WO2017134043A1 - Methods and pharmaceutical composition for neutralising the cytotoxic activity of extracellular histone proteins in subjects suffering from sepsis - Google Patents

Abstract

The present invention relates to methods and pharmaceutical composition for diagnosing patients suffering from sepsis. Inventors have shown that the clusterin interacts with histones and histone-clusterin complexes are detected in higher levels in septic shock patients than in healthy subjects. More particularly, in a prospective study, they quantified the circulating levels of clusterin and of nucleosomes in the serums of patients admitted in the intensive care units (ICU) for sepsis. Results showed that the levels of clusterin were significantly reduced in sepsis patients at their admittance in ICU, compared to the levels in healthy subjects. Thus, the invention relates to neutralize or inhibit the the cytotoxic activity of extracellular histone proteins in patients as diagnosed according to the invention with a therapeutically effective amount of clusterin.

Description

METHODS AND PHARMACEUTICAL COMPOSITION FOR NEUTRALISING THE CYTOTOXIC ACTIVITY OF EXTRACELLULAR HISTONE PROTEINS IN SUBJECTS SUFFERING FROM SEPSIS

FIELD OF THE INVENTION:

The present invention is in the field of infectious disease, more particularly, the invention relates to methods and pharmaceutical composition for neutralising or inhibiting the cytotoxic activity of extracellular histone proteins in subjects suffering from sepsis.

BACKGROUND OF THE INVENTION:

Sepsis is a systemic reaction characterized by arterial hypotension, metabolic acidosis, decreased systemic vascular resistance, tachypnea and organ dysfunction. Sepsis (including septic shock) is characterized by a systemic inflammatory response which results from the activation of a number of host defense mechanisms including the release of cytokines, the activation of immune cells, of the complement system and of the coagulation pathway. Interestingly, Systemic Inflammatory Response Syndrome (SIRS) has been shown not only to be associated with infections in the context of sepsis or septic shock, but also to several other life-threatening conditions including, non-exhaustively, severe burns, polytrauma, acute pancreatitis, and cardiac arrest. Because of the severity of the disease, patients with SIRS are usually referred to Intensive Care Unit where they receive a rapid effective treatment. While enormous efforts have been made in understanding the basic molecular mechanisms that underlie the pathophysiology of sepsis and SIRS, a long list of novel agents have been tested in clinical trials without a single immunomodulating therapy showing consistent benefit. The only agent to successfully complete a phase III clinical trial in human sepsis was human recombinant activated protein C. Unfortunately, this drug has been recently taken off the market after a follow-up placebo-controlled trial that failed to replicate the favorable initial results. With an incidence increasing owing to the ageing population, sepsis is still associated to a high mortality rate, rendering new treatments absolutely necessary.

Clusterin (Clu), also known as apolipoprotein J, is a soluble 80 kDa disulfide-linked heterodimeric glycoprotein, highly conserved during evolution and among mammals. Clusterin is abundant in physiologic fluids (concentrations ranging from 100 to 300 μg/ml in human serum as an example), and is induced in response to a wide variety of cell and tissue injuries. Clusterin has a chaperone activity and is a functional homologue to the intracellular small heat shock proteins (HSP) (10, 11). It binds hydrophobic domains of non-native proteins and targets them for receptor-mediated internalization and intracellular lysosomal degradation (10). This function allows clusterin to interact with a broad spectrum of molecules, such as lipids, components of the complement system, amyloid-forming proteins, and immunoglobulins (12, 13). Although Clu can interact with different molecules implicated in immune function, its potential role in immune responses remains unclear. It has been reported that Clu binds to whole bacteria (Staphylococcus aureus and some Staphylococcus epidermidis strains) and bacterial proteins (such as the Streptococcus pyogenes extracellular protein SIC) (14-16), suggesting that it may modulate anti- microbial responses. Moreover, Clu limits the severity of induced autoimmune myocarditis (17) and pancreatitis (18). Finally, the levels of circulating Clu in systemic lupus erythematosus (SLE) (6), as well as Clu mRNA expression in the synovium of subjects with rheumatoid arthritis, are decreased. (19). These observations suggest a role for Clu in autoimmune disorders but its ability to reduce histone cytoxicity has never been explored in the prior art.

SUMMARY OF THE INVENTION:

The present invention relates to methods and pharmaceutical composition for neutralising or inhibiting the cytotoxic activity of extracellular histone proteins in subjects suffering from sepsis. In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

Clusterin is an extracellular chaperon which expression and activity are enhanced in conditions associated with stress. Clusterin chaperons misfolded proteins, thereby preventing their precipitation/aggregation and favouring their clearance by the liver. Here the inventors report that clusterin chaperons histones in vitro and in vivo. Clusterin interacts with histones and histone-clusterin complexes are detected in higher levels in septic shock patients than in healthy subjects. Clusterin binds to histones and prevents histone-induced endothelial cell death, neutrophil extracellular traps generation and proinflammatory cytokine secretion by monocytes. Importantly, results obtained in vitro using clusterin-depleted human serum or in vivo using clusterin-deficient mice show that the protective effect of clusterin against histone- induced cell activation or cell injury is not redundant. The inventors report that clusterin is stored in platelets and neutrophils and that histones or microbial components (such as LPS) induce its secretion. Histones and LPS also induce the neosynthesis of clusterin by myeloid cells.

Although histones and LPS induce the release of preformed clusterin and the neosynthesis of clusterin (as evidenced by a high expression of clusterin mRNA in circulating leucocytes), the levels of circulating clusterin collapse in septic shock patients in a higher extent in non-survivor than in survivors patients, with a paralleled increase of non-chaperoned circulating histones. These results suggest that, in non-survivor patients, the capacity of clustenn to neutralize histones are overwhelmed (as illustrated by the presence of higher levels of circulating non-chaperoned histones), allowing histones to exert their deleterious activities. Finally, the inventors show that clusterin supplementation increases the survival of mice in histones- and LPS-induced septic shock models and that clusterin-deficient mice are more susceptible than WT mice to cecal and ligation puncture, a model of bacterial sepsis. Thus, clusterin could provide new therapeutic approach to treat patients suffering from sepsis.

Method for neutralising or inhibiting cytotoxic activity of histones A first object of the invention relates to a method for neutralising or inhibiting the cytotoxic activity of extracellular histone proteins in a subject suffering from sepsis comprising administering to the subject a therapeutically effective amount of clusterin.

The term "neutralising or inhibiting" refers to interfering with activation, function or expression of the extracellular histone proteins.

As used herein the term "histone" has its general meaning in the art. Histones are small, basic proteins with a high content of lysine or arginine and function in the packaging of DNA. Histones are highly conserved and can be grouped into five major classes: H1/H5, H2A, H2B, H3, and H4 organised into two super-classes of the core histones (H2A, H2B, H3 and H4) and the linker histones (HI and H5). As described herein a histone protein may be a full length histone, a fragment or variant thereof. A histone variant may be modified by, for example, the deletion, addition and/or substitution of amino acid(s). Alternatively, a histone may be modified by acetylation and/or methylation of lysine and arginine. In general, the modifications do not substantially compromise the polycationic nature of the histone or the ability of the histone to localise in an organ.

The term "subject" refers to a subject that presents one or more symptoms indicative of sepsis (e.g. chills and shivering, breathing difficulty, high temperature...), or that is screened for sepsis (e.g., during a physical examination).

The term "sepsis" has its general meaning in the art and includes without limitation to sepsis associated inflammation, severe sepsis (sepsis associated with hypoperfusion or dysfunction of at least one organ system) and septic shock (abnormal distribution of blood flow in the organs). Thus, the term "subject suffering from sepsis" refers to a subject that presents one or more symptoms indicative of sepsis (e.g. chills and shivering, breathing difficulty, high temperature...), or that is screened for sepsis (e.g., during a physical examination). As used herein, the term "clusterin" has its general meaning in the art and refers to the glycoprotein originally derived from ram rete testes, and to homologous proteins derived from other mammalian species, including humans, whether denominated as clusterin or an alternative name. The sequences of numerous clusterin species are known. An exemplary amino acid sequence of clusterin is SEQ ID NO: 1. Typically the clusterin is recombinantly produced.

SEQ ID NO:l Clusterin_ homo sapiens

mmktlllfvg llltwesgqv lgdqtvsdne lqemsnqgsk yvnkeiqnav ngvkqiktli ektneerktl lsnleeakkk kedalnetre setklkelpg vcnetmmalw eeckpclkqt cmkfyarvcr sgsglvgrql eeflnqsspf yfwmngdrid sllendrqqt hmldvmqdhf srassiidel fqdrfftrep qdtyhylpfs lphrrphfff pksrivrslm pfspyeplnf hamfqpflem iheaqqamdi hfhspafqhp ptefiregdd drtvcreirh nstgclrmkd qcdkcreils vdcstnnpsq aklrreldes lqvaerltrk ynellksyqw kmlntsslle qlneqfnwvs rlanltqged qyylrvttva shtsdsdvps gvtevvvklf dsdpitvtvp vevsrknpkf metvaekalq eyrkkhree

In some embodiments, clusterin has at least 70% of identity of SEQ ID NO: l. According to the invention, a first amino acid sequence having at least 70% of identity with a second amino acid sequence means that the first sequence has 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; or 99% of identity with the second amino acid sequence and conserving biological properties of said second amino acid sequence. Amino acid sequence identity is preferably determined using a suitable sequence alignment algorithm and default parameters, such as BLAST P (20). In particular, variant is a functional conservative variant of. As used herein the term "function- conservative variants" are those in which a given amino acid residue in a protein or enzyme has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like). Accordingly, a "function-conservative variant" also includes a polypeptide which has at least 70 % amino acid identity and which has the same or substantially similar properties or functions as the native or parent protein to which it is compared (i.e. charperon histones).

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 patient 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. By a "therapeutically effective amount" is meant a sufficient amount of clusterin for reaching a therapeutic effect (e.g. treating sepsis). It will be understood, however, that the total daily usage of clusterin 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, half-life and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidential with the specific polypeptide 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 medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically 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.

In some embodiments, the method of the present invention comprises the steps consisting of i) determining the level of clusterin, circulating histones, circulating nucleosomes, histones-clusterin complexes, dsDNA or ssDNA in a biological sample obtained from the subject, ii) comparing the levels determined at step i) with predetermined reference values and iii) administering to said subject a therapeutically effective amount of clusterin when the level determined at step i) is lower than the predetermined reference value.

As used herein, the "circulating histones" refers to the histones which are circulating without clusterin. Typically, the presence of circulating histones indicates that the level of clusterin is exhausted. Thus, there is a need to administer the subject with a therapeutically effective amount of clusterin.

As used herein, the term "histone-clusterin complex" refers to the complex formed when clusterin binds to a histone protein. The term "biological sample" has its general meaning. A biological sample is generally obtained from a subject. Frequently, a sample will be a "clinical sample", i.e., a sample derived from a patient. Such samples include, but are not limited to, bodily fluids which may or may not contain cells, e.g., blood (e.g., whole blood, serum or plasma), synovial fluid, saliva, tissue or fine needle biopsy samples, and archival samples with known diagnosis, treatment and/or outcome history. Biological samples may also include sections of tissues such as frozen sections taken for histological purposes. The term "biological sample" also encompasses any material derived by processing a biological sample. Derived materials include, but are not limited to, cells (or their progeny) isolated from the sample, or proteins extracted from the sample. Processing of a biological sample may involve one or more of: filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, and the like. In some embodiments of the invention, the biological sample is a blood sample i.e. a whole blood, a serum sample or a plasma sample obtained from a subject. In some embodiments, the biological sample is any sample liable to contain histones and clustenn.

In some embodiments, the step i) consists in determining the level of H4-clusterin complex in the biological sample obtained from the subject suffering from sepsis.

According to the invention, the measure of the level of clusterin or histone-clusterin complexes can be performed by different techniques. Typically, the methods may comprise contacting the biological sample with a set of binding partners capable of selectively interacting with the histone-clusterin complexes in the biological sample. In some aspects, the binding partners are antibodies, such as, for example, monoclonal antibodies or even aptamers. Typically, the aforementioned assays may combine the use of binding partners specifically for clusterin optionally with the use of binding partners specifically for histone.

In some embodiments a couple of a first binding partner specific for clusterin, (or alternatively specific for the histone protein, nucleosomes, dsDNA, or ssDNA) and second binding partner specific for the histone protein (or alternatively specific for the clusterin protein, nucleosomes, dsDNA, or ssDNA) is used to create a "sandwich" assay. For instance, a first binding partner (ie. an antibody or aptamer specific for the histone protein or clusterin, or nucleosomes, ssDNA, or dsDNA) is bound to a solid support. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like. The histone- clusterin complex is then revealed with the second binding partner (ie. antibody or aptamer specific for the histone protein or clusterin). Typically, the second binding partner is an enzyme-conjugated specific antibody. The level of histone-clusterin complexes may be measured by using standard immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays. Such assays include, but are not limited to, agglutination tests; enzyme-labelled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; Immunoelectrophoresis; immunoprecipitation. An exemplary biochemical test for identifying specific proteins employs a standardized test format, such as ELISA test, although the information provided herein may apply to the development of other biochemical or diagnostic tests and is not limited to the development of an ELISA test (see, e.g., Molecular Immunology: A Textbook, edited by Atassi et al. Marcel Dekker Inc., New York and Basel 1984, for a description of ELISA tests). It is understood that commercial assay enzyme-linked immunosorbant assay (ELISA) kits for various plasma constituents are available.

A predetermined reference value can be relative to a number or value derived from population studies, including without limitation, subjects of the same or similar age range, subjects in the same or similar ethnic group, and subjects having the same severity of the disease. Such predetermined reference values can be derived from statistical analyses and/or risk prediction data of populations obtained from mathematical algorithms and computed indices of the disease. In some embodiments, the predetermined reference values are derived from the level of clusterin or histone-clusterin complexes in a control sample derived from one or more subjects who were not subjected to the disease. Furthermore, retrospective measurement of the level of clusterin or histone-clusterin complexes in properly banked historical subject samples may be used in establishing these predetermined reference values.

In some embodiments, the predetermined reference value is correlated with the duration of the disease-free survival (DFS) and/or the overall survival (OS). Accordingly, the predetermined reference value may be typically determined by carrying out a method comprising the steps of:

a) providing a collection of biological samples from subject suffering from the sepsis; b) providing, for each biological sample provided at step a), information relating to the actual clinical outcome for the corresponding subject (i.e. the duration of the disease-free survival (DFS) and/or the overall survival (OS));

c) providing a serial of arbitrary quantification values; d) determining the level of histone-clusterin complexes for each biological sample contained in the collection provided at step a);

e) classifying said biological samples in two groups for one specific arbitrary quantification value provided at step c), respectively: (i) a first group comprising biological samples that exhibit a quantification value for level that is lower than the said arbitrary quantification value contained in the said serial of quantification values; (ii) a second group comprising biological samples that exhibit a quantification value for said level that is higher than the said arbitrary quantification value contained in the said serial of quantification values; whereby two groups of biological samples are obtained for the said specific quantification value, wherein the blood samples of each group are separately enumerated; f) calculating the statistical significance between (i) the quantification value obtained at step e) and (ii) the actual clinical outcome of the subjects from which biological samples contained in the first and second groups defined at step f) derive;

g) reiterating steps f) and g) until every arbitrary quantification value provided at step d) is tested;

h) setting the said predetermined reference value as consisting of the arbitrary quantification value for which the highest statistical significance (most significant) has been calculated at step g).

For example the level of clusterin or histone-clusterin complexes has been assessed for 100 biological samples of 100 subjects. The 100 samples are ranked according to the level of clusterin of histone-clusterin complexes. Sample 1 has the highest level and sample 100 has the lowest level. A first grouping provides two subsets: on one side sample Nr 1 and on the other side the 99 other samples. The next grouping provides on one side samples 1 and 2 and on the other side the 98 remaining samples etc., until the last grouping: on one side samples 1 to 99 and on the other side sample Nr 100. According to the information relating to the actual clinical outcome for the corresponding subject, Kaplan Meier curves are prepared for each of the 99 groups of two subsets. Also for each of the 99 groups, the p value between both subsets was calculated. The predetermined reference value is then selected such as the discrimination based on the criterion of the minimum p value is the strongest. In other terms, the level of clusterin or histone-clusterin complexes corresponding to the boundary between both subsets for which the p value is minimum is considered as the predetermined reference value. It should be noted that the predetermined reference value is not necessarily the median value of levels of clusterin or histone-clusterin complexes. Thus in some embodiments, the predetermined reference value thus allows discrimination between a poor and a good prognosis with respect to DFS and OS for a subject. Practically, high statistical significance values (e.g. low P values) are generally obtained for a range of successive arbitrary quantification values, and not only for a single arbitrary quantification value. Thus, in one alternative embodiment of the invention, instead of using a definite predetermined reference value, a range of values is provided. Therefore, a minimal statistical significance value (minimal threshold of significance, e.g. maximal threshold P value) is arbitrarily set and a range of a plurality of arbitrary quantification values for which the statistical significance value calculated at step g) is higher (more significant, e.g. lower P value) are retained, so that a range of quantification values is provided. This range of quantification values includes a "cut-off" value as described above. For example, according to this specific embodiment of a "cut-off" value, the outcome can be determined by comparing the level of clusterin or histone-clusterin complexes with the range of values which are identified. In certain embodiments, a cut-off value thus consists of a range of quantification values, e.g. centered on the quantification value for which the highest statistical significance value is found (e.g. generally the minimum p value which is found). For example, on a hypothetical scale of 1 to 10, if the ideal cut-off value (the value with the highest statistical significance) is 5, a suitable (exemplary) range may be from 4-6. Therefore, a subject may be assessed by comparing values obtained by measuring the level of clusterin or histone-clusterin complexes, where values lower than 5 reveal a poor prognosis and values greater than 5 reveal a good prognosis). In some embodiments, a subject may be assessed by comparing values obtained by measuring the level of clusterin or histone-clusterin complexes and comparing the values on a scale, where values above the range of 4-6 indicate a good prognosis and values below the range of 4-6 indicate a poor prognosis, with values falling within the range of 4-6 indicating an intermediate prognosis.

Pharmaceutical composition

According to the invention, clusterin is typically administered to the subject suffering from sepsis in the form of a pharmaceutical composition.

Typically, clusterin may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer 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 the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal, intraarterial and intranasal administration forms and rectal administration forms. Typically, 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 pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. Clusterin can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. 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: Decreased serum levels of clusterin in septic shock patients. Circulating levels of clusterin were quantified by ELISA in the serums of healthy subjects (n=36) and septic shock patients (n=50) (A), or in the serum of surviving (n=26) and non surviving (n=24) septic shock patients (B). Horizontal bars represent mean + SEM. C, Western blotting analysis of clusterin in the serums of septic shock patients (n=8) and healthy subjects (n=4). 75 μg of proteins were loaded in each lane. At the bottom, the corresponding serum levels of clusterin (CLU) quantified by ELISA. D, Clusterin mRNA expression was analyzed by RT- qPCR in peripheral blood mononuclear cells of 10 healthy subjects and 5 septic shock patients. Results are expressed in relative expression compared to GAPDH mRNA (mean + SEM). E, Circulating levels of nucleosomes were quantified by ELISA in the serums of healthy subjects (n=36) and septic shock patients (n=50). Horizontal bars represent mean + SEM. A, B, D & E, results are analyzed with the Mann Whitney U test.

Figure 2: Elevated levels of clusterin-H4 complexes in septic shock patients. A, Clusterin-H4 complexes were quantified by ELISA in the serums of septic shock patients (n=50) and healthy subjects (n=36). Horizontal bars are mean ± SEM. B, Serums from healthy subjects (n=5) were supplemented with the indicated doses of recombinant H4 and clusterin-H4 complexes were quantified by ELISA. C, Serums from two septic shock patients (lanes 1 and 2) were immunoprecipitated with an anti-clusterin mAb and the presence of clusterin-histone complexes was revealed by Western-Blotting with a pan anti-Histone Ab. D, The binding of 1.5 nM biotinylated clusterin (CLU), CRP and HSA to plates coated with recombinant H2A, H2B, H3, H4 or with calf thymus histones was analyzed by ELISA. A, B & D, results are analyzed with the Mann Whitney U test and expressed in optical density (OD) values as mean + SEM.

Figure 3: Clusterin prevents histone-induced inflammation and cell death. A&B, Monocytes from 5 healthy subjects were incubated in serum-free medium with increasing concentrations of calf thymus histones. C&D, Monocytes (n=5) were stimulated or not with 50 μg/ml calf thymus histones, 50 ng/ml LPS or 50 ng/ml IL-Ιβ in the absence or presence of the indicated concentrations of clusterin (CLU) or a2-macroglobulin (a2M). E&F, Monocytes were incubated or not with 25 μg/ml clusterin, 50 μg/ml calf thymus histones, or 200 ng/ml activated protein C (APC), in serum- free medium supplemented with 10% serum from healthy subjects (n=4) or septic shock patients (n=4). A-F, IL-6 (A, C, E) and TNFa (B, D, F) were quantified in supernatants after 24 h. Results are expressed as mean + SEM (Mann Whitney U test). G&H, Endothelial cells, in medium containing 10% of serum from 6 healthy subjects (G) or septic shock patients (H), were stimulated or not with 50 μg/ml calf thymus histones or clusterin. Percentages of Annexin V⁺ apoptotic cells were analyzed after 24 h. I, Neutrophils were stimulated or not with 30 nM PMA or 50 μg/ml calf thymus histones in the presence or not of 25 μg/ml clusterin. Extracellular DNA was labeled using SYTOX green and fluorescence were quantified after 1 h incubation. Results are expressed as mean + SEM, n=6 (Mann Whitney U test).

Figure 4: Histones induce clusterin expression. A, Monocytes were stimulated with

25 μg/ml histone H4 or calf thymus histones, with IL-Ιβ plus TNFa (both used at 50 ng/ml), or with 100 pg/ml LPS. Clusterin was quantified by ELISA in the 24-hour supernatants. B, PBMC were stimulated with 25 μg/ml calf thymus histones, and clusterin and IL-6 mRNA were analyzed at the indicated time-points by RT-qPCR. C, Human neutrophils were stimulated with 25 μg ml calf thymus histones or 200 ng/ml LPS and clusterin was quantified by ELISA in the 3-hour supernatants. A, B, D & E. Results are expressed as mean + SEM, n=5.

Figure 5: Clusterin protects mice from histone-induced septic shock. Mice were injected intravenously with PBS (n=7), 100 mg/kg calf thymus histones (n=7) or with 100 mg/kg calf thymus histones plus 30 mg/kg clusterin (CLU) (n=7). Results are expressed in percentage of mice survival. Survival curves were done using Kaplan-Meir method and compared using the Log-rank test.

Figure 6: Clusterin protects mice from LPS-induced septic shock. Mice were injected intraperitoneally with 100 mg/kg LPS and, one hour later, were injected intravenously with PBS or with 10 mg kg murine clusterin (CLU). Results are expressed in percentage of mice survival and are representative of 2 different experiments. Survival curves were done using Kaplan Meir method and compared used the Log-rank test.

Figure 7: Clusterin-deficient mice are more sensitive to CLP than wild type mice. Wild type (WT) and clusterin-deficient (CLU^{" _}) mice were subjected to cecal ligation and puncture (CLP), a model of bacterial sepsis. Mice survival was monitored daily until day 15. * p<0.05; Wilcoxon text.

Figure 8: Analysis of clusterin levels in patients suffering from sepsis. Circulating clusterin and nucleosomes were quantified by ELISA in the serums of patients admitted in intensive care units (ICU) of the University Hospital of Angers and of Bordeaux for sepsis. Serums were collected at their admittance and every 3 days until leaving the ICU. The levels of clusterin ^g/ml) and of nucleosomes (AU/ml) were determined in patients surviving (survivors; white histograms) or not (non survivors; grey histograms) from sepsis. As control, clusterin and nucleosomes were also quantified in healthy subjects (n=10; black histograms). Results are expressed as mean + SEM; the numbers of serums from sepsis patients tested at day 0, day 3 and day 7 are mentioned. * p<0.05; ** p<0.01; Mann Withney U test.

EXAMPLE: NON REDUNDANT ROLE OF THE EXTRACELLULAR CHAPERONE CLUSTERIN IN HOST TOLERANCE AGAINST EXTRACELLULAR HISTONE-MEDIATED TOXICITY

Circulating histone-clusterin complexes in septic shock patients. We observed that septic shock patients exhibited lower levels of circulating clusterin, as assessed by ELISA, than healthy subjects (Fig. 1A), with a more profound decrease in non-surviving than in surviving patients (Fig. IB). As clusterin-client complexes may lead to underestimate clusterin quantification by ELISA, we compared clusterin expression by western blotting in septic shock patients and healthy subjects. Results confirmed a decrease in the level of circulating clusterin in some septic shock patients, in the absence of clusterin degradation (Fig. 1C). In contrast, clusterin mRNA expression was enhanced in peripheral blood mononuclear cells from patients compared to healthy subjects (Fig. ID).

As a result of massive cell death, septic shock patients display higher levels of circulating nucleosomes than healthy subjects (Fig. IE). We thus hypothesized that clusterin forms complexes with client molecules released by dying cells, thereby enhancing their clearance and clusterin consumption. As histone H4 is a major mediator of injury in sepsis, we evaluated whether clusterin-H4 complexes exist in human serums. Clusterin-H4 complexes were detectable by ELISA in most of the shock septic patients, at higher levels than in the serums of healthy subjects (Fig. 2A). As a control, the supplementation of healthy serums with recombinant H4 also dose-dependently induced the generation of clusterin-H4 complexes (Fig. 2B). Co-immunoprecipitation performed with an anti-clusterin mAb, revealed by a pan-histone Ab, confirmed the presence of circulating clusterin-histone complexes in septic shock patients (Fig. 2C). Clusterin bound to immobilized recombinant H2A, H2B, H3, H4 and purified calf thymus histones by ELISA (Fig. 2D). As expected, the short pentraxin CRP also bound histones, although its affinity with H3 and H4 appeared lower than with H2A and H2B (Fig. 2D). Biacore analysis revealed an affinity of clusterin for H4 of Kda=3.9 nM. Clusterin reduces histone-induced pro-inflammatory cytokines. Septic shock is associated with an unabated secretion of pro-inflammatory cytokines, such as IL-6 and TNFa, that contributes to host injury and multiorgan failure. In mice, the injection of low doses of histones triggers a dramatic increase in circulating levels of IL-6 and TNFa. As monocytes produce pro-inflammatory cytokines in response to TLR2/4 ligands, we tested the sensitivity of human peripheral blood monocytes to histones. Histones induce a dose-dependent secretion of IL-6 and TNFa by monocytes (Fig. 3A-B). The preincubation of histones with different doses of clusterin dose-dependently reduced histone-induced IL-6 and TNFa production with a major effect using 12 μg/ml of clusterin, without affecting LPS- or IL- 1 β-induced IL-6 and TNFa secretion (Fig. 3C-D). Clusterin alone did not induce IL-6 or TNFa secretion by monocytes (Fig. 3C-D).

We therefore evaluated whether clusterin supplementation may neutralize histones present in septic shock serums. Serums from septic shock patients induced IL-6 and TNFa secretion by monocytes, whereas serums from healthy controls did not, unless supplemented with histones (Fig. 3E-F). This potential pro-inflammatory effect of septic shock serums was prevented by supplementation with clusterin (Fig. 3E-F). The inhibitory effect of clusterin was abolished by depleting histones from serums from shock septic patients using APC, suggesting that, among DAMPs, histones are the major client DAMP in septic shock serums (Fig. 3E-F). These data demonstrate for the first time that clusterin limits the proinflammatory properties of histones.

Clusterin protects endothelial cells and neutrophils from histone-induced cell death. Sepsis is characterized by endothelial injury and a hypercoagulant state that leads to microvascular thrombosis and tissue necrosis. Histones reproduce a sepsis-like syndrome with lung capillaritis leading to microvacular thrombosis and are major inducers of endothelial cell cytotoxicity in vitro and in vivo. We thereby investigated whether clusterin may protect endothelial cells from histone-induced endothelial toxicity. As expected, serums from septic shock patients, but not from healthy subjects, were cytotoxic for endothelial cells (Fig. 3G-H). The supplementation of serums from septic shock patients with recombinant clusterin completely abrogated the endothelial cell cytotoxicity (Fig. 4H). In contrast, supplementation of healthy human serums with calf thymus histones renders them cytotoxic for endothelial cells (Fig. 3G). Thus, clusterin protects endothelial cells against histone-induced cytotoxicity in vitro, thereby suggesting that clusterin may have protective properties on vascular endothelial injury and thrombosis, two hallmarks of septic shock. Clusterin prevents histone-induced NETosis. During septic shock, an excessive NETosis may occur, resulting in host cell injury. As histones are NETs inducers, we analyzed whether clusterin may neutralize histone-induced NETs. The fluorescence microscopy images of SYTOX green stained human neutrophils incubated with medium (None), PMA (30 nM), calf thymus histones (50 μg/ml) or calf thymus histones (50 μg/ml) plus clusterin (25 μg/ml). The incubation of neutrophils with histones or with PMA, used as a positive control, induced the formation of NETs (Fig. 31). Incubation of clusterin with histones, but not with PMA, resulted in a huge decrease of NETs formation (Fig. 31). Microscopy analysis confirmed NETs formation, as opposed to unspecific cell death (data not shown). These results indicate that clusterin appears as the first described endogenous inhibitor of NETs formation that acts by a specific inhibition of histone-induced NETs.

Histones boost clusterin secretion as a protective feed-back mechanism. Clusterin synthesis in response to histones may represent a powerful mechanism to neutralize histone cytotoxicity. We report that H4, calf-thymus histones, LPS and pro-inflammatory cytokines induced clusterin secretion by monocytes (Fig. 4A). Whereas histone-induced IL-6 mRNA peaked at 4 hours (Fig. 4B), histone-induced clusterin mRNA upregulation peaked at 20 h, showing a time window for histones to exert their toxicity (Fig. 4B). We thus evaluated whether preformed stocks of clusterin exist in innate cells. The expression of clusterin in human neutrophils was analyzed by confocal microscopy using a fluorescent anti-clusterin mAb. No fluorescence was observed with an isotype control rriAb (not shown). Insert, expression of clusterin are analyzed by Western Blotting (in non reducing conditions) in neutrophils. Results are representative of one of 5 independent experiments. Immunohistochemistry evidenced preformed clusterin in granules of freshly purified neutrophils (data not shown). Western blotting confirmed constitutive clusterin stock in neutrophils (data not shown). Neutrophil stimulation with histones or LPS resulted in a rapid release of clusterin (Fig. 4D). In addition, supporting results showing that platelets store preformed clusterin, histones also induced a prompt release of clusterin by human platelets. These results show that histones trigger a prompt release of preformed clusterin by platelets and neutrophils and a delayed synthesis of clusterin by myeloid cells, thereby evidencing a potent feedback loop to dampen the cytotoxic activity of circulating histones.

Non-redundant role of clusterin in histone neutralization. Proteomic analysis have identified numerous proteins in human plasma that interact with histones (Pemberton 2010). We performed different set of experiments to determine the physiological place of clusterin in histone neutralization. First, we compared the proinflammatory response of clusterin knock- out (CLU ) and wild-type (WT) mice to intravenous injection of a sublethal concentration of histones (50 mg/kg). CLU^{_ "} mice exhibited higher serum concentrations of IL-6 and TNFoc than WT mice. Second, we evaluated the impact of clusterin depletion on the ability of human serum to protect cells against histones. As expected, histone-induced monocyte activation was more potent in serum- free medium than in medium supplemented with 10% human serum. This neutralizing property of human healthy serum was significantly abolished by clusterin depletion. Similarly, the serum from WT mice was more efficient that the serums from CLU ' mice in preventing histone-induced monocyte activation. Third we report that, contrary to clusterin, alpha2-macroglobulin, one of the main extracellular chaperons (present in 1.5-2 mg/ml in serum) which co-precipitates with histones, did not prevent histone-induced proinflammatory cytokine by monocytes. Finally, we failed in detecting a binding of histones to the extracellular chaperone HSP70. These results demonstrate a central and non-redundant in vivo role of clusterin in histone neutralization, highlighting the deleterious impact of reduced circulating levels of clusterin and suggesting a therapeutic benefit of clusterin repletion in sepsis.

Clusterin protects mice from sepsis-like diseases. In order to evaluate in vivo the capacity of clusterin to neutralize the cytotoxicity of histones, we analyzed whether clusterin may protect mice against histone-induced sepsis-like disease. Histones injected at 100 mg/kg weigh into C57BL/6 mice reproduce a lethal sepsis-like disease and all mice died within 15 minutes of injection (Fig. 5). Co-injection of an equimolar mixture of clusterin and histones rescued 28.5 % of mice (Fig. 5). These results show that clusterin supplementation contributes to protect mice from extracellular histone toxicity.

Before analyzing the benefit of clusterin injection in a murine model of LPS-induced sepsis like disease, we evaluated whether LPS injection in mice mimics the decrease in circulating clusterin observed in septic shock patients. C57BL/6 mice injected intraperitoneally with a suboptimal dose of LPS (50 mg/kg weigh to avoid mice death) exhibited an increase of circulating nucleosomes and clusterin-H4 complexes, whereas clusterin levels decreased, as assessed by ELISA and western blotting. Interestingly, clusterin protein expression was enhanced in the peritoneum 6 h post-challenge and in the kidney and liver 12 and 24 h after injection. In parallel, clusterin mRNA expression was enhanced in the spleen, liver and kidney. Reinforcing in vitro and ex vivo observations, these results show that, although LPS and histones contribute to upregulate clusterin expression, circulating clusterin levels tend to decrease in LPS-injected mice. Mice were injected intraperitoneally with LPS and one hour later first injected intravenously with clusterin or PBS before an intraperitoneal injection of LPS. We observed that mice injected with clusterin were rescued from death more frequently than mice injected with PBS (Fig. 6).

Finally, we compared the survival of WT and CLU^_/" mice after cecal and ligature punction, a reliable model of bacterial sepsis. Results showed that the death of CLU7- mice was significantly higher at day 5 and day 15 post-CLP (p<0.05; Wilcoxon test) compared to WT mice (Fig. 7).

Collectively, these data demonstrate that clusterin protects against LPS-induced and CLP lethality in vivo and supports that the clusterin protective effect is driven by decreased histone cytotoxicity.

The levels of clusterin remain decreased in non-surviving patients with sepsis. In a prospective study, we quantified the circulating levels of clusterin and of nucleosomes in the serums of patients admitted in the intensive care units (ICU) of the University Hospital of Angers and of Bordeaux for sepsis. Serums were collected at admittance and every 3 days until leaving the ICU. Results showed that the levels of clusterin were significantly reduced in sepsis patients at their admittance in ICU, compared to the levels in healthy subjects (Fig. 8).

In parallel, the levels of nucleosomes were significantly elevated in sepsis patients, compared to healthy subjects (Fig. 8). Interestingly, the levels of clusterin remained decreased in non- surviving patients while, in contrast, they progressively returned to normal levels in surviving patients (Fig. 8). The levels of circulating nucleosomes remained elevated in both groups, at each time point analysed (Fig. 8).

These results demonstrate that the levels of clusterin constitute a potent predictive marker to discriminate non-surviving versus surviving patients with sepsis. These results also suggest that restoring normal levels of clusterin may have a therapeutic benefit in sepsis patients.

Discussion:

Clusterin is an extracellular chaperon which expression and activity are enhanced in conditions associated with stress. Clusterin chaperons misfold proteins, thereby preventing their precipitation and favouring their clearance by the liver. We show here that clusterin chaperons histones in vitro and in vivo and neutralizes, in a non redundant manner, their proinflammatory and cytotoxic properties. Although histones and LPS induces clusterin synthesis and the release of preformed clusterin storage, circulating clusterin levels collapse in septic shock patients, in a higher extent in non surviving patients, suggesting an accelerated clearance of histones with an overwhelmed clusterin capacity. Finally, we show that clusterin supplementation increases the survival of mice in histones- and LPS-induced septic shock models. Clusterin therefore appears as a natural molecule with anti-histone properties and thus represents a powerful mechanism to combat the toxicity of circulating histones. Clusterin supplementation thus would prevent tissues damages resulting from massive liberation of nucleosome components in the circulation and thereby appear as a promising approach in the in the treatment of inflammatory conditions.

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.

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Claims

CLAIMS:

1. A method for neutralising or inhibiting the cytotoxic activity of extracellular histone proteins in a subject suffering from sepsis comprising administering to the subject a therapeutically effective amount of clusterin.

2. The method of claim 1 wherein clusterin consists of SEQ ID NO:l.

3. The method of claim 1 wherein clusterin has at least 70% of identity with SEQ ID NO: l.

4. The method of claim 1 which comprises the steps consisting of i) determining the level of clusterin, circulating histones, circulating nucleosomes, histone-clusterin complexes, dsDNA or ssDNA in a biological sample obtained from the subject, ii) comparing the level determined at step i) with a predetermined reference value and iii) administering to said subject a therapeutically effective amount of clusterin when the level determined at step i) is lower than the predetermined reference value.

5. The method of claim 4 wherein the step i) consists in determining the level of H4- clusterin complex in the biological sample obtained from the subject suffering from sepsis.