USE OF HAEMOGLOBIN FROM ANNELIDS FOR TREATING ACUTE RESPIRATORY DISTRESS SYNDROME

Abstract

The present invention relates to the use of a molecule selected from an Annelid globin, an Annelid globin protomer, and an Annelid extracellular haemoglobin, to treat acute respiratory distress syndrome.

Claims

1. A method for treating acute respiratory distress syndrome in patients with a loss of blood oxygen capacity of at least 3 ml O2/dL of blood, comprising administering to said patients at least one molecule selected from an Annelid globin, an Annelid globin protomer and an Annelid extracellular haemoglobin.

2. The method according to claim 1, characterised in that the molecule is selected from extracellular haemoglobins of the family Lumbricidae, extracellular haemoglobins of the family Arenicolidae and extracellular haemoglobins of the family Nereididae, preferably from the extracellular haemoglobin of Lumbricus terrestris, the extracellular haemoglobin of Arenicola sp and the extracellular haemoglobin of Nereis sp, more preferably from the extracellular haemoglobin of Arenicola marina and Nereis virens.

3. The method according to claim 1, characterised in that the molecule is the extracellular haemoglobin of Arenicola marina.

4. The method according to claim 1, characterised in that the acute respiratory distress syndrome is caused by infection with the coronavirus SARS-Cov-2.

5. The method according to claim 1, characterized in that the acute respiratory distress syndrome is present in patients with a loss of blood oxygen-carrying capacity of at least 4 ml O2/dL of blood, preferably at least 5 ml O2/dL of blood.

6. The method according to claim 1, characterised in that the acute respiratory distress syndrome is present in patients with low oxygen saturation, i.e. less than 85%, preferably less than 80%.

7. The method according to claim 1, characterised in that the molecule is formulated in a composition comprising a buffer solution.

8. The method according to claim 7, characterized in that the buffer solution is an aqueous solution comprising salts, preferably chloride, sodium, calcium, magnesium and potassium ions, and gives the composition a pH between 5 and 9, preferably between 5.5 and 8.5, preferably between 6.5 and 7.6, and preferably also comprises at least one stabilizing agent, preferably selected from disaccharides, polyols, antioxidants, maltodextrins and mixtures thereof.

9. The method according to claim 7, characterized in that the molecule is present in the composition in a concentration of between 1 and 200 g/L, preferably between 5 and 100 g/L, more preferably between 10 and 80 g/L.

10. The method according to claim 1, characterised in that the molecule is formulated in a composition in powder form.

11. The method according to claim 1, characterised in that the molecule is in a form adapted to be administered enteral or parenterally, preferably by injection, preferably intramuscularly, subcutaneously, intra-arterially or intravenously, more preferably intravenously.

Description

EXAMPLE

Combating Hypoxaemia in COVID-19 Patients Using Extracellular Haemoglobin From Arenicola marina

[0097] The hypothesis is that intravenous injection of a composition comprising Arenicola marina extracellular haemoglobin (HEMO2Life®) in ARDS due to COVID-19 would improve oxygen transport to the tissues, and that this could prevent progression to multi-organ failure if hypoxaemia persists or worsens.

[0098] This molecule has been administered to humans for transplantation, as an additive in a preservative solution, but never directly by intravenous route.

[0099] The use of extracellular haemoglobin from Arenicola marina is also interesting because of its antioxidant effect, which prevents the cytokine storm induced by SARS-CoV-2. Indeed, the extracellular haemoglobin of Arenicola marina has a superoxide dismutase activity that can solve this problem.

[0100] Extracellular haemoglobin from Arenicola marina can improve tissue oxygenation without altering ventilation for COVID-19 patients. This extracellular haemoglobin has an oxygen binding capacity 40 times greater than vertebrate haemoglobin. Moreover, the size of this molecule is 250 times smaller than a human red blood cell, which allows it to diffuse into all areas of the microcirculation without diffusing out of the vascular sector. This molecule is composed of 156 globin chains and 42 linker chains with a molecular weight of 3.6 MDa. The quaternary structure of this molecule is a hexagonal bilayer with a dimension of 25 nm (front view) and 15 nm (side view). Each globin chain has a heme group similar to human, and the linker chains have an antioxidant property due to a copper and zinc-based superoxide dismutase (SOD)-like activity. Thus, this haemoglobin can carry up to 156 molecules of O2. Oxygen is released against a gradient in the absence of an allosteric effector, supplying the environment with the right amount of O2. It is active over a wide temperature range (4° C. to 37° C.). This molecule has no immunogenic or allergenic effect. It has an oxygen affinity (p50) of 7.5 mm Hg (i.e. similar to that of haemoglobin A (HbA) within the red blood cell), has a cooperativity of 2.5 and does not require a cofactor to release oxygen. In addition, the p50 of myoglobin is 2.6 mm Hg, which is less than 7.5 mm Hg. The release of O2 simply takes place in an oxygen gradient: when the pO2 is lower than the p50, O2 is passively released to the tissues, and consumed by the cells or tissues, avoiding oxidative damage. There is no interaction between the extracellular haemoglobin of Arenicola marina and haemopexin, an important plasma protein in the clearance of haemoglobin.

[0101] SARS-CoV-2 is an enveloped single-stranded RNA virus that replicates in the nuclei of target cells. The DNA in the nucleus of red blood cells is therefore probably one of the targets of the virus, and this explains the leukoerythroblastic reaction described in a patient with COVID-19. To date, few studies have provided data on the use of blood in patients with COVID-19. It has been argued that patients hospitalised with COVID-19 required fewer blood transfusions than other hospitalised patients. Data from Italy showed that 39% of patients required transfusion (median hospital stay 15 days) mainly for anaemia (without bleeding), with very few patients requiring platelets or plasma.

[0102] The extracellular haemoglobin of Arenicola marina is not contained in the cell nucleus and therefore cannot be a target for the virus as SARS-CoV-2 will not recognise this non-red blood cell oxygen carrier. It seems that the virus must attach to the red cell with more affinity for blood group AB, which will not be possible with extracellular haemoglobin. Therefore, this molecule seems well suited to deliver oxygen and avoid hypoxia responsible for dyspnoea, while avoiding being targeted by the virus.

[0103] Another reason to use extracellular haemoglobin from Arenicola marina is related to its oxidative stress reducing properties. SARS-CoV-2 acts on the angiotensin converting enzyme 2 (ACE2) receptor. By binding to the ACE2 receptor, the virus inhibits the conversion of angiotensin II to angiotensin 1,7. The latter is fundamental to NADPH oxidase: this enzyme catalyses the oxidation reaction of NADPH by oxygen, which creates reactive oxygen species (ROS), which are toxic and generate endothelial dysfunction. The extracellular haemoglobin of Arenicola marina, through its SOD-like properties, can reverse this phenomenon by changing O2∘ to O2 or H2O2.

[0104] The extracellular haemoglobin of Arenicola marina also has an action on iron, and may potentially stimulate catalase. COVID-19 causes hypoxia due to anaemia, coagulopathy, thrombosis and multiple organ failure. Lung damage observed on radiographic scans may be caused by the release of oxidative iron from heme groups, overwhelming natural defences against pulmonary oxidative stress; elevated ferritin levels are also found in non-surviving COVID-19 patients compared to surviving patients. The function of catalase is to detoxify free circulating heme, which can cause severe inflammation. Indeed, when iron ions are depleted of haemoglobin, intubation to ventilate is useless as it does not treat the cause of the disease, and iron in free form could be responsible for the cytokine storm due to its very high pro-oxidant activity. The fact that patients return for re-hospitalisation days or weeks after recovery and suffer delayed post-hypoxic leukoencephalopathy reinforces the fact that COVID-19 patients suffer from hypoxia despite no signs of respiratory fatigue or exhaustion.

[0105] Tissue hypoxia, although rarely assessed in the literature, could be an interesting complementary assessment measure.

[0106] Red blood cells carry oxygen from the lungs to all the organs and the rest of the body through haemoglobin. This protein consists of four “hemes”, which contain a special type of iron ion, which is usually quite toxic in its free form, and enclosed in a porphyrin at its centre. In case of COVID-19 infection, the lungs are overwhelmed with oxidative stress, the organs need a lot of O2 and the liver does its best to eliminate and store iron. However, this organ also needs O2, and releases an enzyme called alanine aminotransferase. The patient's immune system cannot fight the virus until the oxygen saturation is too low, and the organs begin to shut down. To avoid this, a maximum supply of oxygen is necessary. The extracellular haemoglobin of Arenicola marina can provide this O2.

[0107] It may also play a role in the treatment of microthrombosis in SARS-CoV-2 infections. Histological analyses of skin and lung patients have shown microvascular lesions and thrombosis associated with severe forms of COVID-19, and a retrospective study of 183 patients shows abnormal coagulation results, in particular high levels of D-dimer and fibrin degradation products in COVID-19 deaths. This microthrombosis is due to a cascade of events causing the destruction of the vascular endothelium by ROS. This microphenomenon of thrombosis can lead to acute respiratory failure and systemic coagulopathy, which are critical to the morbidity and mortality of SARS-CoV-2 infection. As the extracellular haemoglobin of Arenicola marina is 250 times smaller than red blood cells and extracellular, it can cross the thrombus generated by SARS-CoV-2. This hypothesis is also supported by the fact that, in a rat model affected by head trauma, and therefore highly susceptible to intravascular micro-thrombosis, extracellular haemoglobin from Arenicola marina could rapidly reduce acute cerebral hypoxia tissue, avoiding the classical reduction in vessel size without inducing vasoconstriction itself.

[0108] Indeed, the extracellular haemoglobin of Arenicola marina has no vasoconstrictor effect compared to other first or second generation oxygen carriers.

[0109] The extracellular haemoglobin of Arenicola marina is well tolerated and does not induce toxicity. It is pyrogen-free, non-mutagenic, non-cytotoxic and non-irritating. When administered intravenously to hamsters and rats, it showed no acute effects on heart rate and blood pressure, and did not induce microvascular vasoconstriction.

[0110] In another study, fluorescently labelled Arenicola marina extracellular haemoglobin was administered to mice (60 mg/kg, 600 mg/kg, 1200 mg/kg) and was found to be safe, the animals showed no abnormal clinical signs and the half-life of the product was 2.5 days.

[0111] The extracellular haemoglobin of Arenicola marina was evaluated in the human kidney in the OXYOP study (NCT02652520). This study, the first in humans, demonstrated that the addition of extracellular haemoglobin from Arenicola marina to a kidney transplant preservation solution is safe. Although this study was not designed to show the superiority of Arenicola marina extracellular haemoglobin, analysis of the secondary efficacy endpoints shows significantly less delayed graft function recovery and better renal function in recipients of kidneys preserved with this haemoglobin. This study calls for the use of extracellular haemoglobin from Arenicola marina in organ preservation. This also shows the relevance of using this haemoglobin in diseases related to ischaemia-reperfusion injury and hypoxia.

[0112] Some oxygen transporters have been studied and shown to be effective in a preclinical model of ARDS.

[0113] For example, in 2004, Henderson et al. evaluated whether a cross-linked and polymerised bovine haemoglobin (HBOC-201 from Biopure) is an alternative to donor blood for extracorporeal oxygenation in a pig model of ARDS. HBOC-201 appears to be an effective alternative for extracorporeal membrane oxygenation, offering the advantages of rapid availability and reduced exposure to donor blood cells.

[0114] Extracellular haemoglobin from Arenicola marina has not yet been studied in preclinical studies for this condition, but superior efficacy can be expected, as it did not induce vasoconstriction as demonstrated in comparison with first- and second-generation oxygen carriers. Deeply hypoxaemic patients admitted to the ICU under COVID-19 may be a population that could benefit from intravenous administration of Arenicola marina extracellular haemoglobin.

[0115] Given its oxygen-carrying and oxidative stress reduction properties, extracellular haemoglobin from Arenicola marina may be effective in combating hypoxia and oxidative stress caused by SARS-CoV-2.

[0116] It is estimated that the intake of 5 g of this haemoglobin for a 70 kg subject (70 mg/kg), whose blood volume is estimated to be 5 L, represents an increase in arterial O2 content of 1 ml of O2 per 100 mL of blood (or 5% of the “physiological” oxygen content of arterial blood (CaO2) or 7% if the partial pressure of oxygen (PAO2) is 80 mmHg). Administration may be started with a “test dose” of 10 mg to check for anaphylaxis. Then each 1 g dose can be administered intravenously. An assessment of tolerance can be made after each dose, looking for rashes, bronchospasm, hypotension or tachycardia during the next 5 minutes before proceeding to the next dose. If the administration of 70 mg/kg haemoglobin (i.e. 1.4 ml/kg) does not significantly improve tissue oxygenation parameters, and if the dose is well tolerated, then 70 mg/kg haemoglobin can be administered for a total of 140 mg/kg, which corresponds to a 10% increase in CaO2. As this haemoglobin has a 40-fold higher carrying capacity than HbA, it could increase the arterial O2 content in a situation where the pulmonary exchanger is no longer functional, whereas O2 binding and release occurs passively in a simple O2 gradient in the absence of an allosteric effector.

[0117] Extracellular haemoglobin from Arenicola marina could improve survival of COVID-19 patients, avoid tracheal intubation, shorten oxygen supplementation and treat more patients when ventilators are not available.