Therapeutic Potential of Hemoglobin Derived from the Marine Worm Arenicola marina (M101): A Literature Review of a Breakthrough Innovation

Oxygen (O2) is indispensable for aerobic respiration and cellular metabolism. In case of injury, reactive oxygen species are produced, causing oxidative stress, which triggers cell damaging chemical mediators leading to ischemic reperfusion injuries (IRI). Sufficient tissue oxygenation is necessary for optimal wound healing. In this context, several hemoglobin-based oxygen carriers have been developed and tested, especially as graft preservatives for transplant procedures. However, most of the commercially available O2 carriers increase oxidative stress and show some adverse effects. Interestingly, the hemoglobin derived from the marine lugworm Arenicola marina (M101) has been presented as an efficient therapeutic O2 carrier with potential anti-inflammatory, anti-bacterial, and antioxidant properties. Furthermore, it has demonstrated promise as a supplement to conventional organ preservatives by reducing IRI. This review summarizes the properties and various applications of M101. M101 is an innovative oxygen carrier with several beneficial therapeutic properties, and further research must be carried out to determine its efficacy in the management of different pathologies.


Introduction
The existence and maintenance of human life is dependent on oxygen (O 2 ) as it is vital for life-sustaining aerobic respiration in humans [1]. At the cellular level, O 2 acts as the terminal electron acceptor at the end of the electron transport chain within the mitochondrial inner membrane, whereby oxidative phosphorylation results in the synthesis of adenosine triphosphate (ATP), which is necessary for all active metabolic processes [2]. O 2 is also vital for protein synthesis, maturation of extracellular matrices such as collagen and nitric oxide synthesis, eventually, playing a major role in the regulation of vascular tone and angiogenesis [3]. However, in the presence of O 2 , normal metabolism can also generate reactive oxygen species (ROS). In a wound microenvironment, large amounts of molecular O 2 are partially reduced to form ROS [3]. ROS are small and highly reactive molecules including superoxide ions and peroxides [4] that influence cell migration, proliferation and angiogenesis [3]. Exacerbation of hypoxic injury after restoration of oxygenation (reoxygenation) is an important mechanism of cellular injury, especially, in transplantation procedures. Cellular hypoxia and reoxygenation are two essential elements of ischemiareperfusion injury (IRI) [5]. (Table 1). However, the p50 of M101 is closely related to the p50 of human HbA inside the red blood cell and its cooperativity is also similar [37].

Properties
Hb of Arenicola marina References Molecular weight 3600 kDa [35,44,45] Size 15 by 25 nm [44,46] Shape of central piece Ellipsoid (hexagonal bilayer) [46] O 2 binding sites 156 [35,44] Heme pockets large [36,47] Auto-oxidation rate k ox (h −1 ) 0.014 [35,48] Redox potential E o (mV) −50 [48] p50 7.05 ± 0.93 mmHg [40,49] n50 2.54 ± 0.23 [29] Arenicola marina inhabits the intertidal area in a sulfide-rich environment and is exposed to pronounced fluctuations in environmental conditions [50]. The manufacturing of the product HEMO 2 life ® is done under GMP and starts by freezing the worms to create a hemorrhagic shock and to release its extracellular Hb (M101). After successive steps of solid/liquid extraction, purification, filtration and gamma irradiation, the final result is a class III medical device containing M101 [51]. Preclinical studies in rats and hamsters using M101 have shown reduced microvascular vasoconstriction and no significant impact on mean arterial blood pressure compared with other HBOCs that contain bovine or human Hb [40]. Moreover, the biocompatibility and absence of toxicity of HEMO 2 life ® has also been evaluated according to international standards (ISO 10993).
In view of the beneficial properties exhibited by M101, the aim of this review is to summarize the outcomes of M101 application to several in vitro, in vivo and human trials and to assess its potential therapeutic and clinical relevance. To perform this review, only studies published in the English language were included and the last search was carried out in August 2020. The following keywords were used for the search: ("M101" OR "HEMO 2 life" OR "Arenicola marina" OR "extracellular hemoglobin" OR "HEMOXYCarrier") and a systematic literature search was performed in the Pubmed/Medline database. One hundred ninety-one articles were found and after they were reviewed for relevance and repetition, only 16 articles were included in this review.

Structure of M101
The description of the quaternary structure of M101 and an assessment of its heme content was first carried out in 1996. Multi-angle laser-light scattering electrospray-ionization mass spectrometry [50] gave a molecular mass of 3648 ± 24 kDa and a gyration radius of 11.3 ± 1.7 nm. According to Zal et al., [45].
Thus, the Arenicola marina Hb would be composed of 198 polypeptide chains with 156 globin chains and 42 linker chains, each twelfth being in contact with 3.5 linker subunits, providing a total mass of 3682 kDa [50]. The phylogenetic analyses of the Arenicola marina globin chains confirmed a more intricate structural organization of this extracellular globin compared to the previous description [50] and have allowed DNA sequencing of Arenicola marina globin genes, which assigned the five Arenicola marina sequences to the different globin sub-families. The cDNA-derived amino acid sequence exhibits 12 cysteine residues, which is in agreement with previous studies on Arenicola marina's Hb [52]. However, two of the globins were found to be A2 globin chains lacking the cysteine residues proposed to be involved in the binding of hydrogen sulfide by such Hb, which has been linked to the evolution of the Hb sulfide binding function in annelids inhabiting sulfide-rich environments [45,53]. In summary, M101's quaternary structure comprises two overlapping hexagons, which are approximately 25 nm between parallel sides (face view) and 15 nm thick (profile view) ( Figure 1).

Properties of M101
The Arenicola marina Hb M101 can carry up to 156 O 2 molecules (versus 4 for human hemoglobin) in a saturated state, hence, it exhibits a greater O 2 -binding capacity and antioxidative properties. M101 releases O 2 according to a simple gradient [35] and without co-factors. M101 can completely inhibit reduction of nitroblue tetrazolium in the presence of superoxide radicals, thereby, demonstrating intrinsic SOD-like activity linked to Cu/Zn metals and an ability to ameliorate oxidative stress [54].
Sulfide concentration in the pore water of the sediment (lugworm burrow) can reach several hundred µmol/L and is considered an important environmental factor for a sediment-dwelling animal such as Arenicola marina [55,56]. In the presence of sulfide, vertebrate oxyhemoglobin forms sulfhemoglobin. The inability of Arenicola marina's Hb to form this compound might indicate a biochemical adaptation that is necessary for Arenicola marina during exposure to high concentrations of sulfide. The free cysteine on the polypeptide d chains plays a role in this mechanism by inhibiting the formation of sulfhemoglobin and allowing sulfide oxidation. The oxidation product, defined as the brown pigment, and which possesses higher sulfide oxidation activity than hexagonal bilayer Hb, could be due to some modification or breakage of the prosthetic-group-protein linkage during the reaction leading to its formation. However, for Arenicola marina, this would be a detoxification mechanism (i.e., sulfide immobilization by the Hb), and not an adaptation to symbiotic life as is the binding of sulfide by vestimentiferan Hb [50]. Interestingly, M101 is able to work in a broad range of temperatures (4 • C to 37 • C) as opposed to other therapeutic oxygen carriers manufactured using vertebrate Hb, which only work at 21 • C to 37 • C [19,57]. In vivo, studies carried out on mice and rats showed that the half-life of M101 molecule in circulation is 2.5 days [38]. The important properties of M101 have been listed below ( Table 2).

Applications of M101
M101 has already demonstrated its efficacy as a supplement to the solution for graft/organ preservation under static and perfusion conditions during organ transplant. Several in vivo preclinical trials involving different vital organs, for instance, heart [62], kidneys [58,63], liver [64], pancreas [65], lungs [66] have exhibited its ability to maintain and improve graft viability and function. Furthermore, its safety and efficiency has also been established in the human trial OXYOP (Clinical Trial Registry No. NCT 02652520) involving a kidney transplant procedure where HEMO 2 life ® as the class III medical device containing M101 (1 g/L) was added to a preservation solution [51]. Interestingly, no immunological, allergic or prothrombotic effects were observed with M101 application. Furthermore, addition of M101 decreased the delayed graft function (DGF) and improved the renal function [51]. This confirmed the safety and efficacy of M101 for its potential clinical applications. M101 has also been proposed as an instant blood substitute for wounded US army soldiers [67].

M101 and Conventional Preservative Techniques
Hypothermic cold storage (CS) is a conventional strategy based on the principle of metabolism reduction with temperature to minimize ischemic injuries during organ preservation [68], however, even slow metabolism requires O 2 [69,70]. Furthermore, the delayed O 2 delivery to ischemic tissue during CS could also aggravate oxidative stress if the cells fail to restore oxidative respiration [71]. Therefore, the use of an O 2 carrier during the entire preservation procedure could be protective, allowing cold-preserved cells to maintain their ATP reserve, thus, establishing a balanced energy metabolism at reperfusion and coping with the sudden influx of O 2 without causing oxidative stress.
In vitro, M101 supplementation to a range of solutions used in the clinic has demonstrated utility in cold-stored cultured cells. Furthermore, in vivo, M101 addition to University of Wisconsin (UW) and histidine-tryptophane-ketoglutarate (HTK) solution [72] has shown an improvement of graft function and reduction in DGF [57,63].
However, the tissue oxygenation ability of M101 is in accordance with the physiological needs as O 2 binding and release occurs passively in a simple O 2 gradient. The intrinsic Cu/Zn-SOD activity of M101 is crucial for the prevention and management of IRI [58,63,73]. Thus, the simple addition of M101 to CS presents excellent potential for clinical translation. Besides static CS, machine preservation (MP) of grafts has attracted mounting interest as it exhibits improvement in graft quality and function [74][75][76]. Its ability to get rid of metabolites and cellular waste produced during ischemia has been proposed as the reason for its beneficial effects. However, the presence of these products at reperfusion is most likely associated with the intense activation of the innate immune pathway [77,78]. In this context, M101 supplementation to MP for uniform and efficient O 2 delivery to the entire graft could promote organ protection because of the synergistic beneficial effects of M101 and MP [79,80].

M101 as an Extracellular O 2 Carrier
Conventional preservative techniques provide O 2 in excess, which could induce oxidative stress. On the contrary, M101 has the unique property of providing O 2 against a gradient, according to the physiological needs of the cell, thus, eliminating the risk of hyperoxia and oxidative stress [19,35,38]. M101 supplementation increases cellular ATP production, thus, it efficiently maintains the metabolic processes, and protects mitochondria by decreasing the need to switch from mitochondrial respiration to anaerobic glycolysis. The ability of M101 to maintain high ATP levels during the preservation process may also benefit the restoration of energy homeostasis upon reperfusion due to less metabolic stress on oxidative pathways [57]. M101 remains stable in different organ preservation solutions of varying ionic compositions and osmolarities and demonstrates its O 2 carrying and antioxidant (SOD) properties [35]. So far, M101 application in vitro, in vivo and clinically has shown great promise (Tables 3 and 4).
Hence, M101 improves graft vitality by promoting tissue oxygenation, maintaining tissue integrity, reducing IRI, inflammation and fibrosis ( Figure 2).  1 g/L. Cells were exposed to M101 for up to 24 h and cytocompatibility was determined. EC exposed to lipopolysachharide of P. gingivalis (Pg-LPS) or infected with P. gingivalis to mimic inflammatory state were treated with M101 and inflammatory markers were studied. P. gingivalis biofilms grown on glass surface were treated with M101.
P. gingivalis growth. Commercial preservative solutions alone were deleterious to cells. M101 protects cells in vitro against cold preservation lesions. M101 is an effective antioxidant. [57] Human primary aortic endothelium cells (HAECs) In vitro model of cold hypoxia and reoxygenation simulating reperfusion on cold-preserved cells. HAECs were subjected to 24 h hypoxia at 4 • C in UW solution mimicking cold ischemia during organ preservation. M101 (0, 1, 2.5, 5, 10 g/L), was added to UW solution. UW was removed and cells were re-cultured at 37 • C to mimic organ reperfusion. Necrosis assay, lactate dehydrogenase (LDH) release.

Application Concentration &Study Type Results Conclusion Reference
Isolated Langendorf-perfused rat hearts (n = 12/group) Evaluation of M101 (1 g/L) as a protective additive to Celsior solution for static storage of donor hearts prior to transplantation. Heart function in Celsior solution, either alone (control) or with the addition of M101 was measured by intra-ventricular balloon before arrest with cold (7.5 • C). Cold storage (CS) lasted 8 h prior to reperfusion (60 min). Hearts (minced and homogenized) were also assessed by 2,3,5-triphenyltetrazoliumchloride (TTC) staining as a measure of viability and infarct size.

M101
: recovery of left ventricular developed pressure. Recovered heart rate to pre-ischemic value (final recovery between 84 and 89% pre-ischemic value).
The addition of M101 to Celsior preservation solution significantly improved post-ischemic recovery of heart function. [62] Rat pancreas preservation Preservation solution with or without M101 at 2 g/L was injected into the pancreas via the pancreatic duct. Pancreas was removed and placed in the preservation solution at 4 • C, and cold ischemia kinetics were then determined. Samples were taken at 0, 2, 4, 6, 8, 12 or 18 h (n = 4-6). Metabolite extraction was performed on fresh tissue, while protein extraction and OCT slide analysis were performed on snap-frozen tissue at each time-point. Islet isolation was performed after cold ischemia (30 min, 4, 6, 8, 12 or 18 h). For the experiments with M101 perfusion, 2 mL of preservation solution with or without M101 at 2 g/L was injected into the pancreas via the pancreatic duct. Pancreases were preserved for 6 h at 4 • C in the presence of M101, before islet isolation process. 12 h of rat pancreas preservation in the presence of M101.

M101:
maintenance of mitochondrial complex 1 pancreas activity throughout ischemia kinetics compared to controls; Lactate levels compared to the control; variability of active caspase-3 levels; transient phosphorylation of p38, which is observed in the control, after 4 h (p = 0.002) and 6 h (p = 0.008) of ischemia; oxidative stress ROS (25%) compared to control; p38 activity in islets; necrosis (HMGB1); cellular stress pathway (p38 MAPK) activity.
Safe, efficient, anti-oxidant. Freshly isolated islets had improved function when M101 injected in the pancreas.
[65] AKT phosphorylation in tissue after 3 h of exposure to M101 (p = 0.08); increased complex 1 activity; cleaved caspase-3 in tissue; islet yield and function of pancreases relative to the control. insulin secretion from islets (both in basal and in stimulated conditions); overall insulin content; complex 1 mitochondrial activity; activation of AKT activity (a cell survival marker); variability of caspase 3; oxidative stress (ROS); necrosis (HMGB1); cellular stress pathway (p38 MAPK) activity; islet yield and function.
Despite the absence of M101 during the first period of ischemia, positive effects were observed in human pancreas during preservation. [65] Pig pulmonary preservation and post-transplant lung function 36 h cold preservation with 1 g/L of M101. Normothermic ex vivo lung perfusion (EVLP) followed by lung transplant (4 h reperfusion).
M101-treated lungs improved physiologic parameters: oxygenation than in the controls significantly; edema formation significantly; circulating IL-6 within recipient plasma after transplantation.
M101 during an extended pulmonary preservation period led to significantly superior early post-transplant lung function. [82] Inflammatory calvarial abscess model in mice M101 (1 g/L). P. gingivalis (5 × 10 8 CFU) was injected subcutaneously on the calvaria to induce inflammatory abscess which was treated with M101 (1 g/L) subcutaneous injection. The healing response was followed for 5 days.
Proof of concept that M101 may be beneficial in TBI treatment [39] Mice: in vivo oxygenation potency of M101 toward HT29 human colorectal adenocarcinoma subcutaneous tumors M101 I.V. injection at 600 mg/kg and 1200 mg/kg. Single I.V. injection of M101 in mice bearing human-derived subcutaneous tumors. Expression of hyperoxia marker anti-glucose transporter Glut-1 (immunohistochemistry).
Rapid diffusion of M101 in brain, liver, lungs and ovaries: size of carcinomatous areas; dissociation of tumor; intensity of Glut-1 staining ∼20% after 1 h when M101 was injected at 1200 mg/kg; tumor hypoxia of ∼23% (even 5 h after the same treatment); hypoxia in poorly vascularized tissues. Lower M101 doses (60 or 600 mg/kg), no or intermediate hypoxia reductions. No side effects.
Potential oxygen carrying therapeutic product. Ability of M101 to diffuse within poorly vascularized tissues and to behave as a potent O2 carrier towards vertebrate tissues, without inducing obvious side effects. [38]  HEMO 2 life improved early graft function after prolonged cold ischemia. [66] Mice: testing immunogenicity of M101 Antibody response after 1 or 2 I.V. administrations of M101 in hyper-responsive strain (BP/2 mice) that easily produce antibodies. Plasma levels of IgE and IgG2a measured by ELISA. Comparison with a negative control groups (M101 buffer) and Ovalbumin as a positive control.
After single intravenous injection, M101 increased IgE levels very slightly, but this effect did not reach statistical significance when compared to the vehicle-treated group and on the contrary treatment with ovalbumin led to a 3.4-fold increase in IgE level. After two administrations of M101 7 days apart, a slight and non-significant increase of IgE and IgG2a levels was observed at D14 when compared to vehicle treated group. The second administration of M101 at D7 did not induce any mortality. On the contrary, in the ovalbumin-treated group, three mice out of nine died 15 min after the challenge at D7. The remaining mice showed a marked increase in IgE level after the second injection (D14).
Beneficial use of M101 in 2 of the most used preservation solutions (better short-term function recovery and reduced development of fibrosis, the main cause of graft loss). [57] Pig: kidney autotransplantation model Kidney was harvested, cold flushed and preserved for 24 h at 4 • C before transplantation. Dose-ranging study: 1, 2, and 5 g/L M101 supplementing UW solution. Pigs placed in metabolic cages for diuresis and serum creatinine measurements. Biopsies for conventional histology and fibrosis evaluated on sacrifice by Sirius red staining.
intensity of IRI; interstitial fibrosis; graft outcome. Area under the curve of serum creatinine for the first 2 weeks after transplantation showed that the 1 g/L and 2 g/L groups were efficient and nearly identical.
HEMO2Life improved kidney graft function. Beneficial use of M101 at lower dosage. [63] Kidney machine preservation in a porcine transplantation model  In hamster: absence of microvascular vasoconstriction and no significant effect on mean arterial blood pressure. In rat: minor effects on mean arterial pressure (differences not statistically significant compared to control), heart rate and myocardial contractility. M101 appears to have no vasoactivity at the microvascular level. [40] Human safety and proof-of-principle study First-in-human use of M101 (1 g/L) for organ preservation.
Open-label study investigating the safety of M101 used ex vivo as an additive to the preservation solution in kidney transplantation (Clinical Trial Registry No. NCT 02652520). Grafts were preserved either in cold storage (standard donor) or on machine perfusion (extended criteria donor).
60 graft kidneys from 60 deceased donors were preserved with M101. M101 is safe for the graft and for the recipient. No allergic or hypersensitivity reactions or infections related to the product were reported. Less DGF (at least one dialysis) in the M101 group was reported, and the difference between the two groups (23% vs. 33%) was clinically relevant but not statistically significant. When a more stringent definition (more than one dialysis) is used, the difference (7% vs. 26%) was statistically significant (p = 0.038).
This study demonstrated that the addition of the oxygen carrier M101 to preservation solution is safe. Although the study was not designed to show the superiority of M101, the analysis of the secondary efficacy end points is highly promising, with better renal function in recipients of the kidneys preserved with M101. [51]

Perspectives
Owing to its interesting O 2 carrying capacity, the potential of M101 as an O 2 transporter for numerous pathologies involving hypoxia is worth exploring. Furthermore, its prospects as a therapeutic O 2 carrier for conditions involving massive hemorrhage, for instance, accidents, terrorist attacks and war injuries should be investigated. M101's potential application as a temporary or intermittent use alternative for reducing the adverse side-effects associated to pathologies requiring frequent blood transfusions should be further investigated. Additionally, with the recent evolution of its anti-inflammatory and anti-bacterial properties, further research is essential to examine its therapeutic potential for the treatment of inflammatory and infectious diseases. Besides, its incorporation into scaffolds should be tested to establish its feasibility in clinical applications.
Interestingly, the product HEMO 2 life ® has been used to preserve a face transplant during a full-face re-transplantation. The patient was transplanted in January 2018 with a graft preserved with HEMO 2 life ® . The surgery was a success and 30 months post-surgery, the patient is safe and the graft has not been rejected [83]. Recently, M101 has also been proposed as a "molecular respirator", a potential tool to help COVID-19 patients in their struggle with hypoxemia [84]. Testing this hypothesis further could be instrumental in developing a potential therapeutic strategy to combat the COVID-19 crisis.

Conclusions
Therapeutic O 2 carriers could be used as graft/organ preservatives and could also present an alternative in cases requiring frequent blood transfusion. However, extensive investigation is warranted to establish their safety and efficacy, especially for their potential clinical application. Moreover, M101, being an efficient O 2 transporter, may be a promising medical product with therapeutic potential for the management of several pathologies.