Heme is an iron-containing tetrapyrrole with dual biological functions. While it serves as an essential prosthetic group in various hemoproteins, heme is cytotoxic in its 'free', non-protein-bound, form. Labile heme (LH) denotes the intracellular fraction of bioavailable heme that is readily exchangeable for incorporation into hemoproteins. To investigate the regulatory role of this heme fraction in inflammatory activated macrophages, we applied the selective fluorescent small molecule H-FluNox for LH detection in lipopolysaccharide (LPS)-stimulated murine bone marrow-derived macrophages (BMDMs). Studies with H-FluNox and its cell-permeable derivative acetylated (Ac)-H-FluNox revealed a time-dependent decrease of LH levels in living BMDMs upon treatment with LPS. Expression of δ-aminolevulinate synthase 1, the rate-limiting enzyme of heme synthesis, was up-regulated in parallel to decreased LH. Studies in subcellular organelles of BMDMs demonstrated that LH concentrations were markedly higher in mitochondria compared to cytosol and nuclei. Furthermore, expression of inducible nitric oxide synthase (iNOS), a heme-containing pro-inflammatory enzyme, was linked to intracellular LH concentrations. Specifically, LPS-dependent iNOS induction was attenuated in BMDMs displaying decreased LH, either after treatment with pharmacological heme synthesis inhibitors, or with genetic deficiency of the nuclear heme sensor BACH1. By contrast, inducibility of iNOS by LPS was markedly higher in BMDMs exhibiting increased levels of LH following treatment with the heme synthesis substrate δ-aminolevulinate. Finally, pharmacological inhibition of succinate dehydrogenase, which enhances intracellular levels of δ-aminolevulinate and LH, was also associated with higher inducibility of iNOS by LPS. In conclusion, the data indicate that intracellular LH is modulated by inflammatory stimulation in mouse macrophages and is critical for heme incorporation into the hemoprotein iNOS. Thus, heme availability may serve as a regulatory link between metabolic and inflammatory pathways.

Nitric oxide (NO) is a key signaling gas that is involved in a wide range of physiological and pathophysiological processes. NO signaling is closely linked to its interactions with heme, an abundant iron-containing tetrapyrrole in the organism. While heme plays vital roles as a prosthetic group in hemoproteins, it can be toxic in its ‘free’, non-protein-bound form. The chemical and structural characteristics of NO-heme binding in heme-nitrosyl complexes have been extensively characterized in earlier research. Recent studies have provided novel insights on how NO-heme interactions affect key functions of the cell and activities of subcellular organelles such as mitochondria. Notably, the NO-heme network plays a crucial immunomodulatory role in inflammatory responses of macrophages, a major cell population of the innate immune system. Upon immunological activation, these cells generate large amounts of NO through activation of the inducible nitric oxide synthase (iNOS), which contributes to killing of bacteria and modulating of inflammation. NO generates microbicidal pro-oxidant peroxynitrite, which in turn activates feedback loops that provide autoprotection to macrophages. Interestingly, the dynamic interaction between NO and heme adds to the complex control of various heme-containing enzymes involved in inflammation and cellular oxidative stress adaptation. NO interacts with heme both directly and indirectly through multiple biochemical reactions, such as S-nitrosylation of cysteine residues and allocation of heme into specific hemoproteins. The current review summarizes key aspects of the regulatory interplay between NO and heme, highlighting its functional consequences in health and disease. A particular emphasis is on the significance of the NO-heme network in inflammation, especially its role in macrophages.

BACKGROUND:Periodontitis is a chronic inflammatory disease characterized by excessive production of pro-inflammatory mediators, oxidative stress, and extracellular matrix degradation, ultimately leading to alveolar bone loss. Carbon monoxide (CO), an endogenous gasotransmitter produced during heme degradation, exhibits anti-inflammatory, antioxidant, and cytoprotective properties. CORM-401, an orally active CO-releasing molecule, can deliver CO systemically with high efficiency, and its ability to regulate energetic metabolism, reduce inflammation, and limit oxidative stress has been documented. However, its potential therapeutic effects in the treatment of periodontal disease have never been investigated. OBJECTIVE:To evaluate whether orally administered CORM-401 attenuates alveolar bone loss and gingival inflammation in a rat model of ligature-induced periodontitis. METHODS:Adult rats were subjected to ligature placement of the first molar to induce periodontitis and treated orally with CORM-401 or vehicle. Alveolar bone loss was quantified by CEJ-BC measurements. Gingival tissues were analyzed for cytokines (TNF-α and IL-1β), nitric oxide metabolites (NOx), and MMP-2 activity. Systemic inflammation was assessed by measuring plasma TNF-α. RESULTS:CORM-401 significantly reduced alveolar bone loss compared with vehicle-treated periodontitis rats. Treatment markedly decreased gingival levels of TNF-α and IL-1β. CORM-401 also reduced gingival NOx accumulation, indicating attenuation of oxidative and proteolytic stress. Plasma TNF-α was lower in CORM-401-treated animals, demonstrating a systemic anti-inflammatory effect. CONCLUSION:Oral administration of CORM-401 effectively mitigates periodontal inflammation and protects against alveolar bone loss in ligature-induced periodontitis. These findings suggest that CO-based pharmacological modulation may represent a promising host-directed therapeutic strategy for controlling periodontal tissue destruction.

Metabolic reprograming has been linked to epithelial-to-mesenchymal transition (EMT) in cancer cells, but how it influences EMT in normal cells remains largely unknown. Here we explored how metabolism impacts delamination and migration of avian trunk neural crest cells, an important progenitor cell population of the vertebrate embryo. We report that delamination exhibits a quiescent metabolic phenotype whereas migration is characterized by OXPHOS-driven metabolism coupled to distinct expression of metabolic, EMT and developmental genes. While glucose and glutamine are required for delamination and migration, we uncover a specific role for glutamine and its catabolizing enzyme glutaminase in the unfolding of NCC delamination. Namely, glutamine is required for nuclear accumulation of glutaminase, which interacts and cooperates with Wnt signaling to regulate EMT gene expression and cell cycle during delamination. Our data indicate that similarly to cancer cells, embryonic cells engage metabolic enzymes for non-canonical signaling functions to connect metabolism with EMT.

Carbon monoxide (CO), a gas endogenously produced in mammalian tissues, exerts vasodilatory, anti-ischemic, and anti-inflammatory effects. These properties have prompted the development of CO-releasing molecules (CO-RMs) for therapeutic purposes. Among this class of compounds is CORM-A1, a boron-based carboxylic acid, which generates controlled amounts of CO under physiological conditions. In this proof-of-principle study we explored the potential of CORM-A1 to protect kidneys from warm ischemia and reperfusion (WI/R) injury in rat and swine models. We found that intravenous administration of CORM-A1 significantly increased blood carboxyhemoglobin (COHb) levels while facilitating CO accumulation in renal tissue, thus confirming its ability to deliver CO to peripheral organs. In rats subjected to 45- and 60-min WI/R, administration of CORM-A1 improved renal function at reperfusion, as shown by decreased serum creatinine and urea levels. Histopathological analysis revealed substantial protection against tubular damage, cell infiltration, and inflammation, especially after 60-min ischemia. Protection was dose-dependent, with higher doses offering enhanced effects. In a swine kidney auto-transplantation model, CORM-A1 significantly improved graft function, reduced fibrosis and necrosis, and extended graft survival. These findings position CORM-A1 as a promising CO prodrug, with translational relevance for clinical applications in kidney transplantation and other ischemia-related conditions.

Inflammation is vital for defence against injury and infection, but excessive inflammation can lead to tissue damage and disease. The central nervous system (CNS) helps regulate immune responses through neuroendocrine pathways, such as the hypothalamic-pituitary-adrenal axis and the anti-inflammatory reflex, which limit systemic inflammation. Immune responses require significant metabolic energy and the liver adapts by increasing glucose production and mobilizing fatty acids. This immune-metabolic coordination is mediated by cytokines and metabolic regulators. This review explores how the anti-inflammatory reflex modulates the interplay between inflammation and metabolism during endotoxaemia, with a focus on the haem oxygenase 1/carbon monoxide (HO1/CO) pathway. Carbon monoxide, a byproduct of HO1 activity, acts as a key signalling molecule that reduces inflammation, supports mitochondrial function and protects tissues. Understanding this pathway provides new insights into potential therapeutic strategies for treating inflammatory and metabolic disorders by targeting neuroimmune-metabolic communication networks.

HYCOs are hybrid molecules consisting of activators of the transcription factor Nrf2 conjugated to carbon monoxide (CO)-releasing moieties. These 'dual action' compounds (HYCOs) have been designed to mimic the activity of heme oxygenase-1 (HO-1), a stress inducible cytoprotective enzyme that degrades heme to CO which expression is regulated by Nrf2. HYCOs have recently shown efficacy in ameliorating experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. However, the mechanism(s) of action of HYCOs still remains to be fully investigated. Here, we assessed the effects of HYCO-3, a prototype of these hybrids, on myeloid-derived cells, microglial cells and T lymphocytes obtained from EAE-immunized mice. HYCO-3 exerted immunomodulatory effects on all the examined cell populations by inhibiting the generation of pro-inflammatory cytokines and nitric oxide, and downregulating antigen-presenting capacity of these cells. The observed effects support the view that HYCOs are promising candidates to be developed for the treatment of autoimmune and chronic inflammatory disorders.

Carbon monoxide (CO) is an endogenous gasotransmitter with proven anti-inflammatory, anti-apoptotic, and antiproliferative effects. CO-releasing molecules (CO-RMs) have been developed to deliver controlled doses of CO, differing in their CO-release kinetics and chemical structure, showing promise as therapeutic agents in various diseases, including cancer. The main goal of our study is to identify the effectiveness of CO-RMs as a therapeutic strategy to counteract prostatic carcinogenesis. This study aimed to assess the effects of three water-soluble CO-RMs—CORM-3, CORM-401, and CORM-A1—on key hallmarks of prostate cancer (PCa). Using the PCa cell line PC3 we evaluated cell viability across a range of CO-RM concentrations (25-150 µM; 6 h) to identify doses that do not compromise cell viability for further analyses. Confocal microscopy with the DCFH-DA probe revealed that CO-RM treatments significantly reduced reactive oxygen species (ROS) levels (p<0.001). Furthermore, CO-RMs enhanced cell adhesion (p<0.001) and inhibited cell migration (p<0.01). Moreover, mitochondrial integrity, mitochondrial mass and mitochondrial DNA copy numbers were significantly altered (p<0.0001), as shown by MitoGreen, TMRE staining and RT-qPCR. Metabolically, PC3 cells treated with CO-RMs exhibited significant reductions in ATP content (p<0.05), LDH activity (p<0.01) and lactate amounts (p<0.01). At the molecular level, CO-RMs repressed markers of oxidative stress (NFkB, SOD-2), metabolism (PKM2, PDHB, ACO2), proliferation (Ki67) (p<0.01) and angiogenesis (VEGF) (p<0.05), as assessed by RT-qPCR. Additionally, analyzing stem-like characteristics, we observed that CO-RMs reduced spheroid size in a hanging drop assay (p<0.0001), as well as colony formation in a clonogenic assay (p<0.01). As PCa predominantly metastasizes to bone, we further analyzed the potential of CO-RMs to modulate the communication between tumor and bone cells using indirect co-cultures of PC3 cells with osteoblasts precursor cells (MC3T3). CO-RMs decreased osteoblastic marker levels in bone cells (COL1A1, SPP1, RUNX2) (p<0.01) and increased OPG expression in PCa cells (p<0.01) demonstrating that this molecule impaired the crosstalk between both cell types, probably affecting the osteotropism of PC3 cells. This study demonstrates that CO-RMs has a strong effect on modulating key biological processes in PCa cells, highlighting its potential as a therapeutic agent for PCa. Gastón Pascual, Pablo Sanchis, Agustina Sabater, Rocio Seniuk, Javier Cotignola, Elba Vazquez, Ayelen Toro, Roberta Foresti, Roberto Motterlini, Geraldine Gueron. Carbon monoxide-releasing molecules as top-notch candidates for prostate cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5657.

Diabetic retinopathy (DR) is a microvascular complication of diabetes mellitus and a leading cause of blindness in the working-age population. Current pharmacological treatments counteract DR’s later stages without targeting the earlier disease phases. Using computational approaches, our group previously identified the α1D and α2C adrenoceptors (α1DR and α2CR) as new putative drug targets for DR. Therefore, the aim of this work was to validate the role of these receptors in an in vitro model of DR, i.e., retinal pigmented epithelial cells (ARPE-19) challenged with high glucose (HG, 50 mM). We examined the effects of selective α1DR and α2CR agonists and antagonists on hyperglycemia-induced mitochondrial dysfunction and blood retinal barrier breakdown. Seahorse XFe was employed to assess the oxygen consumption rate and extracellular acidification rate. The integrity of the ARPE-19 barrier was evaluated through transepithelial electrical resistance measurements and a sodium fluorescein permeability test. α1DR pharmacological modulation through the α1DR antagonist BMY 7378 (0.1–1 µM, 24 h), but not α2CR, significantly attenuated HG-induced mitochondrial dysfunction. BMY 7378 (0.1–1 µM, 48 h) also prevented HG-mediated damage to retinal epithelial integrity. In contrast, the α1DR agonist phenylephrine (1–10 μM, 24 h) further reduced ARPE-19 mitochondrial activity compared to HG, indicating that α1D activation is directly implicated in DR-mediated mitochondrial dysfunction. In conclusion, the current in vitro study validated α1DR as a pharmacological target for DR.

Origanum majorana L. (O. majorana) (Lamiaceae) is an aromatic Mediterranean plant widely used in food, cosmetics, and traditional medicine due to its aroma and rich content of bioactive compounds. While its leaves and flowers are commonly utilized, lignified stems are often discarded. This study compared hydroalcoholic extracts from the leaves and flowers (valuable fraction, VF) and stems (by-product, BP). Phytochemical analysis revealed qualitatively similar profiles, identifying 20 phenolic compounds, with Rosmarinic acid and Salvianolic acid B as the most and second most abundant, respectively. Antioxidant activity was evaluated in vitro using DPPH (IC50 [µg/mL]: VF 30.11 ± 3.46; BP 31.72 ± 1.46), H2O2 (IC50 [µg/mL]: VF 103.09 ± 4.97; BP 119.55 ± 10.58), and O2•− (IC50 [µg/mL]: VF 0.71 ± 0.062; BP 0.79 ± 0.070). Both extracts (20 µg/mL) fully restored oxidative balance in hemin-stressed AC16 cardiomyocytes, without altering the expression of catalase, heme-oxygenase 1, superoxide dismutase 2, or ferritin. Anti-inflammatory activity in LPS-stimulated RAW 264.7 macrophages showed that VF (IC50 400 µg/mL) reduced •NO release to control levels, while BP achieved a ~60% reduction. Cytotoxicity was assessed on cancer cell lines: CaCo-2 (IC50 [µg/mL]: VF 154.1 ± 6.22; BP 305.2 ± 15.94), MCF-7 (IC50 [µg/mL]: VF 624.6 ± 10.27; BP 917.9 ± 9.87), and A549 (IC50 [µg/mL]: VF 720.8 ± 13.66; BP 920.2 ± 16.79), with no cytotoxicity on normal fibroblasts HFF-1 (IC50 > 1000 µg/mL for both extracts). Finally, both extracts slightly inhibited only CYP1A2 (IC50 [µg/mL]: VF 497.45 ± 9.64; BP 719.72 ± 11.37) and CYP2D6 (IC50 [µg/mL]: VF 637.15 ± 14.78, BP 588.70 ± 11.01). These results support the potential reuse of O. majorana stems as a sustainable source of bioactive compounds for nutraceutical and health-related applications.

Carbon monoxide (CO), a gaseous signaling molecule, has shown promise in preventing body weight gain and metabolic dysfunction induced by high fat diet (HFD), but the mechanisms underlying these effects are largely unknown. An essential component in response to HFD is the gut microbiome, which is significantly altered during obesity and represents a target for developing new therapeutic interventions to fight metabolic diseases. Here, we show that CO delivered to the gut by oral administration with a CO-releasing molecule (CORM-401) accumulates in faeces and enriches a variety of microbial species that were perturbed by a HFD regimen. Notably, Akkermansia muciniphila, which exerts salutary metabolic effects in mice and humans, was strongly depleted by HFD but was the most abundant gut species detected after CORM-401 treatment. Analysis of bacterial transcripts revealed a restoration of microbial functional activity, with partial or full recovery of the Krebs cycle, β-oxidation, respiratory chain and glycolysis. Mice treated with CORM-401 exhibited normalization of several plasma and fecal metabolites that were disrupted by HFD and are dependent on Akkermansia muciniphila's metabolic activity, including indoles and tryptophan derivatives. Finally, CORM-401 treatment led to an improvement in gut morphology as well as reduction in inflammatory and metabolic profiles. Our findings provide therapeutic insights on the efficacy of CO as a potential prebiotic to combat obesity, identifying the gut microbiota as a crucial target for CO-mediated pharmacological activities against metabolic disorders.

The cytoprotective transcription factor NRF2 regulates the expression of several hundred genes in mammalian cells and is a promising therapeutic target in a number of diseases associated with oxidative stress and inflammation. Hence, an ability to monitor basal and inducible NRF2 signalling is vital for mechanistic understanding in translational studies. Due to some caveats related to the direct measurement of NRF2 levels, the modulation of NRF2 activity is typically determined by measuring changes in the expression of one or more of its target genes and/or the associated protein products. However, there is a lack of consensus regarding the most relevant set of these genes/proteins that best represents NRF2 activity across cell types and species. We present the findings of a comprehensive literature search that according to stringent criteria identifies GCLC, GCLM, HMOX1, NQO1, SRXN1 and TXNRD1 as a robust panel of markers that are directly regulated by NRF2 in multiple cell and tissue types. We assess the relevance of these markers in clinically accessible biofluids and highlight future challenges in the development and use of NRF2 biomarkers in humans.

Acute hyper-hemolysis is a severe life-threatening complication in patients with sickle cell disease (SCD) that may occur during delayed hemolytic transfusion reaction (DHTR), or vaso-occlusive crises associated with multi-organ failure. Here, we developed in vitro and in vivo animal models to mimic endothelial damage during the early phase of hyper-hemolysis in SCD. We then used the carbon monoxide (CO)-releasing molecule CORM-401 and examined its effects against endothelial activation, damage, and inflammation inflicted by hemolysates containing red blood cell membrane-derived particles. The in vitro results revealed that CORM-401: 1) prevented the up-regulation of relevant pro-inflammatory, and pro-adhesion controlled by the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and 2) abolished the expression of the nuclear factor erythroid-2-related factor 2 (Nrf2) that regulates the inducible antioxidant cell machinery. We also show in SCD mice that CORM-401 protects against hemolysate-induced acute damage of target organs such as the lung, liver, and kidney through modulation of NF-kB pro-inflammatory and Nrf2 antioxidant pathways. Our data demonstrate the efficacy of CORM-401 as a novel therapeutic agent to counteract hemolysate-induced organ damage during hyper-hemolysis in SCD. This approach might be considered as possible preventive treatment in high-risk situations such as SCD patients with history of DHTR.

Activation of inflammation is tightly associated with metabolic reprogramming in macrophages. The iron-containing tetrapyrrole heme can induce pro-oxidant and pro-inflammatory effects in murine macrophages, but has been associated with polarization towards an anti-inflammatory phenotype in human macrophages. In the current study, we compared the regulatory responses to heme and the prototypical Toll-like receptor (TLR)4 ligand lipopolysaccharide (LPS) in human and mouse macrophages with a particular focus on alterations of cellular bioenergetics. In human macrophages, bulk RNA-sequencing analysis indicated that heme led to an anti-inflammatory transcriptional profile, whereas LPS induced a classical pro-inflammatory gene response. Co-stimulation of heme with LPS caused opposing regulatory patterns of inflammatory activation and cellular bioenergetics in human and mouse macrophages. Specifically, in LPS-stimulated murine, but not human macrophages, heme led to a marked suppression of oxidative phosphorylation and an up-regulation of glycolysis. The species-specific alterations in cellular bioenergetics and inflammatory responses to heme were critically dependent on the availability of nitric oxide (NO) that is generated in inflammatory mouse, but not human macrophages. Accordingly, studies with an inducible nitric oxide synthase (iNOS) inhibitor in mouse, and a pharmacological NO donor in human macrophages, reveal that NO is responsible for the opposing effects of heme in these cells. Taken together, the current findings indicate that NO is critical for the immunomodulatory role of heme in macrophages.

RATIONALE:Lung fibroblast senescence is involved in the pathophysiology of chronic obstructive pulmonary disease (COPD). However, the mechanisms underlining this phenomenon are still poorly understood. Secreted phospholipases (sPLA2, a subclass of phospholipases) are secreted by senescent cells and can in turn induce senescence. However, their role in fibroblasts senescence in COPD is unknown. OBJECTIVES:The aim of this study was to analyze the role of sPLA2 in pulmonary fibroblast senescence. METHODS:Fibroblasts were isolated from patients with COPD and control subjects, and senescence markers and inflammatory profile was analyzed. sPLA2 levels were quantified in serum of COPD and controls. MAIN RESULTS:In comparison with non-smokers and smoker controls, senescent lung COPD fibroblasts exhibited a higher mRNA and protein expression of the sPLA2 isoform XIIA and of syndecan 4 (one of its receptors). sPLA2 XIIA induced in turn senescence of non-senescent pulmonary fibroblasts via a pathway involving consecutively syndecan 4, activation of MAPK and p-serine 727 STAT-3, increased mitochondrial ROS production, and activation of AMPK/p53. This pathway was associated with a specific inflammatory secretome (IL-10, IL-12 and TNFα), globally suggesting occurrence of a mitochondrial damage-induced senescence. COPD fibroblasts were more susceptible to this sPLA2 XIIA effect than cells from controls subjects. sPLA2 XIIA levels were significantly higher in serum from COPD patients as compared to controls. CONCLUSION:sPLA2 XIIA is involved in senescence in COPD and could be a potential target to dampen this process.

Bioenergetic metabolism is a key regulator of cellular function and signaling, but how it can instruct the behavior of cells and their fate during embryonic development remains largely unknown. Here, we investigated the role of glucose metabolism in the development of avian trunk neural crest cells (NCCs), a migratory stem cell population of the vertebrate embryo. We uncovered that trunk NCCs display glucose oxidation as a prominent metabolic phenotype, in contrast to what is seen for cranial NCCs, which instead rely on aerobic glycolysis. In addition, only one pathway downstream of glucose uptake is not sufficient for trunk NCC development. Indeed, glycolysis, mitochondrial respiration and the pentose phosphate pathway are all mobilized and integrated for the coordinated execution of diverse cellular programs, epithelial-to-mesenchymal transition, adhesion, locomotion, proliferation and differentiation, through regulation of specific gene expression. In the absence of glucose, the OXPHOS pathway fueled by pyruvate failed to promote trunk NCC adaptation to environmental stiffness, stemness maintenance and fate-decision making. These findings highlight the need for trunk NCCs to make the most of the glucose pathway potential to meet the high metabolic demands appropriate for their development.

Metal carbonyls have been developed as carbon monoxide-releasing molecules (CO-RMs) to deliver CO for therapeutic purposes. The manganese-based CORM-401 has been recently reported to exert beneficial effects in obese animals by reducing body weight gain, improving glucose metabolism and reprogramming adipose tissue towards a healthy phenotype. Here, we report on the synthesis and characterization of glyco-CORMs, obtained by grafting manganese carbonyls on dextrans (70 and 40 kDa), based on the fact that polysaccharides facilitate the targeting of drugs to adipose tissue. We found that glyco-CORMs efficiently deliver CO to cells in vitro with higher CO accumulation in adipocytes compared to other cell types. Oral administration of two selected glyco-CORMs (5b and 6b) resulted in CO accumulation in various organs, including adipose tissue. In addition, glyco-CORM 6b administered for eight weeks elicited anti-obesity and positive metabolic effects in mice fed a high fat diet. Our study highlights the feasibility of creating carriers with multiple functionalized CO-RMs.

Simultaneous poisoning by carbon monoxide (CO) and hydrogen cyanide is the major cause of mortality in fire gas accidents. Here, we report on the invention of an injectable antidote against CO and cyanide (CN-) mixed poisoning. The solution contains four compounds: iron(III)porphyrin (FeIIITPPS, F), two methyl-β-cyclodextrin (CD) dimers linked by pyridine (Py3CD, P) and imidazole (Im3CD, I), and a reducing agent (Na2S2O4, S). When these compounds are dissolved in saline, the solution contains two synthetic heme models including a complex of F with P (hemoCD-P) and another one of F with I (hemoCD-I), both in their iron(II) state. hemoCD-P is stable in its iron(II) state and captures CO more strongly than native hemoproteins, while hemoCD-I is readily autoxidized to its iron(III) state to scavenge CN- once injected into blood circulation. The mixed solution (hemoCD-Twins) exhibited remarkable protective effects against acute CO and CN- mixed poisoning in mice (~85% survival vs. 0% controls). In a model using rats, exposure to CO and CN- resulted in a significant decrease in heart rate and blood pressure, which were restored by hemoCD-Twins in association with decreased CO and CN- levels in blood. Pharmacokinetic data revealed a fast urinary excretion of hemoCD-Twins with an elimination half-life of 47 min. Finally, to simulate a fire accident and translate our findings to a real-life scenario, we confirmed that combustion gas from acrylic cloth caused severe toxicity to mice and that injection of hemoCD-Twins significantly improved the survival rate, leading to a rapid recovery from the physical incapacitation.