional attention in designing proper controls is needed in future biological experiments using these CO-RMs for their CO-donating functions. Further, much more work is needed to understand the true implications of the chemical reactivity of these CO-RMs in cell-culture and animal-model studies of CO biology.

Carbon monoxide (CO) gas therapy has recently emerged as a promising strategy for cancer treatment. However, the uncontrolled release of CO and challenges in accumulating it at tumor sites have limited its therapeutic efficacy. In this work, a stepwise targeted delivery and a controlled release strategy for CO gas is proposed. By encapsulating a mitochondria-targeting CO prodrug (TPP-CORM) in biodegradable hybrid hollow mesoporous silica nanoparticles and coating tumor-targeting ligands on the surface of the mesoporous silica via host-guest interactions, targeted delivery of CO gas from tumor tissue to tumor cells and ultimately to mitochondria is achieved. After safely and efficiently delivering CO to the mitochondria of tumor cells, CO induces mitochondrial damage, leading to metabolic disruption, a reduction in adenosine triphosphate (ATP) levels, mitochondrial membrane depolarization (ΔΨ reduction), and subsequently, the induction of apoptosis in tumor cells. This stepwise targeted CO delivery strategy provides a novel approach for targeted tumor therapy using CO gas.

Antiproliferative effects of carbon monoxide on pancreatic cancer

Inhaled carbon monoxide (CO) gas has therapeutic potential for patients with acute respiratory distress syndrome if a safe, evidence-based dosing strategy and a ventilator-compatible CO delivery system can be developed. In this study, we used a clinically relevant baboon model of Streptococcus pneumoniae pneumonia to 1) test a novel, ventilator-compatible CO delivery system; 2) establish a safe and effective CO dosing regimen; and 3) investigate the local and systemic effects of CO therapy on inflammation and acute lung injury (ALI). Animals were inoculated with S. pneumoniae (10(8)-10(9) CFU) (n = 14) or saline vehicle (n = 5); in a subset with pneumonia (n = 5), we administered low-dose, inhaled CO gas (100-300 ppm × 60-90 min) at 0, 6, 24, and/or 48 h postinoculation and serially measured blood carboxyhemoglobin (COHb) levels. We found that CO inhalation at 200 ppm for 60 min is well tolerated and achieves a COHb of 6-8% with ambient CO levels ≤ 1 ppm. The COHb level measured at 20 min predicted the 60-min COHb level by the Coburn-Forster-Kane equation with high accuracy. Animals given inhaled CO + antibiotics displayed significantly less ALI at 8 days postinoculation compared with antibiotics alone. Inhaled CO was associated with activation of mitochondrial biogenesis in the lung and with augmentation of renal antioxidative programs. These data support the feasibility of safely delivering inhaled CO gas during mechanical ventilation and provide preliminary evidence that CO may accelerate the resolution of ALI in a clinically relevant nonhuman primate pneumonia model.