EP4125997A1

EP4125997A1 - New method to treat the hepatotoxicity induced by amanitins

Info

Publication number: EP4125997A1
Application number: EP21715260.2A
Authority: EP
Inventors: Vincent Lagente; Thomas GICQUEL; Brendan LE DARE; Eric DELPY; Franck Zal
Original assignee: University of Rennes; Institut National de la Santé et de la Recherche Médicale; Hemarina SA; Centre Hospitalier Universitaire de Rennes
Current assignee: University of Rennes; Institut National de la Santé et de la Recherche Médicale; Hemarina SA; Centre Hospitalier Universitaire de Rennes
Priority date: 2020-03-31
Filing date: 2021-03-30
Publication date: 2023-02-08
Also published as: WO2021198216A1

Abstract

The present invention relates to the treatment of intoxication induces by amanitins. The inventors studied the effect of M101 on the amanitins hepatotoxicity. Particularly, by testing the M101 molecule in vitro with the appropriate models, they showed that M101 could exert its hepatoprotective effect against amanitins, at least in part, through its antioxidant properties. Thus, the present invention relates to a haemoglobin for use in the treatment of hepatotoxicity induced by an amatoxin in a subject in need thereof.

Description

NEW METHOD TO TREAT THE HEPATOTOXICITY INDUCED BY AMANITINS

FIELD OF THE INVENTION:

The present invention relates to a haemoglobin for use in the treatment of hepatotoxicity induced by an amatoxin in a subject in need thereof.

BACKGROUND OF THE INVENTION:

In 2017, 1386 cases of mushroom poisoning were recorded in the information system of French poison control centers. Among phalloid syndromes, cases of severe liver damage had required transplantation. The toxicity of this syndrome, attributable to amanitins (a- and b- amanitin), is partially explained by oxidative stress generation (1). The main antidote currently available is silymarin (Legalon®), which enhances the antioxidant systems, including superoxide dismuthase (SOD) (2 and 3). However, since the use of silymarin is still associated with approximately 10% of deaths, the development of new antidotes remains a challenge in toxicology (3).

Ml 01 is an extracellular haemoglobin extracted from the marine worm Arenicola marina, currently used as an oxygen carrier in organ preservation solutions. In addition, in vitro studies have demonstrated its intrinsic SOD activity (4 and 5).

SUMMARY OF THE INVENTION:

In this context, the objective of the inventors was to study the effect of Ml 01 on the amanitins hepatotoxicity. Particularly, by testing the M101 molecule in vitro with the appropriate models, the inventors showed that Ml 01 could exert its hepatoprotective effect against amanitins, at least in part, through its antioxidant properties. Thus, the present invention relates to a haemoglobin for use in the treatment of hepatotoxicity induced by an amatoxin in a subject in need thereof. Particularly, the invention is defined by its claims.

DETAILED DESCRIPTION OF THE INVENTION:

The present invention relates to a haemoglobin for use in the treatment of hepatotoxicity induced by an amatoxin in a subject in need thereof. In a particular embodiment, the haemoglobin is an extracellular haemoglobin from annelids which is present in the three classes of annelids: the polychaetes, the oligochaetes and the achaetes.

In a particular embodiment, the extracellular haemoglobin is not naturally contained in a cell, and can therefore circulate freely in the blood stream without chemical modification to stabilize it or make it functional.

In a particular embodiment, the extracellular haemoglobin from annelids is a giant biopolymer with a molecular weight of between 2000 and 4000 kDa, consisting of approximately 200 polypeptide chains of between 4 and 12 different types which are generally grouped into two categories. The first category, with 144 to 192 components, groups together the “functional” polypeptide chains which bear an active site of heme type, and are capable of reversibly binding oxygen; these are chains of globin type, the weights of which are between 15 and 18 kDa and which are very similar to the a- and b-type chains of vertebrates.

The second category, with 36 to 42 components, groups together the “structural” or “linker” polypeptide chains which have few or no active sites but enable the assembly of the subunits called one-twelfth subunits or protomers. Each haemoglobin molecule consists of two superposed hexagons which have been named hexagonal bilayer, and each hexagon is itself formed by the assembly of six subunits (or “one-twelfth subunits” or “protomers”) in the form of a drop of water. The native molecule is made up of twelve of these subunits (dodecamer or protomer). Each subunit has a molecular weight of between 200 and 250 kDa, and constitutes the functional unit of the native molecule.

Particularly, the extracellular haemoglobin from annelids is chosen from the extracellular haemoglobins from polychaete annelids, preferably from the extracellular haemoglobins from the family Arenicolidae. Even more particularly, the extracellular haemoglobin from annelids is chosen from the extracellular haemoglobin from Arenicola spmore particularly the extracellular haemoglobin from Arenicola marina.

More information can be found in the patent application WO2013030496. In a particular embodiment, the haemoglobin is an extracellular haemoglobin and particularly an extracellular haemoglobin extracted from the marine worm Arenicola marina (also known as Ml 01).

As used herein, the term “haemoglobin” denotes an iron-containing oxygen-transport protein in the red blood cells (erythrocytes) of almost all vertebrates as well as the tissues of some invertebrates.

As used herein, the term "extracellular haemoglobin" refers to a haemoglobin not contained in a cell and dissolved in the blood.

As used herein, the term “extracellular haemoglobin derived from Arenicola marina ” denotes a natural extracellular (i.e. not in a red blood cell) polymeric Hb of the polychaete annelid Arenicola marina (see for example Rousselot et al. Biotechnol. J. 2006).

The extracellular haemoglobin of Arenicola marina is a giant bipolymer of mass about 3 to 4 million daltons and made up of about 200 polypeptide chains of two types. Three quarters are chains of the globin type capable of reversibly binding oxygen (O2) and the remaining quarter are structural chains (“linkers”) which ensure the maintenance of the quaternary structure and are thought to be responsible for the antioxidant activity of this molecule. The functional unit of this molecule is the dodecamer which has a mass lying between 200 and 250 kDa. More information can be found in the patent application W02009050343.

According to the invention, the haemoglobin of the invention can be used in a composition comprising a buffer solution. In this case, the haemoglobin is present in the composition at a concentration of between 0.20 and 1.50 mg/ml.

As used herein, the term “amatoxin” denote a subgroup of at least eight related toxic compounds found in several genera of poisonous mushrooms, most notably the death cap (Amanita phalloides) and several other members of the genus Amanita, as well as some Conocybe, Galerina and Lepiota mushroom species. Amatoxins are lethal at even low doses, as little as half of a mushroom. The amatoxins presents in this subgroup are: the a-amanitin, the b-amanitin, the g-amanitin, the d-amanitin, the e-amanitin, the amanullin, the amanullinic acid, the amaninamide, the amanin and the proamanullin. Particularly, according to the invention, the term the “amatoxin” denotes the “alpha-Amanitin” (a-amanitin) which denotes a cyclic peptide of eight amino acids which is possibly the most deadly of all the amatoxins, toxins found in several species of the mushroom genus Amanita, one being the death cap (Amanita phalloides) as well as the destroying angel, a complex of similar species, principally A. virosa and A. bisporigera. More particularly, according to the invention, the term the “amatoxin” also denotes the “beta-Amanitin” (b-amanitin) which is a cyclic peptide comprising eight amino acids. It is part of a group of toxins called amatoxins, which can be found in several mushrooms belonging to the genus Amanita.

As used herein the term “hepatotoxicity induced by an amatoxin” denotes hepatitis with centrolobular necrosis, apoptosis and hepatic steatosis, as well as acute tubulointerstitial nephropathy, which altogether induce severe liver and kidney failure.

In a particular embodiment, the invention relates to a haemoglobin for use as an antidote against the hepatotoxicity induced by an amatoxin in a subject in need thereof.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

According to the invention, the term “subject” denotes a mammal, such as a rodent, a feline, a canine, and a primate. Particularly, the subject according to the invention is a human. Particularly, the subject denotes human having hepatotoxicity induced by an amatoxin.

The present invention also relates to a method for treating a hepatotoxicity induced by an amatoxin in a subject in need thereof comprising administering to said subject a therapeutically effective amount of a haemoglobin for use in the treatment of hepatotoxicity induced by an amatoxin.

Therapeutic composition

Another object of the invention relates to a therapeutic composition comprising a haemoglobin for use in the treatment of a hepatotoxicity induced by an amatoxin in a subject in need thereof.

Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.

"Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. In a particular embodiment, the compound can be administered orally, intra-nasally, parenterally, intraocularly, intravenously, intramuscularly, intrathecally, intracerebroventricularly, in-utero or subcutaneously to subject in need thereof.

In one embodiment, the compound of the invention is administrated chronically.

As used herein the terms "administering" or "administration" refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., a compound which modifies microglia) into the subject, such as by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.

A “therapeutically effective amount” is intended for a minimal amount of active agent which is necessary to impart therapeutic benefit to a subject. For example, a "therapeutically effective amount" to a subject is such an amount which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with or resistance to succumbing to a disorder. It will be understood that the total daily usage of the compounds of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A drug typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.

The pharmaceutical compositions of the invention can be formulated for a topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular, or subcutaneous administration and the like.

Particularly, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

The doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.

In addition, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used.

Pharmaceutical compositions of the present invention may comprise a further therapeutic active agent. The present invention also relates to a kit comprising an activator according to the invention and a further therapeutic active agent.

For example, compounds used to treat diseases (like hepatotoxicity) induced by the amatoxin (like a-amanitin or b-amanitin) may be added to the pharmaceutical composition as described below. Such molecules may be N-acetylcysteine, benzylpenicillin, cimetidine, thioctic acid, polymyxin B, vitamin C, steroids, silymarin and silybin.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: Effect of a- and b-amanitin on different hepatocyte cell lines viability a- or b-amanitin (0.2 to 100 pM) or medium (CTL) were incubated during 24 h on different hepatocyte cell lines. Cell viability of (A) differentiated HepaRG (B) Progenitors HepaRG and (C) HepG2 cell line was measured by an extracellular ATP assay and expressed relative to the value determined after medium treatment alone (arbitrary set to 100%). The data are quoted as the mean ± SEM from three independent experiments performed in triplicate for HepaRG cells (differentiated and progenitors) and one experiment performed in triplicate for HepG2 cell line. * p < 0.05 ** p < 0.01 *** p < 0.001: the control condition compared with a- or b-amanitin at different concentrations.

Figure 2: Effect of Ml 01 on amanitin hepatotoxicity. a- or b-amanitins (2 pM) or medium alone were incubated on differentiated and progenitors HepaRG during 24 h with or without Ml 01 (1 g/L). Cell viability of (A) differentiated HepaRG and (B) progenitors HepaRG cells was measured by an extracellular ATP assay and expressed relative to the value determined after medium treatment alone (arbitrary set to 100%). The data are presented as the mean ± SEM from three independent experiments performed in triplicate. ** p < 0.01: b- amanitin compared to b-amanitin + M101. # p < 0.05: a-amanitin compared to a-amanitin + M101.

Figure 3: Kinetic of M101 hepatoprotective effect. In (A) differentiated and (B) progenitors HepaRG, a- or b-amanitin (2 mM) or medium alone were incubated during 24 h without or with Ml 01 added up to 6 h after amanitins treatment. Cell viability was measured by an extracellular ATP assay and expressed relative to the value determined after medium treatment alone (arbitrary set to 100%). The data are presented as the mean ± SEM from three independent experiments in triplicate in differentiated HepaRG and one experiment performed in triplicate in progenitor HepaRG. * p < 0.05 ** p < 0.01 *** p < 0.001: a- or b-amanitin compared to a- or b-amanitin + Ml 01 added at different time. # # # p < 0.001: Control condition compared with a- or b-amanitin alone.

Figure 4: Oxidative stress assessment in differentiated HepaRG cells after amanitin treatment with or without M101. (A) a- or b-amanitin (0.2 to 20 mM) or medium (0) were incubated on differentiated HepaRG without Ml 01 (1 g/L) for 24 h at 37°C with 5% C02. (B) a- or b-amanitin (2 pM) or medium (0) were incubated on differentiated HepaRG with or without M101 (1 g/L) for 24 h at 37°C with 5% C02. Mitochondrial ROS 24 h after amanitin treatment were detected using MitoSox (5 pM) probe. The results are expressed as mean fluorescence ± SEM from three independent experiments performed in triplicate and normalized using a protein assay. ** p < 0.01 *** p < 0.001: a- or b-amanitin compared to control condition. # # p < 0.01 # # # p < 0.001: a- or b-amanitin alone compared to a- or b- amanitin + M101.

Figure 5: Oxidative stress assessment in differentiated HepaRG cells after amanitin treatment with or without Ml 01 added up to 6 h after amanitin. a- or b-amanitin (2 pM) or medium alone were incubated on differentiated HepaRG with or without Ml 01 (1 g/L) for 24 h at 37°C with 5% C02. Mitochondrial ROS 24 h after amanitin treatment were detected using MitoSox (5 pM) probe. The results are expressed as mean fluorescence ± SEM from three independent experiments performed in triplicate and normalized using a protein assay. ** p < 0.01 *** p < 0.001: a- or b-amanitin compared to a- or b-amanitin + M101 added at different time. # # # p < 0.001 : Control condition compared with a- or b-amanitin alone

EXAMPLE:

Material & Methods

Test article information

- Denomination: M101

• Supplier: Hemarina®

• Batch: S208/ORGAN/FC003

• Quantity : 20 mL/vial (concentration ~50 mg/ml)

• Vehicle: none

• Storage: between -20°C and -80°C Test system

In order to choose the most suitable model for studying the amanitins hepatotoxicity, four hepatic cell models were used:

• A progenitor HepaRG hepatocyte cell line

• A differentiated HepaRG hepatocyte cell line

• A HepG2 cell line from American Tissue Culture Collection (ATCC, Los Altos, CA, USA)

Materials

• PBS, William’s E medium, Hanks' balanced salt solution (HBSS), penicillin- streptomycin, L-glutamine (Life Technologies, Eugene, OR, USA).

• Fetal Calf Serum (FCS) (Hyclone, Logan, UT, USA).

• Insulin from Sigma Aldrich (St. Louis, MO, USA).

• Hydrocortisone from Sigma Aldrich (St. Louis, MO, USA).

• a- and b-amanitin from Sigma Aldrich (St. Louis, MO, USA).

• Luminescent ATP Detection Assay Kit (Abeam, Cambridge, MA, USA).

• Pierce BCP Protein Assay Kit (ThermoFisher Scientific, Germany)

• MitoSOX red mitochondrial superoxide indicator and cell-permeant 2', 7'- dichlorodihydrofluorescein diacetate (H2-DCFDA) reactive oxygen species (ROS) indicator (ThermoFisher Scientific, Waltham, MA, USA).

Cell culture and treatment

HepaRG progenitor cells were seeded at a density of 3 ^c 104 cells/cm2. These cells were cultured in William’s E medium supplemented with 10% of FCS, 5 pg/mL insulin, 2 mM L-glutamine, 50 units/mL penicillin, 50 pg/mL streptomycin and 50 pM Hydrocortisone hemisuccinate sodium salt. Cells were used 4 days after plating. These HepaRG progenitor cells were exposed to various concentrations of a- and b-amanitin (0.2 to 100 pM) for 24 h . The viability was performed by extracellular ATP assay according to the manufacturer’s instructions.

Differentiated HepaRG were obtained from HepaRG progenitor cells. Two weeks after plating, the cells were maintained for 2 more weeks in the same William’s E medium further supplemented with 2% dimethylsulfoxide (DMSO) in order to obtain the full hepatocyte differentiation. Cells will be used 4 days after plating. Differentiated HepaRG cells were exposed to various concentrations of a- and b-amanitin (0.2 to 100 pM) for 24 h . The viability was performed by extracellular ATP assay.

HepG2 cells were seeded at a density of 6.6x104 cells/cm² in 96-well plates in MEM Eagle medium supplemented with 10% FBS, 50 UI/mL penicillin, 50 pg/mL streptomycin and 4 mM L-glutamine. Cells were used 4 days after plating and exposed to various concentrations of a- and b-amanitin (0.2 to 100 pM) for 24 h. The viability was performed by extracellular ATP assay.

These three cell types were also incubated in the presence of amanitins (2 pM in accordance to literature data and our preliminary results (6 and 7)) with or without Ml 01 (1 g/L) for 24h in order to investigate its antidotic effect. Cell viability was also assessed following the addition of Ml 01 lh, 3h and 6h after amanitin administration (2 pM) by extracellular ATP assay.

Measurement of oxidative stress

The cell-permeant 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) (ThermoFisher Scientific, MA, USA) and the MitoSOX Red Mitochondrial Superoxide Indicator (ThermoFisher Scientific, MA, USA) were used to measure the total ROS production and mitochondrial ROS production respectively. Briefly, after 24h treatment as previously described, cells are incubated with 100 pL of MitoSox (5 pM) or 100 pL of H2-DCFDA (2 pM) probes during 30 minutes at 37°C and 5% C02 in the dark. After the incubation time, medium is removed and cells are washed once with 100 pL of HBSS at room temperature. Multi-wells are then quickly read using a fluorescence microplate reader (POLARstar Omega®, BMG labtech®, Ortenberg, Germany) and data are analyzed using MARS software (BMG labtech®, Ortenberg, Germany). Results are normalized using a protein concentration assay (Pierce BCA protein assay Kit®, ThermoFisher Scientific®, Germany).

Statistical analysis

Data were expressed as the mean ± standard error of the mean (SEM). Intergroup differences as a function of the treatment were probed in a one-way analysis of variance (ANOVA), with a Bonferroni post hoc test for group comparisons. All analyses were performed using Prism software (version 5.0, GraphPad Software, La Jolla, CA, USA). All tests were two- sided, and the threshold for statistical significance was set to p<0.05. Results

Three types of cells were used in these preliminary results: A progenitor HepaRG hepatocyte cell line, a differentiated HepaRG hepatocyte cell line and a HepG2 hepatocyte cell line. The results are presented in the figure 1 to 5.

The a- and b-amanitin dose-dependently decreased cell viability observed in our differentiated and progenitor HepaRG cells. Considering the 50% decrease in cell viability in the differentiated HepaRG cells using 2 mM amanitin (Figure 1A), this concentration was used for the following experiments. Furthermore, these results are consistent with literature data, were 2 pM of amanitin is the most frequently used concentration. Progenitor HepaRG cells (Figure IB) were less sensitive to the amanitins toxicity since only 25% decrease in cell viability was found using 2 pM. HepG2 cells viability was not affected by increasing amanitins concentrations, which is consistent with literature data showing an absence of hepatic amanitin transporters (particularly OATP1B3) in this cell line (Figure 1C). This cell model was therefore not used for the following experiments.

The Ml 01 treatment (1 g/L) significantly decreased hepatotoxicity after a- and b- amanitin exposure during 24 h in differentiated HepaRG cells (Figure 2A). However, M101 did not significantly decrease amanitin hepatotoxicity in progenitor HepaRG cells (Figure 2B).

In differentiated HepaRG, Ml 01 induced a decrease in the amanitins toxicity when added up to three hour after a-amanitin and six hours after b-amanitin (Figure 3A). In progenitor HepaRG, although cell viability was not significantly altered under any conditions, a tendency was noted for Ml 01 to have a protective effect when incubated at the same time than amanitins (Figure 3B). This effect decreased as M101 is added later, suggesting a protective effect rather than a cellular repair effect.

In order to evaluate whether Ml 01 exerts its protective effect against amanitins hepatotoxicity through an antioxidant action, mitochondrial ROS production was measured using MitoSox probe.

Using MitoSox probe, amanitins (0 to 20 pM) dose-dependently increased mitochondrial ROS production at 24 h (Figure 4A). Furthermore, a decrease in amanitin- induced mitochondrial ROS production was observed in the presence of M101 after 24 h of amanitin treatment (Figure 4B). Taken together, these results suggest that Ml 01 exerts its effects through an antioxidant action.

In order to investigate the Ml 01 antioxidant effect limits, mitochondrial ROS production using MitoSox probe was measured after amanitin treatment with or without Ml 01 added up to 6 h after amanitins. Mitochondrial ROS production was higher as M101 was added later. Thus, mitochondrial ROS production was significantly lower when Ml 01 was added together with a-amanitin. In addition, mitochondrial ROS production was found to be significantly lower when Ml 01 was added 6 h after b-amanitin (Figure 5). Interestingly, these results are inversely related to cell viability data obtained in the same conditions (Figure 3A- B). Taken together, these results suggest that M101 could exert its hepatoprotective effect against amanitins toxicity through its antioxidant property.

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

1: Garcia et al. Amanita phalloides poisoning: Mechanisms of toxicity and treatment. Food Chem Toxicol. 2015 Dec;86:41-55.

2: Ye Y, Liu Z. Management of Amanita phalloides poisoning: A literature review and update. J Crit Care. 2018 Aug;46: 17-22.

3: Wellington K, Jarvis B. Silymarin: a review of its clinical properties in the management of hepatic disorders. BioDrugs. 2001;15(7):465-89.

4: Thuillier et al. Supplementation with a new therapeutic oxygen carrier reduces chronic fibrosis and organ dysfunction in kidney static preservation. Am J Transplant. 2011 Sep;l 1(9): 1845-60.

5 : Roussel ot Morgane, Eric Delpy, Christophe Drieu La Rochelle, Vincent Lagente, Ralph Pirow, Jean-Francois Rees, Agnes Hagege, Dominique Le Guen, Stephane Hourdez and Franck Zall. Arenicola marina extracellular hemoglobin: a new promising blood substitute. Biotechnol. J. 2006, 1, 333-345.

6: Magdalan J et al. alpha-Amanitin induced apoptosis in primary cultured dog hepatocytes. Folia Histochem Cytobiol. 2010 Jan l;48(l):58-62. 7: Magdalan J et al. Influence of commonly used clinical antidotes on antioxidant systems in human hepatocyte culture intoxicated with alpha-amanitin. Hum Exp Toxicol. 2011 Jan;30(l):38-43.

Claims

CLAIMS:

1. A haemoglobin for use in the treatment of hepatotoxicity induced by an amatoxin in a subject in need thereof.

2. A haemoglobin for use according to claim 1 wherein the haemoglobin is an extracellular haemoglobin from annelid.

3. A haemoglobin for use according to claim 2 wherein the extracellular haemoglobin from annelids is chosen from the extracellular haemoglobins from the family Arenicolidae.

4. A haemoglobin for use according to claim 3 wherein the haemoglobin is an extracellular haemoglobin extracted from the marine worm Arenicola marina also known as M101.

5. A therapeutic composition comprising a haemoglobin for use in the treatment of a hepatotoxicity induced by an amatoxin in a subject in need thereof.

6. A method for treating a hepatotoxicity induced by an amatoxin in a subject in need thereof comprising administering to said subject a therapeutically effective amount of a haemoglobin for use in the treatment of hepatotoxicity induced by an amatoxin.