WO2007031100A1

WO2007031100A1 - Active immunotherapy of life-threatening systemic inflammation

Info

Publication number: WO2007031100A1
Application number: PCT/EP2005/009866
Authority: WO
Inventors: Roberto Ringhini
Original assignee: Ostini, Marco
Priority date: 2005-09-14
Filing date: 2005-09-14
Publication date: 2007-03-22

Abstract

This invention describes the use of HMGB1 or immunogenic fragments thereof for the active immunoprophylaxis and treatment of systemic inflammatory conditions, such as sepsis, endotoxic shock, hemorrhagic shock and other related syndromes. The administration of HMGB1 or of peptides derived therefrom induces an immune response which is characterized by the generation of anti-HMGB1 antibodies capable of neutralizing the activity of the circulating HMGB1 by inhibiting its ability to promote the secretion of the pro-inflammatory cytokine TNF-α. The efficacy of the prophylactic treatment can be measured by a method which measures the neutralizing activity of the antibodies against the biological effect of HMGB1 on human monocytes.

Description

ACTIVE IMMUNOTHERAPY OF LIFE-THREATENING SYSTEMIC INFLAMMATION

The present invention relates to the field of clinical immunology and in particular to the field of immunoprophylaxis and therapy of degenerative disease associated with high expression of pro-inflammatory cytokines. In particular, the invention is based on the discovery that prophylactic treatment with the protein known as HMGBl is capable of inducing, in a human subject, the production of circulating antibodies which neutralise the biological activity of the endogenous HMGBl in biological fluids.

The HMGBl biological activity, described in the invention, comprises HMGB l 's ability to promote the release of the pro-inflammatory cytokine TNF-ce which is present in an abnormal and uncontrolled amount in numerous systemic inflammatory conditions such as, for example, sepsis, septic shock, hemorrhagic shock, and other related pathologic conditions.

Sepsis, also called septicemia, is a systemic syndrome that develops following the amplification and de-regulation of the immunological response to a massive bacterial infection. Initially, the symptoms of this pathologic condition include fever, mental confusion, hypotension, impaired renal function and thrombocytopenia. Often, it evolves into a clinical stage of severe sepsis which is characterized by non-pharmacologically controllable hypotension, altered coagulation and organ dysfunction affecting in particular the kidney and lungs. A recent epidemiological study of sepsis reported a frequency in the USA of 3 cases every 1.000 inhabitants per year, totaling approximately 750.000 yearly cases, with a mortality rate of 29% (corresponding to 215.000 deaths), mortality rate increasing to 50% in the presence of septic shock (Angus D. C. et al., 2001, Crit. Care Med., 29: 1303-1310). It is important to underline that this very high mortality rate is reported in patients who have access to hospital intensive care units and are treated with appropriate antibiotic therapy, in the absence of which the mortality rate can be greater than 90%. Massive bacterial infections can spread within hospitals during certain therapeutic procedures, as a consequence of accidental exposure in areas characterized by high bacterial contamination.

An experimental model involving the administration of lipopolysaccharide (LPS) endotoxins produced by gram-negative bacteria, has shown that the pathogenic effects of sepsis, and related diseases, are due to the uncontrolled activation of the pro-inflammatory cascade of cytokines with subsequent release by monocytes of high quantities of TNF-α, IL-I and other factors, which are early mediators of acute endotoxemia (Dinarello C. A., 1997, Chest, 112:321S-329S).

In physiological conditions the host's immune defense reacts to contain bacterial aggression with an inflammatory response which is followed by an anti-inflammatory response that limits and modulates the intensity of the initial inflammatory response. In sepsis, massive activation of phagocytes results in the uncontrolled release into the circulation of inflammatory cytokines. This cytokine release is followed by the activation of endothelial cells which amplifies the inflammation by promoting endothelial adhesion of circulating lymphocytes with subsequent oxidative damage and loss of vascular integrity. In the advanced phases of septic shock, pathologic changes occur in the capillary system of organs such as the kidney and lung, with the formation of oedema and reduction in myocardial function, which are the main causes of death (Cohen J., 2002, Nature, 420:885-891).

The therapeutic management of sepsis based on the inhibition of factors released early in the inflammatory process, such as TNF-ce and IL-I, was shown to be moderately effective, but it does not target factors released late in the inflammatory process such as HMGBl which itself can sustain the disease process and lead to a fatal outcome (Yang H. et al., 2001. Shock, 15:247-253).

HMGBl, also known as HMG-I (High Mobility Grouρ-1) (Bustin M.,

2001, Trends Biochem. ScL, 26:152-153) or amphoterin (Parkinnen J., 1993, J. Biol. Chem., 107:19726-19738), is a protein consisting of 215 amino acids, with a molecular weight of 25 kDa approximately, which is highly conserved in mammals. The protein belongs to the protein superfamily called

"HMG-Box". Its structure is organized into three domains two of which are capable of binding DNA and are defined as HMG-box A and HMG box-B. The third domain is located in the C-terminus and comprises 30 residues of aspartic acid and glutamic acid. The two DNA-binding domains comprise approximately 80 amino acids, 29% of which are identical and 65% similar, and are assembled in three alpha-helix structures which form an L (Read et al., 1993, Nucleic Acids res., 21:3427-3436). HMGBl, classified as a chromosomic non-histonic nucleic protein, is stable in acid conditions (perchloric acid 5%) and can be separated from chromatin by saline extraction with sodium chloride 0.35 M. HMGBl is abundant and ubiquitous in the nucleus (106 molecules per nucleus). It binds to DNA with structural non sequential specificity determining a characteristic folding of its helices which facilitates the formation of nucleoprotein complexes capable both of stabilizing the structure and of binding with specific transcription factors.

Initially, the HMGBl protein was called amphoterin due to the bipolar distribution of the electric charges on its surface. In the central nervous system it has been found not only in the nucleus and cytoplasm but also in the extracellular space where it contributes to the neurite outgrowth (Rauwala H. et al., 1987, J. Biol. Chem., 262:16625-16635). This protein was also localized on the membrane of murine erythroleukemia cells from which it is released during the differentiation process (Passalacqua M. al. 1997, FEBS Lett., 400:275-279). Recent studies have demonstrated that HMGBl can be released by necrotic cells or by native immunity cells, for example macrophages and monocytes, following stimulation by bacterial endotoxins and immunomodulatory cytokines (Scaffidi P. et al., 2002, Nature 418(6894): 191-195; Wang H. et al., 1999, Science, 285:248-251). Amongst the specific activities of the molecule HMGB 1 it is important to underline its ability to bind RAGE (Receptor of Advanced Glycosylation End-Products). This receptor is present in numerous cell types, including endothelial cells, smooth muscle cells, mononuclear phagocytes and neurons, and it is involved both in a number of pathological conditions, such as diabetes and atherosclerosis, and in numerous physiological processes, such as inflammation and elongation of neurites during embryonic development (Hutten et al., 1999, J. Biol. Chem., 274: 19919-19924). Another effect correlated with the binding of RAGE by HMGBl is tumor growth and formation of metastases. In fact, the interaction of HMGl with RAGE activates endocellular molecules which act as effectors in the mechanisms of cancer proliferation and invasion (Taguchi A. et al., 2000, Nature, 405:354-360). In addition, in inflammation, the presence of RAGE in the tunica media of large blood vessels of smooth muscles is important for the transmigration through the tunica intima of damaged cells with modified phenotype. Inhibition of binding between HMGBl and RAGE prevents, delays or blocks such migration which has a fundamental role in the physiopathology of atherosclerosis and in restenosis after coronary angioplasty (WO 02/074337). It has now been found that HMGBl stimulates the synthesis and release of TNF-ce subsequent to HMGBl interaction with receptors present in PBMC (peripheral blood mononuclear cells), also defined as mature monocytes (Andersson U. et al., J. Exp. Med., 192:565-570). Current evidence indicates that the interaction between HMGBl and RAGE promotes the synthesis and release of the cytokine TNF-o; by monocytes and macrophages. The uncontrolled secretion of this pro-inflammatory cytokine is one of the pathogenic mechanisms which underlies sepsis, as previously described, and related systemic diseases such as endotoxic shock and hemorrhagic shock. The poor prognosis associated with these conditions is due to the release, into the vascular compartment, of an abnormally high amount of HMGB 1 which leads to secretion of the cytokine TNF-α. Such release of TNF-α induces further release of HMGBl creating an inflammatory loop that is self-promoting. In septic patients high blood levels of HMGBl were reported to be directly correlated with the severity of their pathologic condition, whereas in the blood of normal subjects this protein is not present (Wang H. et al., 1999, Science 285:248-251). It is also well known that anti-HMGBl antibodies neutralize the biological activity of HMGB 1 by blocking its interaction with RAGE and eliminating one of the conditions necessary for the expression of the cytokine cascade that supports the inflammatory process.

This finding is supported by the demonstration that it is possible to decrease the severity of sepsis and/or prevent its fatal outcome by administering xenogenic anti-HMGBl antibodies (passive immunotherapy) while the administration of HMGB 1 increases the lethality of this pathological condition (Van Der Poll T., 2001, Lancet Infectious Diseases 1 :165-174).

Various pharmaceutical compounds capable of inhibiting the interaction between HMGB 1 and RAGE have been proposed for the treatment of sepsis and of related conditions. These compounds utilize fragments of HMGBl as direct inhibitors of HMGBl biological activity (WO 02/092004; US 2004/0156851) or exogenous antibodies with affinity for the entire molecule or its parts to be used as passive immunotherapy (WO 00/47104, US 6,448,223). EP 1079849 discloses the cytotoxic activity of HMG and HMG-I proteins. The present invention describes instead a pharmaceutical composition comprising as the active ingredient HMGBl, or an immunogenic peptide thereof, and its use to induce the production of specific endogenous antibodies

(active immunotherapy) capable of blocking RAGE binding in circulating monocytes.

In the description, the term "HMGBl" refers to a protein with a degree of sequence identity of at least 70%, preferably greater than 80%, and even more preferably greater than 90%, with the protein identified in the NCBI-Entrez data-base by the access number P09429 (GI: 123369). The term "peptide" is used according to its conventional meaning, to indicate a sequence of amino acids forming a part of a native protein, not limited in terms of number of constituting residues or presence of post-transcriptional modifications, such as glycosylation, acetylation, phosphorylation and other available natural or artificial modifications. In particular, the term refers to linear sequences of amino acids containing from 10 to 40 residues, preferably from 10 to 30 residues, and more preferably from 10 to 20 residues, which constitute an immunogenic portion of the protein from which they derive.

The term "immunogenic" defines the peptide's capacity to induce in the receiving host a specific antibody response to the fragment itself, or to the molecule from which the fragment was derived.

The term "peptide variant" refers typically to a peptide that differs from the peptide described by one or more deletion, substitution or insertion of amino acids.

Peptides variants included in the present invention are typically those that show, in their sequence length, an identity of 70%, preferably of 80%, and more preferably of 90% with the peptides derived from the active molecule.

The substitutions considered in this invention are those defined as

"conservative", in other words obtained from a substitution of amino acids with similar properties. In this case and on the basis of the current knowledge of peptides chemistry, after substitution the secondary structure and hydrophobic characteristics remain substantially the same.

Generally, the animal model of endotoxic shock consisting in the administration of lipopolysaccharide (LPS) to mice is considered as an adequate experimental model to study the pathophysiology of sepsis because it provokes in mice haematological changes and a clinical evolution similar to those observed in humans.

The intraperitoneal administration of an LPS dose of 25 mg/kg in male Balb/C mice weighing 20-25 grams induces lethargy status, pilo erection and diarrhea followed by death within 48 hours. In these mice, increased serum levels of the cytokine TNF-α have been reported which reach maximum values 90 minutes after LPS administration and then decrease to non significant levels in the subsequent 10 hours (Van der Poll T. et al., 1999, Infect. Dis. Clin. North Am. 13:413-426). Furthermore, the protein HMGBl can be detected in the serum 8 hours after LPS administration and its concentration increases steadly reaching a plateau 16 to 32 hours after endotoxin administration (Wang H. et al., 2001, Am. J. Crit. Care Med. 164:1768-1773). In an in vitro experimental model based on the use of murine macrophage cell line RAW 264.7 (ATCC, Cat. N. TIB-71) adding LPS to the cell culture induces after 8 hours the release and accumulation in the medium of HMGB 1. An identical cellular response can be obtained using TNF-α as stimulant instead of LPS (Wang H. et al., 1999, Science 285:248-251). Finally, an experimental model based on the use of immunocompetent cells has shown that HMGB 1 modulates, in a dose-dependent manner, the release of TNF-ce from peripheral blood mononuclear cells (PBMC) but not from lymphocytes, and that this effect is lost using peptides derived from the fragmentation of HMGBl (Anderson U. et al., 2000, J. Exp. Med. 192;565-570). Although the mechanism of interaction between HMGBl and the monocytes is not fully known, there is scientific evidence suggesting the active role of RAGE in the release of TNF-α (Schmidt A.N. et al., 2001, J. Clin. Invest. 108:949-955). More complete information exists in the literature on the mechanism of HMGBl release by macrophages which involves the autocrine function of TNF-ce secreted after stimulation with IFN-γ (Rendon-Mitchell B. et al., 2003, J. Immunol., 170:3890-3897).

It has now been found that it is possible to reduce or eliminate the lethal effect of sepsis, and of related conditions, by administering HMGBl or fragments thereof, inducing, in the treated subject, the formation of circulating antibodies having neutralizing activity against HMGB 1 circulating in blood.

The invention accordingly provides the use of HMGB 1 or of a protein or peptide having an identity with the human protein of at least 50% for the preparation of a medicament for the treatment of diseases associated with the presence of an abnormal and excessive amount of HMGBl protein in the biological fluids.

The selection of peptides capable of inducing the production of neutralizing antibodies may be performed by analyzing the characteristics of the amino acids present in the primary structure of HMGB 1. In particular, well known methods allow the characterization of the antigenic profile of the molecule and of related peptides.

In this invention, the antigenic profiles were generated using the software ANTHEPROT 2000 Version 5.2 (institute of Biology and Protein Chemistry UMR 5086 CNRS-UCBL, Lyon, France) and the software ProtScale present in the database ExPASy (Swiss Institute of Bioinformatics, Basel University, Basel, Switzerland). The analysis of the antigenic profiles has allowed the selection of 9 peptides, containing from 13 to 15 amino acids each, having the sequences reported in the Sequence Listing. Other peptides may be prepared by the same method.

Said peptides are a further object of the invention. A further embodiment of the invention refers to pharmaceutical compositions comprising HMGBl or peptides derived therefrom. Although some peptides can be naturally immunogenic, it is often necessary or preferable to conjugate the peptide to high molecular weight molecules in order to induce or enhance the host's immunological response. According to current knowledge, various known molecules such as Bovine Serum Albumin (BSA), hemocyanin (KLH) and Ovalbumin (OVA) may be used as peptide carriers after chemical conjugation with a bi-functional reagent (e.g. glutaraldehyde, carbodiiimide). An alternative to this type of conjugation is the preparation of multiple antigenic peptides (MAPs) by known methods.

Usually, the macromolecule which forms the complex consists of 8 identical peptides covalently bound to an internal heptameric structure (core) of lysine which is non immunologically reactive and has a molecular weight adequate to make the complex highly immunogenic. In the present invention, an appropriate formulation would consists of the native forms of the peptides derived from HMGBl, preferably conjugated to a protein carrier, and preferably assembled as MAPs. The compositions of the invention may be administerd by systemic, inhalation or parenteral route to an animal species, preferably to humans.

Examples of suitable compositions include saline or parenteral solutions for injectable drugs, e.g. as prescribed by International Pharmacopeias.

In the present invention, the quantity of immunogenic substance present in the pharmaceutical composition must be sufficient to guarantee therapeutic benefit to the subject who receives it. In other words, such quantity should be sufficient to induce the production of circulating antibodies endowed with neutralizing activity against the biological/receptorial activity of HMGBl present in circulation. The dose of immunogenic substance depends on the animal species treated. Based on currently used pharmaceutical methods, a dose between 5 μg/kg and 2 mg/kg of body weight, preferably between 20 μg/kg and 500 mg/kg of body weight, and even more preferably between 20 μg/kg and 100 μg/kg of body weight should be adequate in man.

Another aspect of the invention concerns compositions that contains not only the immunogenic substance but also an immunostimulant capable of enhancing the immune response (cell-fixed or circulating) induced by it (Powell M.F., Newman M.J., 1995. "Vaccine Design. The subunit and adjuvant approach", Plenum Press Ed., New York). The immunostimulant to be used in this invention is classified in the category of the adjuvants, including both substances that protect the antigen against a rapid catabolism, such as aluminum hydroxide or mineral oil, and those that stimulate the immune response in a non-specific way, such as lipid A and proteins derived from the microorganism Bordetella pertussis.

The adjuvant can be selected from those already commercially available for pharmaceutical use, for example the products ISA-51 and ISA-720 (Seppic, France), SAF and MF-59 (Chiron, USA), SBAS-2 and SBAS-4 (SmithKline Beecham, Belgium), Detox and RC-529 (Corica, USA) or others. Furthermore, the compositions of the invention can contain physiologic buffers, carbohydrates, mannitol, stabilizing proteins, anti- oxidants, bacteriostatics, chelating agents or other substances described in International Pharmacopeias which are generally used for the preparation of drugs or vaccines for warm-blooded animals and preferably for humans. The evaluation of the neutralizing capacity of the endogenous antibodies induced by the administration of the claimed product can be performed using an analytical method and calculating a parametric value known as the Neutralization Index (NI), whose determination is a useful condition for the use of the invention.

This assay measures the capacity of the induced antibodies to inhibit the interaction between HMGBl and RAGE on the surface of mononuclear cells of peripheral blood using donor's PBMC obtained applying well known centrifugation methods such as those described in Example 1. Briefly, after separation the cells are incubated with a fixed amount of recombinant protein HMGBl (HMGBIr) and a pre-established volume of complement- free serum obtained from a subject treated with the product claimed in this invention. The presence of anti-HMGBl antibodies in the analyzed serum blocks the interaction between the recombinant molecule and its receptors on the cells thereby inhibiting the release of TNF-a.

Finally, the TNF-α concentration in the cellular growth medium is measured using a commercially available immunometric assay, such as, for example, the human TNF-ce Quantikine immunoassay produced by R&D SYSTEM (Cat. N. DTA50). The ratio between the TNF-α amount present in the sample and the maximum release of this cytokine obtained by the addition of a non specific hyperimmune serum gives a normally distributed value comprised between 0 and 1. The clustering of the measured ratio in a numeric scale allows the determination of the sample's neutralization index (NI), which is an index of the assayed serum capacity to neutralize the biological activity of the circulating HMGBl. In particular, a value of this ratio greater than or equal to 0,9 gives a NI value equal to zero; a ratio greater than or equal to 0,75 but smaller than 0,9 gives NI = +1; a ratio greater than or equal to 0,5 but smaller than 0,75 gives a NI = +2; a ratio greater than or equal to 0,25 but smaller than 0,5 gives a NI = +3; a ratio smaller than 0,25 gives a NI value equal to +4. Based on the value assigned to the sample, the neutralizing capacity of the analyzed serum is considered null for a +1 index, moderate for a +2 index, high for +3 index and very high for a +4 index. This neutralizing capacity is directly correlated with the efficacy of the treatment with the claimed product (active immunotherapy). In fact, a NI value greater than 1 unit indicates a good prophylactic effect, indicating the presence in the serum of the treated subject of a significant amount of neutralizing antibodies, whereas indexes equal to or smaller than 1 unit suggest the need for boosters enhancing the anti-HMGBl activity of the serum.

The possibility of using an immunoprophylactic approach with the claimed product in order to fight a severe infection caused by Gram negative microorganisms was investigated in the murine model of endotoxic shock. In a preliminary step, mice were treated according to a specific protocol with a prophylactic product containing an adjuvant mixed to a peptide derived from HMGB in the form of MAP or the complete HMGBl recombinant molecule. Subsequently, animals were inoculated with a quantity of LPS sufficient to provoke lethal effects within three days. The two groups treated with the prophylactic agents had a survival rate which was at least 50% greater than that of the reference group treated with adjuvant only.

The results obtained with this validated murine model of systemic lethal inflammation, support the possibility of using the claimed product for the active prophylactic treatment of pathological conditions associated with inflammation and modulated by the presence of large quantities of the cytokine TNF-α, such as sepsis, septic shock, and haemorrhagic shock . Although similar survival rates have been obtained using the recombinant molecule or its peptides, the use of the peptides is preferable because the native protein could induce the release of pro-inflammatory cytokines into the blood. The following examples further elucidate the invention.

Example 1

Use of the product for active immunoprophylaxis in mice

This example shows a model of active immunoprophylaxis against endotoxic shock in Balb/C mice and the evaluation of the neutralizing capacity of mice sera after treatment.

16 Balb/C male mice aged 6-7 weeks (weighing 20-25 grams) received an intramuscular injection of the recombinant HMGBl (HMGBIr) protein (SIGMA-ALDRICH, Cat. N. H-4652) or of the peptide SEQ. ID. No.l in the form of octameric MAP supplied by PRIMM srl (Italy). The injection was prepared by adding 10 μg of HMGBIr (35 μl physiologic solution) or 25 μg of MAP (50 μl phosphate buffer) to 50 μl of ImmunEasy Mouse Adjuvant (QIAGEN AG, Basel). The reagents were mixed thoroughly and incubated for 5 minutes at room temperature (18-25°C) prior to the intramuscular administration into the quadriceps.

Half of the animals received the entire protein (Group 1) whereas the other half were immunized with the peptide complex (Group 2). The treatment protocol for both groups consisted of three administrations each with a two- week interval period, i.e. primer 38 days, first booster 24 days, and second booster 10 days, before blood sampling, in order to evaluate the presence of neutralizing antibodies. The control group consisted of 8 Balb/C male mice (Group 3), with the same age and average body weight, treated according to the protocol described above with 50 μl of ImmunEasy Mouse Adjuvant diluted with an equal volume of saline solution. Three days after the last inoculum, blood sampling was performed in order to obtain 150 μl of serum, treated at 56°C for 30 minutes to remove complement and stored at 4 ± 2°C.

The evaluation of the mice sera neutralizing capacity was based on the measure of the TNF-α releases by human monocytes after stimulation with HMGB-I in presence of the mice complement- free sera. Monocytes preparation was obtained by adding 5 ml of phosphate buffer to 3 ml of human venous blood stored in heparin or EDTA vials, mixing by inversion and carefully layering the mixture onto 3 ml of Histopaque-1077 (SIGMA-ALDRICH, Cat. N. H-8889) added to a 5 ml conical centrifuge tube. After centrifugation at 400 x g for 30 minutes the upper layer was removed up to the opaque interface containing the mononuclear cells. This interface was then carefully transferred to a clean conical centrifuge tube, added with 10 ml of phosphate buffer and mixed by inversion. After centrifugation for 10 minutes at 250 x g, the supernatant was aspirated and the pellet was suspended with 5 ml of phosphate buffer mixing by gentle trituration. The cells wash was repeated and the final precipitate suspended in 1 ml of culture medium RPMI 1640 (IRVINE, Cat. N. 9024) added with 10% of Bovine Foetal Serum (SIGMA-ALDRICHT, Cat. N. F2442). The cells were then diluted at the concentration of 106 cells/ml culture medium and 0.5 ml of this suspension was transferred into the well of a sterile polystyrene 24-wells multidish (NUNC, Cat. N. 142475). Each serum sample was analyzed in quadruplicate and further four wells were used totaling 4 for the negative and positive controls (two for each control samples). After distribution of the cells in all the test-wells and incubation for 1 hour at 37°C (5% CO₂), 0.4 ml of culture medium of each well was removed to eliminate the lymphocytes in suspension and was substituted with equal volume of RPMI 1640. Subsequently 100 μl of complement-free murine serum was added in four test-wells while the same volume of complement-free serum obtained from Group 3 mice was transferred in duplicate into the wells labeled as PC (Positive Control) or NC (Negative Control). To promote the release of the cytokine, 55 ng of HMGBIr (50 μl of a 1.1 μg/ml solution in a RPMI 1640 medium) were added to each well with the exception of that labeled as NC in which an equivalent volume of culture medium was pipetted. After incubation for 4 hours (37°C, 5% CO₂), 150 μl culture medium was carefully taken from each well avoiding to remove the cells adhering to the bottom of the well and transferred into a dilution test tube. Following culture medium dilution 1:5 (v:v) with the Diluent RD6-35 contained in the kit human TNF-α Immunoassay Quantikine (R&D SYSTEM, Cat. N. DTAOOC), the sample was assayed with such commercial kit following the supplier instruction. The TNF-α concentration was then measured.

The assay was performed in duplicate and the average concentration of each sample was obtained by multiplying the result by the dilution factor. The mean value of TNF-o; obtained assaying the quadruplicate wells or the duplicate wells associated to the Positive or Negative Controls is reported in the table below.

The neutralization index (NI) of each serum sample was assessed calculating the ratio between the value obtained for the tested sample and that obtained for the positive control sample and by classifying the obtained value according to the numerical scale described previously.

The described serum analytical assay is considered acceptable only if meets established TNF-α concentration criteria for Negative and Positive Controls i.e. TNF-α values <100 ng/ml and >4.000 ng/ml for these samples respectively.

As shown above, the evaluation of the NI related to the serum specimens #1 and #3 suggests, respectively, a poor response or no response to the immunoprophylactic treatment. In fact, the capacity of their sera to neutralize the monocytes TNF-α secretion stimulated by the presence of HMGB-I was very low. In the other assayed animals the treatment efficacy is classified as moderate (#4, #5), good (#2) and very good (#6).

Example 2 Efficacy of active immunotherapy in the murine experimental model of sepsis

The efficacy of the prophylactic use of HMGBl or peptides of the invention was evaluated by administering a lethal dose of LPS endotoxin to animals pretreated with non lethal doses of LPS . In this study the same mice treated in Example 1 were treated and clustered into three groups receiving HMGBIr (Group 1), MAP derived from the peptide labeled as SEQ.ID.No 1 (Group 2) and an adjuvant (Group 3), respectively. After the prophylactic treatment, which was performed according to the protocol described in Example no.l, mice received an intraperitoneal injection of a lethal dose of LPS (SIGMA-ALDRICH Cat. N. L2262) i.e. 1 mg of endotoxin in phosphate buffer solution (PB S), determining progressive multiorgan dysfunction and early death. Mice were housed under standard temperature, humidity, and light and dark cycle conditions. Mice were monitored for 72 hours and the survival percentage was evaluated at the end of this period. Data obtained showed that mortality was significantly lower in the treated groups in comparison with the control group which received the adjuvant only.

Claims

1. The use of HMGB 1 or of a protein or peptide having an identity with the human protein of at least 50% for the preparation of a medicament for the treatment of diseases associated with the presence of an abnormal and excessive amount of HMGBl protein in the biological fluids.

2. The use according to claim 1 wherein HMGBl is a human recombinant HMGBl.

3. The use according to claim 1 of immunogenic HMGBl peptides.

4. The use according to claim 3 wherein the HMGBl peptides are selected from the peptides having Id Sequence 1-9.

5. The use according to any one of claims 1-4, wherein the diseases associated with the presence of an abnormal and excessive amount of HMGBl are sepsis, endotoxic shock, hemorrhagic shock and other related syndromes.

6. A method of treatment of diseases associated with the presence of an abnormal and excessive amount of HMGBl protein in the biological fluids consisting in the administration to patients affected by said diseases of an effective amount of HMGBl or of a protein or peptide having an identity with the human protein of at least 50%.

7. A method according to claim 6 consisting in the administration of a human recombinant HMGB 1.

8. A method according to claim 6 consisting in the administration of immunogenic HMGBl peptides.

9. A method according to claim 8 consisting in the administration of the peptides having Id Sequence 1-9.

10. A method of treatment according to any one of claims from 1 to 9 wherein the diseases are sepsis, endotoxic shock, hemorrhagic shock and other related syndromes.

11. A pharmaceutical composition comprising HMGBl or a protein or peptide having an identity with the human protein of at least 50% and suitable carriers and excipients.

12. A composition according to claim 11 further comprising an adjuvant.

13. A composition according to claim 11 comprising an HMGBl immunogenic peptide.

14. A composition according to claim 11 or 12 comprising as the active ingredient a peptide having Id Sequence 1-9.

15. A composition according to claim 12-14, wherein the peptides are conjugated with, transformed by, or bound to an immunologically inactive protein support.

16. Peptides having Sequence Ids 1-9.

17. A method for measuring the efficacy of the active immunoprophylaxis treatment induced by the administration of the compositions of claims 11-15, comprising the following steps: a) determination of the amount of pro -inflammatory cytokine TNF-ce released by immunocompetent cells after stimulation with HMGBl in presence and absence of serum from a treated subject; b) comparison between the measured concentrations and calculation of a parametric index; c) use of a parametric index in order to determine the efficacy of the prophylactic treatment.

18. A method, as described in Claim 17, in which the immunocompetent cells are human monocytes and the pro-inflammatory cytokine is the molecule TNF-ce.