WO2019063320A1

WO2019063320A1 - Composition comprising bactericidal/permeability increasing protein and hyaluronic acid for the treatment of arthropathies

Info

Publication number: WO2019063320A1
Application number: PCT/EP2018/074889
Authority: WO
Inventors: Leonardo PUNZI; Anna SCANU; Roberto LUISETTO
Original assignee: Universita' Degli Studi Di Padova
Priority date: 2017-09-27
Filing date: 2018-09-14
Publication date: 2019-04-04

Abstract

The present invention relates to a composition comprising Bactericidal/permeability- increasing protein (BPI) and hyaluronic acid. This composition offers significant advantages in the treatment of arthropathies.Pharmaceutical compositions and kits comprising the Bactericidal/permeability-increasing protein (BPI) and hyaluronic acid, in particular for the treatment of arthropathies,also fall within the scope of the present invention.

Description

COMPOSITION COMPRISING BACTERICIDAL/PERMEABILITY INCREASING PROTEIN AND HYALURONIC ACID FOR THE TREATMENT OF

ARTHROPATHIES DESCRIPTION

*****

The present invention relates to the pharmaceutical and biological field.

In particular it relates to pharmaceutical compositions comprising

Bactericidal/permeability increasing protein (BPI) and hyaluronic acid (HA), more specifically to a medicament for use in the treatment of arthropathies.

FIELD OF INVENTION

The term arthropathy comprises more than 100 different conditions that may affect individuals of all ages, genders and ethnicities. All these conditions are characterized by joint pain accompanied by stiffness and swelling and in case of arthritis, by inflammation. All these diseases are the leading cause of disability in the USA and in Europe.

At present, treatments for arthropathies aim at reducing pain, inflammation and preventing permanent joint damage.

However, not all patients benefit from the pharmacological treatments available to date.

Moreover, many drugs have potentially serious side effects.

Although in the last decade the pharmacological therapy for these diseases has significantly changed, there is still the need for new therapeutic targets and new effective drugs at the same time having fewer side effects.

Bactericidal/permeability-increasing protein (BPI) is a cationic protein with high antimicrobial activity. It is produced and released in extracellular fluids mainly by polymorphonuclear cells, but can be also expressed in fibroblasts and epithelial cells (Reichel PH, Seemann C, Csernok E, Schroder JM, M jller A, Gross WL, Schultz H. Bactericidal/permeability-increasing protein is expressed by human dermal fibroblasts and upregulated by interleukin 4. Clin Diagn Lab Immunol 2003;10:473-5; Canny G, Levy O, Furuta GT, Narravula-Alipati S, Sisson RB, Serhan CN, Colgan SP. Lipid mediator-induced expression of bactericidal/permeability-increasing protein (BPI) in human mucosal epithelia. Proc Natl Acad Sci USA 2002;99:3902-7; Schultz H, Weiss JP. The bactericidal/permeability-increasing protein (BPI) in infection and inflammatory disease. Clin Chim Acta 2007;384:12-23).

BPI selectively and specifically acts against infections caused by gram-negative bacteria by damaging bacterial membranes, neutralizing lipopolysaccharide (LPS), and opsonizing bacteria to enhance their phagocytosis by polymorphonuclear leukocytes (PMN) (Mannion BA, Weiss J, Elsbach P. Separation of sublethal and lethal effects of the bactericidal/permeability increasing protein on Escherichia coli. J Clin Invest 1990;85:853-60; Marra MN, Wilde CG, Griffith JE, Snable JL, Scott RW. Bactericidal/permeability-increasing protein has endotoxin-neutralizing activity. J Immunol 1990;144:662-6; lovine NM, Elsbach P, Weiss J. An opsonic function of the neutrophil bactericidal/permeability-increasing protein depends on both its N- and C- terminal domains. Proc Natl Acad Sci USA 1997;94:10973-8).

In addition to its well-known anti-infective properties, BPI inhibits angiogenesis inducing cell apoptosis, inhibiting migration of endothelial cells and complexing the vascular endothelial growth factor (VEGF) (van der Schaft DW, Toebes EA, Haseman JR, Mayo KH, Griffioen AW. Bactericidal/permeability-increasing protein (BPI) inhibits angiogenesis via induction of apoptosis in vascular endothelial cells. Blood 2000;96:176-81 ; van der Schaft DW, Wagstaff J, Mayo KH, Griffioen AW. The antiangiogenic properties of bactericidal/permeability-increasing protein (BPI). Ann Med 2002;34:19-27; Yamagata M, Rook SL, Sassa Y, Ma RC, Geraldes P, Goddard L, Clermont A, Gao B, Salti H, Gundel R, White M, Feener EP, Aiello LP, King GL. Bactericidal/permeability-increasing protein's signaling pathways and its retinal trophic and anti-angiogenic effects. FASEB J 2006;20:2058-67). It has been also hypothesized that BPI may interact with some membrane receptors, such as toll-like receptor 4 (TLR4) and glypican 4 (Canny G, Cario E, Lennartsson A, Gullberg U, Brennan C, Levy O, Colgan SP. Functional and biochemical characterization of epithelial bactericidal/permeability-increasing protein. Am J PhysiolGastrointest Liver Physiol 2006;290:G557-67; Geraldes P, Yamagata M, Rook SL, Sassa Y, Ma RC, Clermont A, Gao B, Aiello LP, Feener EP, King GL. Glypican 4, a membrane binding protein for bactericidal/permeability-increasing protein signaling pathways in retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 2007;48:5750-5). BPI has been detected in high amount in synovial fluid of patients with reactive arthritis and rheumatoid arthritis, and to lower extent in psoriatic arthritis and osteoarthritis however its specific role in these different types of arthropathies has not been defined yet (Punzi L, Peuravuori H, Jokilammi-Siltanen A, Bertazzolo N, Nevalainen TJ. Bactericidal/permeability increasing protein and proinflammatory cytokines in synovial fluid of psoriatic arthritis. Clin Exp Rheumatol 2000;18:613-5). Only recently it has been shown that BPI is able to inhibit the inflammatory processes induced by pathogenic crystals that are found in some arthropathies by reducing the leukocyte recruitment and cytokine production (Scanu A, Luisetto R, Oliviero F, Galozzi P, Ramonda R, Punzi L. Bactericidal/permeability-increasing protein downregulates the inflammatory response to pathogenic crystals in vitro and in vivo. Ann Rheum Dis 2017;76(supplement 2):210). WO9420128 describes some therapeutic uses of BPI, including the treatment of chronic inflammatory diseases, such as arthritis.

However, no pharmaceutical formulation related to the use of BPI in arthritis or similar pathologies has been commercialized.

Hyaluronic acid (HA) is widely used for viscosupplementation of joints. Studies have reported that HA may also exhibit anti-inflammatory, analgesic and chondroprotective effects.

Therefore, there is still the need of an effective treatment of arthropathies with substances that are potentially less harmful than those currently available.

SUMMARY OF THE INVENTION

It has been shown that the combination of Bactericidal/permeability-increasing protein (BPI) and hyaluronic acid has a very advantageous effect in the treatment of arthropathies, in particular there is a greater effect compared to the effect of BPI or hyaluronic acid used individually.

Even more in particular, said combination showed to be significantly effective in reducing inflammation and clinical signs in animals with induced experimental arthritis.

The use of said combination is advantageous compared to the use of the single components since it shows a greater and faster efficacy in the improvement of the arthritic condition. More specifically, a synergistic effect of the two components was observed with respect to their separate use.

Furthermore, the compounds used are not toxic, thus avoiding the side effects that characterize the pharmacological therapies currently used for this type of pathology. It is, therefore, an object of the present invention a composition comprising Bactericidal/permeability-increasing protein (BPI) and hyaluronic acid.

It is also an object of the present invention the use of said composition as a medicament.

It is also an object of the present invention, a composition comprising Bactericidal/permeability-increasing protein (BPI) and hyaluronic acid for use in the treatment of an arthropathy.

Preferably, said arthropathy is selected from the group consisting of degenerative arthropathies, post-traumatic arthropathies, metabolic arthropathies, crystal-induced arthropathies, inflammatory arthropathies, septic arthritis and reactive arthritis.

It is also an object of the present invention a pharmaceutical composition comprising as active agents at least the Bactericidal/permeability-increasing protein (BPI) and hyaluronic acid together with at least one pharmaceutically acceptable carrier and/or excipient.

It is also an object of the present invention a pharmaceutical composition for use in the treatment of an arthropathy comprising as active agents at least the Bactericidal/permeability-increasing protein (BPI) and hyaluronic acid and at least one pharmaceutically acceptable carrier and/or excipient.

It is also an object of the present invention the Bactericidal/permeability-increasing protein (BPI) for use in the treatment of an arthropathy in combination therapy with hyaluronic acid or in a therapeutic regimen comprising hyaluronic acid.

A further object of the present invention is a kit including:

a) Protein Bactericidal/permeability-increasing (BPI), and

b) Hyaluronic acid.

In a preferred embodiment, said kit is for use as a medicament, in particular for the treatment of an arthropathy.

Indeed, it has been found that the administration of Bactericidal/permeability- increasing protein (BPI) and hyaluronic acid in an inflammatory condition, such as monosodium urate and calcium pyrophosphate crystal-induced inflammation, for example in gout and in pseudogout, is able to significantly reduces the total white blood cell count, the percentage of polymorphonuclear cells and inhibit the production of the inflammatory mediators evaluated.

In particular, the use of the combination of BPI and hyaluronic acid was clearly more effective than the treatment with the BPI protein alone, resulting in a synergistic effect.

Furthermore, it has been found that in an experimental model of arthritis, co- treatment with BPI and hyaluronic acid resulted in improved clinical symptoms, reduction of arthritic scores and down-regulation of specific inflammatory mediators. In particular, the improvement of all parameters assessed was significantly greater and faster than that observed after the administration of BPI alone or hyaluronic acid alone.

Furthermore, no toxicity was observed after the combined administration of BPI and hyaluronic acid, in particular no significant changes were observed in the total and differential leukocyte counts. This is a further advantage of this invention that allows obtaining an effective treatment without side effects.

DESCRIPTION OF THE INVENTION

Within the meaning of the present invention, arthropathy means any joint disease. Within the meaning of the present invention, "Bactericidal/permeability-increasing protein'Or "BPI" means a known protein of about 50 kDa, which is produced by the immune system. "Bactericidal/permeability-increasing protein" or "BPI" means any Bactericidal/permeability-increasing protein produced through natural, synthetic or recombinant methods; comprising also its natural, synthetic and recombinant biologically active polypeptide fragments; and biologically active polypeptides or their BPI analogues.

Within the meaning of the present invention biologically active fragments are fragments of BPI protein retaining the biological activity, such as fragment 1 -199, fragment 200-456 (Ooi CE, Weiss J, Elsbach P, Frangione B, Mannion B. A 25-kDa NH2-terminal fragment carries all the antibacterial activities of the human neutrophil 60-kDa bactericidal/permeability-increasing protein. J BiolChem 1987;262:14891-4) fragment 13-191 (Horwitz AH, Ammons WS, Bauer RJ, Dedrick R, Nadell R, Williams RE, Liu PS. rBPI(10-193) is secreted by CHO cells and retains the activity of rBPI21. J Endotoxin Res 2004;10:97-106) and fragment 1 -193 (lovine NM1 , Elsbach P, Weiss J. An opsonic function of the neutrophil bactericidal/permeability- increasing protein depends on both its N- and C-terminal domains, Proc Natl AcadSci U S A. 1997 Sep 30;94(20): 10973-8).

Within the meaning of the present invention biologically active analogues are recombinant analogue of BPI protein retaining the biological activity such as rBPI23 (Jellema WT1 , Veerman DP, De Winter RJ, Wesseling KH, Van Deventer SJ, Hack CE, van Lieshout JJ. In vivo interaction of endotoxin and recombinant bactericidal/permeability-increasing protein (rBPI23): hemodynamic effects in a human endotoxemia model. J Lab Clin Med. 2002 Oct;140(4):228-35) and rBPI21 (Horwitz, A. H., S. D. Leigh, S. Abrahamson, H. Gazzano-Santoro, P. S. Liu,R. E. Williams, S. F. Carroll, and G. Theofan. 1996. Expression and characterization of cysteine-modified variants of an amino-terminal fragment of bactericidal/permeability-increasing protein. Protein Expr. Purif. 8:28-40).

Within the meaning of the present invention, "hyaluronic acid" or "hyaluronan" means the known glycosaminoglycan which is a major component of human and animal connective tissue and comprising a disaccharide polymer composed of glucuronic acid and N-acetyl-glucosamine. Hyaluronic acid is also herein referred by the acronym HA.

Detailed description of the invention

According to the present invention, the composition of the invention comprises a protein known as Bactericidal/permeability-increasing protein (BPI).

This is a known protein (see for example references 1 -13) and can be commercially obtained or produced by methods known in the art. For example, it can be a natural, synthetic or recombinant protein, obtained through techniques common in the field. The invention also comprises biological, synthetic or recombinant biological fragments of BPI, with the proviso that the biological functionality of the protein is maintained.

Biologically active analogs of BPI may also be used, such as BPI in which one or more amino acids have been replaced without alteration of biological activity. For example, the molecule may lack of one or more N-terminal amino acids, one or more internal amino acids and/or one or more carboxy-terminal amino acids. BPI analogs may also be recombinant hybrid fusion proteins comprising BPI or its biologically active fragments and at least a portion of one or more other polypeptides, such as the heavy chain constant region of an immunoglobulin or an allelic variant thereof.

Said fragments or analogs can easily be obtained by the expert in the field based on his common general knowledge. For example, they can be generated synthetically or isolated using methods known in the art.

For example fragments can be fragment 1 -199, fragment 200-456, fragment 13-191. For example recombinant analogues can be rBPI21 , rBPI23.

BPI or its fragments or analogs may also be in the monomeric, dimeric or multimeric form.

According to the invention, the other component of the composition is hyaluronic acid. Hyaluronic acid, also called hyaluronan, is a well-known compound and can be commercially found or synthesized according to common knowledge in the art. According to the present invention hyaluronic acid can be used in any of its known forms, in particular it can be a polymer of any chain length and molecular dimension. For example, it can refer to low molecular weight, high molecular weight, cross- linked and/or esterified hyaluronic acid, and/or a salt thereof (hyaluronate) and/or an amine derivative thereof. All these forms are well known in the art and commercially available or obtainable through common knowledge in the field. As regards, in particular, the use of hyaluronic acid in viscosupplementation, (see for example references Strauss EJ, Hart JA, Miller MD, Altman RD, Rosen JE. Hyaluronic acid viscosupplementation and osteoarthritis: current uses and future directions. Am J Sports Med 2009;37:1636-44 and Hunter DJ. Viscosupplementation for osteoarthritis of the knee. N Engl J Med 2015;372:1040-7).

The two components can be present in the composition of the invention in any ratio. In a preferred embodiment, the ratio between BPI and hyaluronic acid is comprised between 1 :0.001 and 1 :1000. More preferably, said ratio is comprised between 1 :0.005 and 1 :100. Even more preferably, the ratio between the amounts by weight of the two components is about 1 :4 (BPI:HA).

BPI is preferably administered at a dose ranging from 0.01 to 10 μg g of body weight, more preferably at a dose ranging from 0.1 to 5 μg g of body weight, even more preferably at a dose comprised between 0.2 and 1 .5 μg g of body weight. In a preferred embodiment, the dose of BPI is about 1.43 μg g of body weight; in another preferred embodiment, the BPI dose is about 0.29 μg g of body weight. The hyaluronic acid is preferably present in the composition in an amount, expressed as weight/volume%, comprised between 0.01 and 10% with respect to the total composition, more preferably in an amount ranging from 0.1 to 1 % with respect to the total of composition. In a preferred embodiment, hyaluronic acid is in the amount of about 0.02% with respect to the total composition.

The composition described above can be used as a medicament.

Preferably, it is for use in the treatment of arthropathies.

Arthropathies can be, for example, degenerative arthropathies, including osteoarthritis; post-traumatic arthropathies, resulting from physical trauma; arthropathies from infection, including reactive arthritis and septic arthritis; primary inflammatory arthropathies, including rheumatoid arthritis and psoriatic arthritis; crystal-induced arthritis, such as gout and pseudogout; metabolic arthropathies associated with metabolic diseases; para- or extra-articular rheumopathies.

In a preferred embodiment, the composition is for use in the treatment of arthritis. The composition can be administered to a subject affected by the aforesaid pathologies by conventional methods.

Conveniently, said medicament is in the form of a preparation for parenteral administration, but other forms are equally suitable for implementing the present invention. Injection, in particular intra-articular injection, is a preferred route of administration.

However, the person expert in the field can decide to administer the composition by any conventional pharmaceutical composition. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences.

Generally the components of the composition of the invention are administered in a therapeutic effective amount.

The expert in the field will decide the effective timing of administration, depending on the patient's condition, the disease severity, the patient's response and any other clinical parameter included in the general knowledge of this field.

The therapeutically effective dose for each compound can be initially estimated in cell culture assays or in animal models, usually mice, rats, guinea pigs, dogs or pigs. The animal model can also be used to determine the appropriate dose range and the route of administration. This information can be used to determine doses and routes of administration that are useful for humans. It is recommended to use the conversion table provided in the document Guidance for Industry and Reviewers (2002, US Food and Drug Administration, Rockville, Maryland, USA) to calculate the Human Equivalent Dose (HED).

An average dose for human administration can be established during clinical trials, as is customary in the field.

The specific effective dose for a human subject will depend on the disease severity, the general health of the subject, the age, the weight and the sex of the subject, the diet, the time and frequency of administration, the possible combinations of drugs and the tolerance/response to therapy. This amount can be determined by routine testing and is part of the physician evaluation.

It is also an object of the present invention a pharmaceutical composition comprising as active ingredients at least the Bactericidal/permeability-increasing protein (BPI) and hyaluronic acid together with at least one pharmaceutically acceptable carrier and/or excipient.

The pharmaceutical compositions will contain as active agents at least the BPI, or its biologically active fragments or analogs, and hyaluronic acid. The compositions according to the present invention contain, together with the active ingredient, at least one pharmaceutically acceptable carrier or excipient.

It may be particularly useful to utilize formulation adjuvants such as, for example, solubilizing agents, dispersing agents, suspending agents and emulsifying agents.

The pharmaceutical compositions according to the present invention may also contain one or more further active ingredients.

This active ingredient can be, for example, a compound useful for the treatment of arthropathies, for example a compound that reduces pain, reduces inflammation and/or prevents joint damage. Examples of said compounds are anti-inflammatory, such as traditional non-steroidal anti-inflammatory drugs (NSAIDs) or corticosteroids, painkillers, opioid or non-opioid analgesics, which can be appropriately selected by the expert in the field depending on the desired effect.

Average amounts of active compound may vary and in particular should be based on the recommendations and prescription of a qualified physician. The administration regimen, dosage and route of administration will be decided by the physician based on their experience, the disease to be treated and patient conditions. Depending on the route of administration, the form of the compositions will be solid or liquid, suitable for oral or parenteral administration, in particular for intra-articular administration.

In one embodiment of the invention, the pharmaceutical composition of the invention is for use as a medicament. Preferably, it is for use in the treatment of an arthropathy, preferably selected from those described above.

The composition of the invention may also be in the form of a kit comprising as component a) Bactericidal/permeability-increasing protein (BPI) and as component b) hyaluronic acid. In this case, the two components can be administered simultaneously or in any sequence.

It is also an object of the present invention the Bactericidal/permeability-increasing protein (BPI) for use in the treatment of an arthropathy in therapy combined with hyaluronic acid or in a therapeutic regimen with hyaluronic acid. The two substances can be administered by means of a single composition comprising both the active ingredients or by means of two separate compositions, one for each active ingredient, administered simultaneously or sequentially, in any order. The expert in the field will be able to choose the most appropriate therapy, timing and dosage by referring to his general knowledge in the field.

The present invention is further illustrated by the following examples.

EXAMPLES

Example 1

Effect of the combination of BPI and HA on crystal-induced inflammation

Materials and Methods

In vivo model of crystal-induced inflammation

Inflammation was induced by injection of monosodium urate (MSU) or calcium pyrophosphate (CPP) crystals, commonly involved in gout and CPP crystal-induced arthritis, respectively. All animals (mice C57/BI6) used for the experiments were male aged 8-9 weeks and an average body weight of 35 g (n=14 per condition).

Preparation of monosodium urate and calcium pyrophosphate crystals

MSU and CPP crystals were prepared by Denko's (Denko CW, Whitehouse MW.

Experimental inflammation induced by naturally occurring microcrystalline calcium salts. J Rheumatol 1976;3:54-62) and Cheng's method (Cheng PT, Pritzker KP. The effect of calcium and magnesium ions on calcium pyrophosphate crystal formation in aqueous solutions. J Rheumatol 1981 ;8:772-82), respectively. Their shape and birefringence were assessed by compensated polarized light microscopy. Crystals were sterilized by heating at 180°C for 2 h before each experiment. Less than 0.015 EU/ml endotoxin were measured in crystal preparations by Limulus amoebocyte lysate assay.

Induction of inflammation and treatment

2 mg of MSU or CPP crystals in 1 ml of PBS (Phosphate-buffered saline) were injected into the peritoneum of mice according to the method described by Leypoldt et al. (Leypoldt JK, Kamerath CD, Gilson JF. Acute peritonitis in a C57BL/6 mouse model of peritoneal dialysis. AdvPerit Dial 2007;23:66-70) in the presence or absence (50 g/ml) of BPI. In a group of mice, crystals were co-injected with a composition of BPI 50 μg ml (1 .5 μg g body weight) and HA (0.02%). Controls received the same volume of sterile PBS alone or BPI in PBS.

Mice were sacrificed after 3 h by an excess of anesthesia (inhalation of sevoflurane at 8%) and peritoneal fluids were harvested by washing with 2 mL of PBS. Immediately after, lavage fluids, collected with eventually present exudate, were assessed for total and differential white blood cell (WBC) count to determine the inflammation degree. The fluids were then centrifuged at 3000 rpm for 10 min and in supernatants was evuated by ELISA concentration of IL (interleukin)-l β, IL-6, CXCL1 (chemokine(C-X-C motif) ligand 1 ), TNF (tumornecrosisfactor) and VEGF (vascular endothelial growth factor) (Scanu A, Luisetto R, Oliviero F, Gruaz L, Sfriso P, Burger D, Punzi L. High-density lipoproteins inhibit urate crystal-induced inflammation in mice. Ann Rheum Dis 2015;74:587-94).

Results Injection of suspension of MSU or CPP crystals induced an inflammatory response, with a peak after 3 hours, involving leukocyte recruitment, mainly polymorphonuclear cells (PMN), and increased the levels of ΙΙ_-1 β, CXCL1 , IL-6, TNF and VEGF in lavage fluids collected.

5 The administration of BPI and HA together with crystals significantly reduced leukocyte influx, PMN rate, inhibited the production of pro-inflammatory mediators compared with the treatment with crystals alone.

The use of the combination of BPI with HA was better that the treatment with the protein alone, although the results showed the activity of BPI in inflammation i o reduction.

Injection of PBS ore BPI alone di not induced any inflammatory effect.

Results are reported in the following table 1. The grey areas refer to the use of a composition according to the invention.

Table 1 . Results are reported as meanistandard deviation (SD)

During experiments, no specific toxicity was observed.

Supplementary results

A group of mice (n=14 per condition)was injected with crystals in the presence of HA

5 (0.02%). Inflammation induced by the 2 different types of crystals was slightly reduced after treatment with HA.

Results are reported in the following table 2.

Table 2. Results are reported as mean±SD

o Example 2

Effect of the combination of BPI and HA on collagen induced arthritis

Materials and methods

Murine model of collagen-induced arthritis (CIA)

Arthritis induction

s CIA was induced in male C57BI/6 mice (n=12 per condition) aged 9-10 weeks, and an average body weight of 35 g, by immunization with type II collagen (CII)/complete Freund's adjuvant (CFA) emulsion at days 0 and 21 (Bevaart L, Vervoordeldonk MJ, Tak PP. Collagen-induced arthritis in mice. Methods Mol Biol 2010;602:181 -92). Briefly, anesthetized mice were injected intradermally at the base of the tail with 2 mg/ml CM emulsified 1 :1 with 1 mg/ml CFA (70 μΙ; T=0). After 3 weeks (T=21 ) the initial immunization mice received a booster dose. Development and severity of arthritis were monitored by measuring paw swelling with a digital caliper (Kroeplin) and scored from 0 to 5 according to the Bevaart clinical scoring system (Bevaart L, Vervoordeldonk MJ, Tak PP. Collagen-induced arthritis in mice. Methods MolBiol 2010;602:181 -92) every other day. Animal weight was also recorded.

Treatment

Arthritic mice (paw score=2) were intraperitoneally injected with 200 μΙ of BPI (50 μg ml) in PBS in the presence or absence of HA (0.02%). Treatment and arthritis evaluation were carried out twice a week for 2 months. Arthritis severity was assessed as described above. Untreated mice or animals that received HA alone (0.02%) were used as controls. At the experiment endpoints, mice were euthanized by an excess of anesthesia (inhalation of sevoflurane at 8%) and post-mortem examinations were also performed.

Post-mortem examinations

At the sacrifice blood was collected by cardiac puncture. A small quantity of the blood was used to prepare blood smears in order to evaluate the total and differential white blood cell count by May-Grunwald-Giemsa staining.

The remaining blood was centrifuged at 3000 rpm for 10 min to obtain serum and the levels of IL-1 β, IL-6, CXCL1 , TNF and VEGF were measured by ELISA.

Hind paw specimens were fixed in formalin, embedded in paraffin and stained with haematoxylin and eosin for histological analysis. Paws were assessed for cartilage destruction, pannus formation, bone resorption and inflammatory changes based on the number and type of inflammatory cells in ankle joints. The severity of each parameter was scored separately on a scale from 0 to 5 (0= normal, 1 = minimal effect, 2= mild effect, 3= moderate effect, 4= marked effect, 5= severe effect) (Lories RJ, Matthys P, de Vlam K, Derese I, Luyten FP. Ankylosingenthesitis, dactylitis, and onychoperiostitis in male DBA 1 mice: a model of psoriatic arthritis. Ann Rheum Dis 2004;63:595-8).

Results

CIA is a well-established experimental model that through an autoimmune reaction to a cartilage component can lead to a chronic destructive polyarthritis. An increased paw thickness, in particular of the ankle joints, was observed in all mice. Results are reported in the table as mean±SD of the ankle thickness, the difference between the final and the initial ankle thickness, and the Bevaart scores assigned.

Histological analysis confirms the development of arthritis and shows the typical features of CIA, including synovial inflammation, leukocyte infiltration, cartilage damage, bone resorption, pannus formation.

In this model, co-treatment with BPI and HA displayed improvement of clinical symptoms, reduction of arthritic scores and down-regulation of specific inflammatory mediators. In addition, we observed that the combined use of BPI and HA showed a greater and faster reduction of all parameters evaluated than that observed after administration of BPI alone.

All parameters evaluated indicated a tendency towards a decrease in mice injected with HA alone as compared with untreated controls.

During experiments no specific toxicity was observed. Examination of blood smears did not reveal any considerable changes due to toxicity in total and differential white blood cell count as compared to controls.

Results are reported in tables 3 and 4. The grey areas refer to the use of a composition according to the invention.

Table 3. Results are reported as mean±SD

Table 4. Results are reported as mean±SD

Claims

1 . A composition comprising Bactericidal/permeability increasing protein (BPI), or at least a biologically active fragment or a biologically active analogue thereof and hyaluronic acid.

2. The composition according to claim 1 wherein said BPI protein is natural, synthetic or recombinant.

3. The composition according to claim 1 o 2 comprising one or more fragments of the BPI protein, said fragments being natural, synthetic or recombinant.

4. The composition according to anyone of the preceding claims wherein said hyaluronic acid is in one of the following forms: low molecular weight form, high molecular weight form, salt form, cross-linked form, reticulate form, esterified form, amide form.

5. The composition according to anyone of the preceding claims wherein the ratio between the amounts by weight between BPI and hyaluronic acid is comprised between 1 :0.001 and 1 :1000, preferably said ratio is comprised between 1 :0.005 and 1 :100.

6. The composition according to claim 5 wherein the ratio between the amounts by weight between BPI and hyaluronic acid is around 1 :4.

7. The composition according to anyone of the preceding claims wherein the BPI protein BPI is administered at a dose ranging from 0.01 to 10 μg g of body weight, preferably at a dose ranging from 0.1 to 5 μg g of body weight, more preferably at a dose comprised between 0.2 and 1 .5 μg g of body weight.

8. The composition according to anyone of claims 1 -5 for use according to claim 7 wherein the dose of BPI protein is of around 1 ,43 μg g of body weight or of around 0,29 μg g of body weight.

9. The composition according to anyone of claims 1 -5 for use according to claim 7 wherein hyaluronic acid is present in the composition in an amount, expressed as weight/volume%, comprised between 0.01 and 10% with respect to the total composition.

10. The composition according to anyone of claims 1 -5 for use according to claim 9, comprising an amount of hyaluronic acid of around 0,02% with respect to the total composition.

1 1 . The composition according to anyone of the preceding claims for use as a medicament.

12. The composition according to anyone of claims 1 -10 for use for the treatment of an arthropathy.

13. The composition for use according to claim 12 wherein said arthropathy is selected from the group consisting of: degenerative arthropathies, posttraumatic arthropathies, metabolic arthropaties, crystal-induced arthropaties, inflammatory arthropathies, arthropathies from infection, rheumatic diseases, septic arthritis, reactive arthritis.

14. The composition for use according to claim 12 or 13 wherein said arthropathy is selected from the group consisting of: osteoarthritis arthritis, rheumatoid arthritis, psoriatic arthritis, gout, pseudogout, metabolic arthropathy associated with a metabolic disease and para- or extra-articular arthritis.

15. The composition for use according to claims 1 1 -14 wherein said composition is administered by parenteral route, preferably by intra-articular injection.

16. A pharmaceutical composition comprising as active agents the Bactericidal/permeability increasing protein (BPI) and hyaluronic acid together with at least one pharmaceutically acceptable carrier and/or excipient.

17. The pharmaceutical composition according to claim 16 for use for the 5 treatment of an arthropathy.

18. Bactericidal/permeability-increasing protein (BPI) for use in the treatment of an arthropathy in therapy combined with hyaluronic acid or in a therapeutic regimen with hyaluronic acid.

19. A kit comprising :

i o a) a) Bactericidal/permeability-increasing protein (BPI) and

b) hyaluronic acid. In this case, the two components can be administered simultaneously or in any sequence

20. The kit of claim 19 for use for use for the treatment of an arthropathy.