WO2003068245A1

WO2003068245A1 - Use of polyanhydroglucuronic acid comprising microdispersed oxidised cellulose as immunomodulator

Info

Publication number: WO2003068245A1
Application number: PCT/IE2003/000023
Authority: WO
Inventors: Blanka Rihova; Marketa Jelinkova; Jiri Briestensky; Ivan Santar
Original assignee: Alpenstock Holdings Limited
Priority date: 2002-02-14
Filing date: 2003-02-14
Publication date: 2003-08-21
Also published as: AU2003245750A1

Abstract

A biocompatible Ca/Na salt of polyanhydroglucuronic acid (PAGA) or derivative thereof and use thereof in the preparation of a medicament for modulating an immune response in an organism. The polyanhydroglucuronic acid comprises a microdispersed oxidised cellulose or derivative thereof.

Description

YA^NHYDROGLUCURONIC ACID COMPRISING MICRODISPERSED OXID_ISE_D CEL_LULO_SE MODULATOR

The invention relates to stimulating an immune response in an organism.

The immune system can be manipulated specifically by vaccination or non- specifically by immunomodulation. In general, immunomodulators are biological response modifiers (BRM) that affect the immune response in either a positive or a negative fashion.

Numerous polysaccharides of various origins (yeast, bacteria, algae, fungi and higher plants) have been investigated for immunomodulatory activities. One of the most intensively studied polysaccharides is glucan [1, 2, 3]. β-(l,3)-glucan consists of a linear backbone of β-(l,3)-linked D-glucopyranosyl groups with varying degrees of branching from the C6 position. Immunopharmacological activities of glucans vary depending on their physico-chemical characteristics, i.e. molecular weight, degree of branching and conformation. Glucans have been shown experimentally to exert anti- cancer and anti-infective effects and these activities are mediated to a large extent by stimulating effects on the immune system (immunocompetent cells, cytokines, complement and nitric oxide production). β-(l,3)-glucans activate neutrophils [4], macrophages [5] and natural killer (NK) cells [6] to trigger potent tumoricidal activity. These glucans bind to a specific site on polymorphonuclear leucocytes and increase cytokine production (including IL-1, IL-2, IL-6, M/GM-CSF, TNF-α, etc.). The leucocyte complement receptor type 3 (CR3 or CD lib/CD 18) has been reported to bind both paniculate and soluble β-(l,3)-glucan, as well as other polysaccharides [7-9].

Glucans with a linear backbone of β- (1,4) -linked D-glucose or 2-deoxy-2- aminoglucose units are the most abundant natural polysaccharides. Chitin, chitosan and cellulose are members of the same family. β-(l, 4) -linked polyglucuronic acid, which was prepared by oxidation of cellulose at position C6 (C6OXY), has application in medical practice. Oxidized regenerated cellulose (commercially available for example under the Trade Marks Oxycel, Surgicel or Interceed.) is one of the most commonly used bioabsorbable topical hemostatic agents. It has also been used as a wound healing aid and as a synthetic barrier to prevent postoperative adhesions [10-14].

Otterlei et al [23, 24] describe diequatorially bound B-1,4 polyuronates such as cellulose oxidised in the C-6 position (C60XY), chitosans and their use for cytokine stimulation.

It is clear that any method for improving immunopotential in an organism would have valuable therapeutic benefit.

Statements of Invention

According to the invention there is provided use of a biocompatible salt of polyanhydroglucuronic acid (PAGA) or derivative thereof in the preparation of a medicament for modulating an immune response in an organism wherein the PAGA comprises a microdispersed oxidised cellulose or derivative thereof.

In one embodiment of the invention the derivative is an inorganic salt. Preferably the inorganic salt is selected from any one or more of K, Na, Ca, Mg, Zn, Al or Co.

Preferably the derivative is a simple acetate type or complex or mixed salt or intermolecular complex thereof. Most preferably the derivative is a 'mixed' salt containing more than one inorganic cation. Ideally the derivative is a Ca/Na salt.

In one embodiment of the invention the derivative is an organic derivative of the complex salt or intermolecular complex type comprising aminoacids and their metabolites and/or peptidic hydroylsates of proteins selected from for example any one or more of urea, gelatine or arginine.

Most preferably the polyanhydroglucuronic acid and salts thereof comprises a biocompatible intermolecular polymer complex of

- polyanhydroglucuronic acid; and

- a cationic component comprising a linear or branched natural, semi- synthetic or synthetic oligomer or polymer.

Preferably the cationic component is selected from any one or more of derivatives of acrylamide, methacrylamide and copolymers thereof, cationised natural polysaccharide, a synthetic or semi-synthetic polyamino acid, a synthetic anti- fibrinolytic, a natural or semi-synthetic peptide, or an aminoglucane or derivatives thereof.

Most preferably the polyanhydroglucuronic acid contains in their polymeric chain from 8 to 30 per cent by weight of carboxyl groups, at least 80 per cent by weight of these groups being of the uronic type, at most 5 per cent by weight of carbonyl groups, and at most 0.5 per cent by weight of bound nitrogen.

The invention provides use of a biocompatible salt of the invention in the preparation of a medicament for use as an immunosuppressor; as an immunostimulator; stimulating the production of TNF-α; stimulating bone marrow hematopoieses; or in an adjuvant therapy.

Preferably the preparation of a medication is for use in disorders including disorders including malignancies, immunodeficiencies such as AIDS, viral infections such as epidemic influenza or prion infections such as Creutzfeld-Jacob disease, and inflammatory diseases.

Most preferably the invention provides use of a biocompatible salt of the invention in the preparation of a medicament for use in the proplylaxis of recurrent infections or respiratory tract infections.

The invention also provides use of a mixed salt containing more than one inorganic cation, of polyanhydroglucuronic acid (PAGA) or derivative thereof in the preparation of a medicament for stimulating an immune response in an organism.

One preferred embodiment of the invention provides a mixed salt wherein the mixed salt is a Ca/Na salt of polyanhydroglucuronic acid (PAGA) or derivative thereof.

Most preferably the use a biocompatible salt of the invention comprises at least one other biocompatible biologically active substance and/or at least one biologically acceptable adjuvant and/or at least one pharmaceutically active adjuvant.

The invention provides a biocompatible Ca/Na salt of polyanhydroglucuronic acid (PAGA) or derivative thereof. Preferably the polyanhydroglucuronic acid comprises a microdispersed oxidised cellulose or derivative thereof prepared in the microdispersed form by oxidative hydrolysis of raw oxidised cellulose.

The invention also provides a composition comprising a biocompatible Ca/Na salt of polyanhydroglucuronic acid or derivative thereof preferably comprising at least one other biocompatible biologically active substance and/or at least one biologically acceptable adjuvant and/or at least one pharmaceutically active adjuvant. Preferably the polyanhydroglucuronic acid or derivative comprises a microdispersed oxidised cellulose or derivative thereof. Brief description of the drawings

The invention will be more clearly understood from the following description thereof given by way of example only with reference to the accompanying drawings in which; -

Fig. 1 is a graph showing the production of TNF-α by human peripheral blood leucocytes (PBLs) treated with different MDOC derivatives; and

Fig. 2 is a graph showing the production of TNF-α by A/Ph mouse splenocytes treated with different microdispersed oxidised cellulose (MDOC) derivatives.

Detailed description

It was surprisingly found that certain derivatives of polyanhydroglucuronic acid (PAGA) or salts thereof, in the form of a stable microdispersed oxidised cellulose (MDOC) have significant immunostimulation capacity.

MDOC is described in detail in PCT/IE98/00004 the entire contents of which are incorporated herein by reference. In particular, the MDCO is prepared by a process wherein a polyanhydroglucuronic acid-containing material obtained by oxidation with nitrogen oxides is subjected to partial or complete hydrolysis and neutralisation in an aqueous solution of at least one inorganic and/or organic salt and/or base in the presence of at least one suitable oxidising agent. The hydrolysate undergoes fractional coagulation to form a stable microdispersed product.

In the first step of the process a suitable polysaccharide material is oxidised in the presence of nitrogen oxides. The oxidised product is then subjected to partial or complete hydrolysis and neutralisation in an oxidising environment. In this way the secondary products such as aldehydes and ketones and their condensation products inevitably produced as a result of the initial oxidation step are removed. These aldehyde and ketone impurities have a fundamental influence on the stability of the polyanhydroglucuronic acid (PAGA) product.

A stable PAGA product with a reduced degree of crystallinity and a high degree of purity in a microdispersed form is produced. The microdispersed PAGA has easily controllable physicochemical properties which are essential for a product for medical or pharmaceutical use.

Cytokines play a central role in the immune response by promoting the activation of antigen-specific and nonspecific effector mechanisms and tissue repair. Selective cytokine production in particular can determine the outcome of a response by stimulating protective or exacerbative immune mechanisms. CD4⁺ helper T cells that produce IL-2, IFN-γ and TNF-α/β, but not IL-4 are designated Thl and are chiefly responsible for cell-mediated responses (delayed-type hypersensitivity (DTH) and activation of CD8⁺ cells). They can also help B cells to produce IgG_2a, but not much IgGi or IgE, respectively. CD4⁺ Th cells that produce IL-4, IL-5, IL-10 and IL-13, but not IL-2 or IFN-γ, are designated Th2. They are very efficient helper cells for B cell activation and augment humoral responses such as the secretion of antibodies, especially of IgGi and IgE [15].

Native CD8⁺ T cytotoxic (Tc) cells, similar to CD4⁺ Th cells, can differentiate into at least two subsets of cytolytic effector cells with distinct cytokine patterns; Tel cells secrete a Thl -like cytokine pattern, including IL-2 and IFN-γ, while Tc2 cells produce Th2 cytokines including IL-4, IL-5 and IL-10. Tel and Tc2 cells induce similar DTH reactions involving edema and granulocytic infiltration despite their distinct cytokine profiles in vitro and in vivo [16]. The Tel and Tc2 subsets of CD8⁺ T cytotoxic effector cells have similar functions, including perforin- and Fas- dependent cytotoxicity. CD8⁺ Tc cells often produce lower cytokine levels than CD4⁺ Th cells because the CD8⁺ Tc cells kill their antigen-presenting cells before full stimulation can occur. Either CD4⁺ Th or CD8⁺ Tc cells can dominate a DTH response depending on the MHC restriction of the antigens presented to the T cells [17, 18].

We have surprisingly found that derivatives of MDOC and in particular the Ca/Na salt, Na salt and gelatine derivative were able to induce in vitro release of TNF-α but not IFN-γ, IL-4 or IL-6. The most intensive cell proliferation and cytokine release was found with the MDOC Ca/Na salt, in particular the MDOC Ca Na salt was found to stimulate spontaneous proliferation of mouse splenocytes and human peripheral blood leukocytes in vitro, enhance the production of TNF-α in vitro and stimulate bone marrow hematopoiesis in vivo.

The oxidative hydrolytic treatment of polyanhydroglucuronic acid followed by reprecipitation or fractionation as described in PCT/IE98/00004 enables the preparation of glucuronoglucanes in the form of simple and complex salts or intermolecular complexes thereof with a reduced polydiversity. These compounds have surprisingly been found to have enhanced immunomodulatory effect. All the compounds appear to display an amorphous structure

The Ca/Na salt of MDOC has therefore potential as a biological response modifier.

In particular the Ca/Na salt of MDOC has potential as a potent immunostimulator.

In an experimental mouse model, it was found that MDOC Ca/Na salt potentiated in vivo proliferation of bone marrow stem cells measured as CFU-s. No side toxicity of the MDOC Ca Na salt was found in preliminary in vivo experiments. It was also found that the MDOC Ca/Na salt was able to overcome the inhibition of proliferation of mouse splenocyte induced by mitomycin. The salts of MDOC with immunomodulatory capabilities therefore have valuable therapeutic potential and may be used as an adjuvant therapy in a variety of disorders including malignancies, immunodeficiencies such as AIDS, viral infections such as epidemic influenza or prion infections such as Creutzfeld-Jacob disease, and inflammatory diseases. They may also be potentially useful to prevent recurrent infections including infections of the respiratory tract.

The term "mixed" salt is taken throughout the specification to indicate that a number, one or more, cations are present and their nature affects the size and conformation and biological properties of the resulting polysaccharide.

A "mixed" salt may include a complex salt or an intermolecular complex of a simple acetate type or complex salt.

The present invention will be more fully understood from the following description given by way of example.

Methods and Materials

The immunomodulatory capabilities of MDOC were tested in its effect on a) in vitro proliferation of mouse and human immunocompetent cells, b) production of selected cytokines, namely IFN-γ, TNF-α cytokines typical for T lymphocyte helper 1 cells (Thl cells), and IL-4, IL-6, cytokine typical for T lymphocyte helper 2 cells (Th2 cells), and c) bone marrow stem cells evaluated as colony-forming units-spleen (CFU-S).

Preparation of PAGA in the form of Microdispersed oxidized cellulose (MDOC)

MDOC was prepared as described in PCT/IE98/00004 which is herein incorporated by reference. Briefly, MDOC is β- (1,4) -linked D-glucuronic acid prepared by oxidation of cellulose at position C6. The oxidized sample was obtained by the reaction of nitrogen oxides in 60% nitric acid with cotton. The intermediate product contained 16.2 wt% carboxyl groups which corresponded to 63.5% theoretical quantity; the 0.08 wt% residue after hot drying at 800°C, and 0.42 wt% bound nitrogen, was subjected to a controlled hydrolysis (pH and ionic strengths monitored) for 1 h at 90°C in solutions of inorganic (H, K, Na, Ca, Mg, Zn, Al, Co, Ca/Na) or organic (urea, gelatine, arginine) salts in the presence of H₂O₂ (pH 6.5- 7.5). The samples were fractionated using 99.0-99.5% ethyl alcohol, and 50 wt% water-ethanol mixture.

The final products were analysed for cation content using AAS and ICP-AES methods (Jobin Yvon JY 38). The ¹³C-NMR spectra were recorded at 298 K in D₂O solution using a Bruker AM 300 instrument. Acetone was used as an internal standard (CH3 resonance at 31.07 ppm). The molecular weight was determined for each sample by GPC carried out on Sepharon HEM A 1000 column (4.6 x 300 mm), using 0.25M NaCl solution as eluent, flow rate 0.3 ml/min, IR detection. X-ray diffraction, FTIR, and ¹³C-NMR methods were used for further sample characterisation. A summary of samples used for biological evaluation is given in Table 1. The tested samples were apyrogenic and sterile.

Table 1

*Complex salts with organic cation

Although Table 1 disclose a number of bound cations tested, the list is not exhaustive and it is anticipated that other inorganic cations for example Li, Fe, Cr, Bi, Pt, Ag and Cu will also show such activity.

Urea and arginine represent organic complex salts with amino and amido group containing compounds. Other organic complex salts may include for example histamine, purine or pyrimidine bases.

Other peptides may include for example vegetable or fish peptides. Mice

Inbred strains of A Ph (H-2^a) and C57BL/10 (H-2^b) mice, aged 10 weeks, were purchased from the Animal Centre of the Institute of Physiology, Academy of Sciences of the Czech Republic. All the mice were housed in accordance with approved guidelines and were provided with food and water ad libitum. The experimental designs were in accordance with the Czech Republic Act for Experimental Work with Animals (Decrees No. 311/97, 117/87, and Act No. 246/92) which is fully compatible with the corresponding European Community Acts.

Cell culture Mouse splenocytes

The mice were killed by cervical dislocation. The spleens were removed aseptically, stripped of fat and placed in a culture medium. The culture medium was RPMI 1640

(Sigma) supplemented with 4 mM L-glutamine (Gibco BRL) and 10% foetal calf serum (FCS, Gibco BRL). Single-cell suspensions were obtained by gentle homogenisation of mouse spleen in a tissue homogeniser. The spleen lymphocytes were separated from the debris and then washed twice (5 min at 800 x g at 4°C). Isolated cell suspensions were washed three times in the culture medium. The lymphocyte viability was assessed by the trypan blue dye exclusion. The viability of the cells used throughout was > 95%.

Human peripheral blood leukocytes

Blood was obtained from healthy donors. Peripheral blood leucocytes (PBL) were isolated from the heparinized blood by centrifugation over a Ficoll-Hypaque gradient (Amersham Pharmacia Biotech). Lymphocytes were washed and cultured in RPMI 1640 (Sigma) supplemented with 4 mM L-glutamine (Gibco BRL), penicillin- streptomycin solution (Sigma) and 10% FCS. The viability of isolated cells was 96- 99%.

Proliferation assay of mouse splenocytes or human blood leucocytes

To measure cell proliferation, [³H]-thymidine incorporation was measured in 96- well flat-bottomed tissue culture microplates (NUNC) using non-stimulated cells (spontaneous proliferation) or cells exposed to the T cell mitogens (Sigma): lectin from Canavalia ensiformis (ConA, 1.25 μg/well), Phaseolus vulgaris agglutinin (PHA, 2.5 μg/well) or to the B cell mitogen (Sigma): Salmonella typhimurium derived lipopolysaccharide (LPS, 0.5 μg/well). Culture plates were seeded with 0.1 ml of cell suspension (5x10⁶ mouse splenocytes/ml or 2x10⁶ human PBL/ml). Various concentrations of MDOC derivatives were added to the wells to achieve the desired concentrations and a final well volume of 0.25 ml. Pure medium served as a control. At the end of the incubation period (3 days in 5% CO₂ in a humidified 37°C incubator), 1 μCi/50 μl of [³H]-thymidine (Amersham Pharmacia Biotech) was added per well, followed by 6 h incubation. The cells were then collected onto glass- fibre filters (Filtermat, Wallac) using a cell harvester (Tomtec). Upon drying, a sheet of solid scintillator Meltilex (Wallac) was placed in a sample bag together with a filtermat containing 96 samples and run together through a heat sealer (Microsealer,

Wallac). Samples were measured in MicroBeta TriLux (Wallac). The results were calculated as arithmetic means of the c.p.m. in three or six individual wells. The stimulation index (SI) was calculated by the following formula:

SI = mean cpm in experimental cultures / mean cpm in control cultures

Mixed leucocyte reaction (MLR)

Five hundred thousand responder cells (H-2^a) were mixed with lxlO⁶ mitomycin C (50 μg/ml, Sigma) treated stimulatory cells (H-2^b) in triplicate wells. Cultures were maintained at 37°C in 5% CO₂. After 5 days, each culture was pulsed with 1 μCi of [³H]-thymidine per well. Twenty hours later the incorporation of the radiolabel was measured by scintillation counting as described for proliferation assay.

Cytokine secretion assays

Cytokine levels in culture supernatants were determined by a standard sandwich enzyme-linked immunosorbent assay (ELISA). Briefly, after the cells were removed, supernatants from three to six wells were pooled at each time point and frozen at - 70°C for the cytokine assays. Murine commercial kits for IFN-γ and IL-4 were purchased from Diaclone Research (France) and for TNF-α and IL-6 from R&D Systems (USA). Human kits for IFN-γ, IL-4, TNF-α and IL-6 were purchased from Diaclone Research (France). Supernatants were assayed for the presence of cytokines according to manufacturer's instructions. The optical density was measured at 450 nm as the primary wavelength and at 620 nm as the reference wavelength using a

Spectra Rainbow Thermo reader (Tecan). To quantify the amount of cytokine present in test samples, the values were extrapolated from standard curves established by analysing different dilutions of recombinant murine or human IL-4, IL-6, IFN-γ and TNF-α.

Colony-forming unit-spleen (CFU-s)

The method of Till and McCulloch [30] was used. The donor inbred mice A/Ph (5 mice per one experimental group) were injected five times, i.e. on day 1, 3, 5, 7 and 9 intraperitoneally with two derivatives of MDOC (Ca/Na salt; Na salt) in total doses of 1.25 mg/mouse, 0.25 mg/mouse and 0.05 mg/mouse. An equal number of mice served as controls and received injection of phosphate buffer saline (PBS) solution only. The mice were exsanguinated 24 h after the last injection. The bone marrow cells from each group of mice were harvested by washing both femurs with RPMI 1640 medium and pooled. After repeated washing with RPMI 1640 supplemented with 10% fetal calf serum, the concentration of cells was adjusted to 5x10⁵ cells/ml. The viability of cells in all experiments exceeded 95%. Then the donor bone marrow suspension was injected i.v. (0.2 ml containing lxlO⁵ cells) into syngenic recipient mice which had been x-irradiated by °Co (8 Gy). Transplanted mice were sacrificed 8 days after transplantation, their spleens were removed and fixed in Bouin's solution, and the number of CFU-s was enumerated.

Statistics

All results were calculated from mean + standard deviation (SD). Statistical evaluation of the data was performed by Student's t-test to determine a significant variance. A P value < 0.01 was considered significant.

Results

Effect of MDOC Ca/Na salt on spontaneous and mitogen-induced proliferation of splenocytes isolated from high IgG (A/Ph) or low IgG (C57BL/10) responder strains of mice.

A/Ph mice are high IgG responders, while C57BL/10 mice are low IgG responders to many antigens. These two mouse inbred strains have also different responses and cytokine production in vitro [19-22]. MDOC Ca/Na salt under in vitro cultivation had a substantial effect on spontaneous proliferation of mouse splenocytes isolated from both inbred strains of mice (Table 2). Very intensive cell proliferation was detected after cultivation of resting splenocytes with 0.2 - 2 mg MDOC/ml ( <0.001). The mitogen-induced proliferation of A/Ph and C57BL/10 splenocytes was co-stimulated by MDOC Ca/Na salt always at the concentration of 1 mg/ml

(P<0.01). In A/Ph inbred strain, B cell proliferation induced by LPS was also slightly elevated at the concentration of 0.02 - 1 mg MDOC/ml (P<0.01). On the other hand,

ConA-induced proliferation was slightly inhibited if the cells were exposed to lower MDOC concentrations (0.001 - 0.02 mg/ml, P<0.01). It was shown that the proliferation pattern was influenced by the genetic background of the tested organism. In contrast to the A/Ph strain where we have observed cell inhibition during ConA-induced proliferation, C57BL/10 splenocyte inhibition was detected during PHA and LPS-induced proliferation in the highest concentration of 2 mg MDOC/ml ( <0.01). Generally, MDOC Ca/Na salt had the highest stimulatory effect on spontaneous proliferation of A/Ph (SI = 34.73) and C57BL/10 (SI = 15.27) splenocytes at the optimum concentration of 1 mg/ml. Mitogen-induced proliferation was co-stimulated in all cases at the same concentration of 1 mg/ml.

Table 2

Data represent stimulation index (SI), nd = not detected Average proliferation of control A/Ph splenocytes: spontaneous = 329 + 66 cpm, ConA-stimulation = 51048 ± 3162 cpm (SI = 155.16), PHA-stimulation = 10851 + 2004 (SI = 32.98) and LPS-stimulation = 13858 ± 4299 (SI = 42.12). Average proliferation of control C57BL/10 splenocytes: spontaneous = 271 + 52 cpm, ConA-stimulation = 17201 ± 5529 cpm (SI = 63.47), PHA-stimulation = 10521

± 1232 (SI = 38.82) and LPS-stimulation = 10584 ± 2352 (SI = 39.06). *P < 0.01, ** < 0.001

Effect of MDOC Ca Na salt on spontaneous proliferation of human peripheral blood leucocytes

The peripheral blood leucocytes (PBLs) from two healthy donors were exposed to serial concentrations of Ca/Na salt of MDOC in vitro (Table 3). In both cases, cell proliferation was stimulated at a concentration of 1 mg MDOC/ml (P<0.001, P<0. 1). The lowest concentration of MDOC Ca/Na salt had a slightly inhibitory effect on spontaneous proliferation of human peripheral blood leukocytes (P<0.01), but other concentrations were without any significant effect on PBLs. The dose- dependent stimulation was higher (SI = 2.41) in the donor with a lower background proliferation (271 + 41 cpm).

Table 3

Data represent stimulation index (SI).

Average proliferation of nonstimulated PBLs - donor 1 = 271 + 41 cpm, PBL donor 2 = 543 + 41 cpm.

* P < 0.01, ** P < 0.001

Effect of MDOC Ca/Na salt on mixed leucocyte reaction (MLR)

The effect of MDOC Ca/Na salt was tested on the mixed leucocyte reaction (MLR), an in vitro correlation of the in vivo allograft transplantation reaction (Table 4). A/J (H-2^a) splenocytes were used as responding and MMC -treated C57BL/10 (H-2^b) splenocytes as stimulating cells. We have observed a considerable stimulation of MLR at high MDOC concentrations (0.1 - 2 mg/ml, P<0.001). The inhibition of C57BL/10 splenocytes induced by mitomycin C (MMC) can be partly overcome by

MDOC Ca/Na salt in concentrations of 0.02, 1 and 2 mg/ml (P<0.01). Table 4

Data represent stimulation index (SI).

Average proliferation of control MLR (responder cells (H-2^a) mixed with MMC treated stimulatory cells (H-2^b)) = 8394 + 445 cpm; average proliferation of nonstimulated responder cells (H-2^a) = 700 + 130 cpm; average proliferation of MMC treated stimulator cells (H-2^b) = 75 + 9 cpm. * P < 0.01, ** P < 0.001

Effect of various MDOC derivatives on spontaneous proliferation of human peripheral blood leucocytes and A/Ph mouse splenocytes

Twelve different soluble derivatives of MDOC (Acid form, K salt, Na salt, Ca salt, Mg salt, Zn salt, Al salt, Co salt, Ca/Na salt, urea, gelatine and arginine derivatives) were tested for their ability to stimulate in vitro proliferation of human peripheral blood leucocytes and mouse splenocytes (Tables 5, 6, 7, 8). Using the human peripheral blood leucocytes (PBLs), a significant cell stimulation was seen with MDOC acid form (P<0.01), Ca/Na salt (P<0.001, Table 5) and with MDOC urea (P<0.01), gelatine (P<0.01), arginine (P<0.001, Table 6) in a concentration ranging from 0.2 to 2 mg/ml. The strongest stimulatory effect was demonstrated by the MDOC Ca/Na salt at the optimal concentration of 1 mg/ml (SI = 3.55) and arginine derivative at a concentration of 2 mg/ml (SI = 3.19). Ca, Mg, Zn and Co salts induced cell inhibition in dependence on sample concentration (P<0.01, P<0.001) or were without any significant effect on PBLs. The best result, i.e. the most intensive proliferation of PBLs, was obtained with Ca/Na salt (SI = 3.55, P<0.001), while almost no effect was observed with K, Na and Al salts.

Table 5

Data represent stimulation index (SI).

Average proliferation of nonstimulated PBLs = 234 + 63 cpm.

* P < 0.01, ** P < 0.001

Table 6

Data represent stimulation index (SI).

Average proliferation of nonstimulated PBLs = 243 + 27 cpm.

• P < 0.01, ** P < 0.001 In contrast to human leucocytes, A/Ph mouse splenocytes responded to most MDOC derivatives by intensive proliferation (P<0.001, Tables 7, 8). Similarly, as observed on human leucocytes, the most potent stimulator of mouse splenocyte proliferation was Ca/Na salt at 1 mg/ml (SI = 29.04, P<0.001). Among organic derivatives, the highest stimulation index was detected with gelatine (SI = 12.19, P<0.001) at 1 mg/ml (Table 8). On the other hand, Zn salt (P<0.001) and Co salt (P<0.001) significantly reduced the proliferation of mouse splenocytes (Table 7).

Table 7

Data represent stimulation index (SI).

Average proliferation of nonstimulated A/Ph splenocytes = 629 + 75 cpm.

** P < 0.001

Table 8

Data represent stimulation index (SI).

Average proliferation of nonstimulated A/Ph splenocytes = 1073 + 153 cpm.

**P < 0.001 MDOC and its derivatives induce the release of TNF-α from human peripheral blood leucocytes and mouse splenocytes.

These studies evaluated the ability of four MDOC derivatives (Na salt, Ca/Na salt, gelatine and arginine derivatives) to elicit the release of Thl (IFN-γ and TNF-α) and

Th2 (IL-4 and IL-6) cytokines in vitro. Results obtained with human peripheral blood leucocytes and mouse splenocytes were comparable. The stimulation with MDOC derivatives (Na salt, Ca/Na salt and gelatine derivative) resulted in a strong release of TNF-α, while MDOC arginine was without any significant effect (Figs.l, 2).

In both systems, mouse and human, the level of TNF-α was the highest if the supernatants were taken after 24 h of cultivation and cells were exposed to 1 mg/ml of MDOC derivatives. In human lymphocytes, the highest increase of TNF-α production was seen in cell supernatants exposed to the Ca/Na salt (2177+200 pg

TNF-α/ml, Fig.l) while in mouse splenocytes the highest increase of TNF-α production was seen in cell supernatants exposed to MDOC gelatine (599+2 pg TNF-α/ml, Fig.2). The level of the control response to LPS (118+32 pg TNF-α/ml) in mouse cells was lower than that to MDOC Na salt, Ca/Na salt and gelatine (Figs.l, 2). The level of the control response to PHA (770+53 pg TNF-α/ml) in human peripheral blood leucocytes was lower than to the Ca/Na salt (Figs.l, 2). LPS and PHA were used as controls according to manufacturer's instructions.

The levels of IL-4, IL-6 and IFN-γ induced by MDOC derivatives were not significantly greater than those in control cells. These cytokines were not detected after either 48 h or 72 h of cultivation (data not shown). Thus, the above data suggest that selected MDOC derivatives could activate mouse splenocytes and human peripheral blood leukocytes to enhance the production of pro-inflammatory cytokine TNF-α. Detection of Colony-Forming Units-spleen after in vivo application of MDOC Ca/Na salt and MDOC Na salt

The detection of colony-forming unit-spleen (CFU-s) is based on the fact that the intravenous injection of donor bone marrow cells into sublethally irradiated recipient leads to the formation of proliferating cell colonies in all organs that can be easily counted in spleen as CFU-s. The proliferating capacity of donor marrow cells can be used to study the immunomodulating effects of the tested substance on mouse bone marrow cells in vivo [30]. We have tested the effect of two MDOC derivatives (Ca/Na salt and Na salt) injected intraperitoneally in three different doses (total doses were 6.25 mg/mouse, 1.25 mg/mouse and 0.25 mg/mouse) in five separate applications on bone marrow stem cells. It was apparent that both derivatives stimulate the proliferation of hematopoietic stem cells taken from the bone marrow of donor mice twenty four hours after the last injection. The best effect was obtained with Ca/Na salt injected in a total dose of 1.25 mg/mouse, where the amount of CFU-s colonies, measured in the spleen of recipient mice eight days after transplantation, represented 151% of control colonies (Table 9).

Table 9

Data represent percentage of control.

Average CFU-s of non-stimulated control = 17 + 0.8. Effect of MDOC Ca/Na salt on the immune cell subsets of mouse peripheral blood after in vivo application

Two-color direct immunofluorescence method and flow cytometry technology for leukocyte subset were used for identification and immune monitoring. An immunophenotyping analysis was undertaken with normal immune status mice. It was performed in the presence or absence of MDOC Ca/Na salt to determine changes in individual immune cell subsets. Mice were injected intraperitoneally 14 times with 7.15 mg of MDOC Ca/Na salt and the peripheral blood was collected for determination of the effect of this compound on the leukocyte subsets. A summary of the results is presented in Table 10. The results demonstrated that

MDOC Ca/Na salt induced a significant increase of peripheral monocytes (CDllb⁺ from 18.36% to 22.16%) and B lymphocytes (CD19⁺ from 43.01% to 52.94% of total counted cells). The activation of leukocytes (except monocytes) was determined using CD45⁺ CD19^" cell surface markers. It was found that the number of activated leukocytes was increased in mice exposed to MDOC Ca/Na salt from

60.24% to 69.74%. After long-term in vivo application of MDOC, no toxic effects on peripheral blood leukocytes were observed.

Table 10

Data represent percentage of total counted cells + standard deviation. * P < 0.01.

It has been shown that oxidized β-glucan stimulates T and B lymphocytes in the peripheral blood [24]. Form Table 10 above we can see that using MDOC Ca/Na salt, a significant increase of monocytes and B lymphocytes in vivo was observed.

Possible uses of the Invention

The salts of MDOC with immunomodulatory capabilities therefore have valuable therapeutic potential and may be used as an adjuvant therapy in a variety of disorders including malignancies, immunodeficiencies such as AIDS, viral infections such as epidemic influenza or prion infections such as Creutzfeld-Jacob disease, and inflammatory diseases. They may also be potentially useful to prevent recurrent infections including infections of the respiratory tract.

The invention is not limited to the embodiment hereinbefore described but may be varied in detail.

References

[1] Ooi, V.E. and Liu, F. (2000) Immunomodulation and anti-cancer activity of polysaccharide-protein complexes. Curr Med Chem 7, 715-729.

[2] Hofer, M. and Pospisil, M. (1997) Glucan as stimulator of hematopoiesis in normal and gamma-irradiated mice. A survey of the authors' results. International Journal of Immunopharmacology 19, 607-609. [3] Ross, G.D., Vetvicka, V., Yan, J., Xia, Y. and Vetvickova, J. (1999)

Therapeutic intervention with complement and beta-glucan in cancer. Immunopharmacology 42, 61-74.

[4] Morikawa, K., Takeda, R., Yamazaki, M. and Mizuno, D. (1985) Induction of tumoricidal activity of polymorphonuclear leukocytes by a linear beta-l,3-D- glucan and other immunomodulators in murine cells. Cancer Res 45, 1496- 1501

[5] Chen, J.T. and Hasumi, K. (1993) Activation of peritoneal macrophages in patients with gynecological malignancies by sizofiran and recombinant interferon-gamma. Biotherapy 6, 189-194.

[6] Di Renzo, L., Yefenof, E. and Klein, E. (1991) The function of human NK cells is enhanced by beta-glucan, a ligand of CR3 (CD lib/CD 18). Eur J Immunol 21, 1755-1758.

[7] Thornton, B.P., Vetvicka, V., Pitman, M., Goldman, R.C. and Ross, G.D. (1996) Analysis of the sugar specificity and molecular location of the beta- glucan-binding lectin site of complement receptor type 3 (CD lib/CD 18). J Immunol 156, 1235-1246.

[8] Vetvicka, V., Thornton, B.P. and Ross, G.D. (1996) Soluble beta-glucan polysaccharide binding to the lectin site of neutrophil or natural killer cell complement receptor type 3 (CD lib/CD 18) generates a primed state of the receptor capable of mediating cytotoxicity of iC3b-opsonized target cells. J

Clin Invest 98, 50-61.

[9] Vetvicka, V., Thornton, B.P., Wieman, TJ. and Ross, G.D. (1997) Targeting of natural killer cells to mammary carcinoma via naturally occurring tumor cell-bound iC3b and beta-glucan-primed CR3 (CD lib/CD 18). J Immunol

159, 599-605.

[10] Stilwell, R.L., Marks, M.G., Saferstein, L. and Wiseman, D.M. (1997) Oxidized Cellulose: Chemistry, Processing and Medical Applications. In: Handbook of biodegradable polymers. A. Dombs, J. Kostand D. Wiseman (Eds.), Harwood Academic Publishers, Amsterdam, p. 291.

[11] Haney, A.F. and Doty, E. (1992) Comparison of the peritoneal cells elicited by oxidized regenerated cellulose (Interceed) and expanded polytetrafluoroethylene (Gore-Tex Surgical Membrane) in a murine model. Am J Obstet Gynecol 166, 1137-1146.

[12] Reddy, S., Santanam, N., Reddy, P.P., Rock, J.A., Murphy, A.A. and Parthasarathy, S. (1997) Interaction of Interceed oxidized regenerated cellulose with macrophages: a potential mechanism by which Interceed may prevent adhesions. Am J Obstet Gynecol 177, 1315-1320.

[13] Ryan, C.K. and Sax, H.C. (1995) Evaluation of a carboxymethylcellulose sponge for prevention of postoperative adhesions. Am J Surg 169, 154-159.

[14] Sandhu, G.S., Elexpuru Camiruaga, J.A. and Buckley, S. (1996) Oxidized cellulose (Surgicel) granulomata mimicking tumour recurrence. Br J

Neurosurg 10, 617-619.

[15] Mosmann, T.R. and Coffman, R.L. (1989) THl and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu

Rev Immunol 7, 145-173. [16] Li, L., Sad, S., Kagi, D. and Mosmann, T.R. (1997) CD8Tcl and Tc2 cells secrete distinct cytokine patterns in vitro and in vivo but induce similar inflammatory reactions. J Immunol 158, 4152-4161.

[17] Liew, F.Y. (1982) Regulation of delayed type hypersensitivity to pathoges and alloantigens. Immuno Today 3, 18-23.

[18] Kundig, T.M., Althage, A., Hengartner, H. and Zinkernagel, R.M. (1992) Skin test to assess virus-specific cytotoxic T-cell activity. Proc Natl Acad Sci U S A 89, 7757-7761.

[19] Rihova, B. and Riha, I. (1981) Genetic regulation of antibody response to sheep red blood cells: linkage to H-2 complex. Am J Reprod Immunol 1,

168-173. [20] Rihova, B., Riha, I., Rossmann, P. and Vetvicka, V. (1985) Low IgG response of the mouse strain C57BL/10ScSn after immunization with protein antigens. Folia Microbiol Praha 30, 295-301.

[21] Rihova, B. and Vetvicka, V. (1991) Immunological Disorders in mice. Boca Raton: CRC Press, USA, , pp. 153 - 232. [22] Sϊrova, M., Riha, I. and Rihova, B. (1996) Limited T helper cell activity in C57BL/10 (BIO) mice with inherited low IgG responsiveness. Scand J Immunol 44, 453-460..

[23] Otterlei et al (1993) Infect Immun 61, 19171925

[24] Cross GG, Jennings HJ, Whitfield DM, Penney CL, Zacharie B, Gagnon L. Immunostimulant oxidized beta-glucan conjugates. Int Immunopharmacol 2001;1:539 - 50.

Claims

1. Use of a biocompatible salt of polyanhydroglucuronic acid (PAGA) or derivative thereof in the preparation of a medicament for modulating an immune response in an organism wherein the PAGA comprises a microdispersed oxidised cellulose or derivative thereof.

2. Use as claimed in claim 1 wherein the derivative is an inorganic salt.

3. Use as claimed in claim lor 2 wherein the inorganic salt contains any one or more of K, Na, Ca, Mg, Zn, Al or Co.

4. Use as claimed in claims 1 to 3 wherein the derivative is a simple acetate type or complex or mixed salt or intermolecular complex thereof.

5. Use as claimed in any of claims 1 to 4 wherein the derivative is a mixed salt containing more than one inorganic cation.

6. Use as claimed in any of claims 1 to 5 wherein the derivative is a Ca/Na salt.

7. Use as claimed in claim 1 wherein the derivative is an organic derivative selected from any one or more of urea, gelatine or arginine.

8. Use as claimed in claim 1 wherein the microdispersed oxidised cellulose or derivative thereof is formed by a process wherein a polyanhydroglucuronic acid-containing material obtained by oxidation with nitrogen oxides is subjected to partial or complete hydrolysis and neutralisation in an aqueous solution of at least one inorganic and/or organic salt and/or base in the presence of at least one suitable oxidising agent and the hydrolysate undergoing fractional coagulation to form a microdispersed oxidised cellulose.

9. Use as claimed in any preceding claim wherein the polyanhydroglucuronic acid and salts thereof comprises a biocompatible intermolecular polymer complex of

- polyanhydroglucuronic acid; and

- a cationic component comprising a linear or branched natural, semi- synthetic or synthetic oligomer or polymer,

10. Use as claimed in claim 9 wherein the cationic component is selected from any one or more of derivatives of acrylamide, methacrylamide and copolymers thereof, cationised natural polysaccharide, a synthetic or semi- synthetic polyamino acid, a synthetic anti-fibrinolytic, a natural or semi- synthetic peptide, or an aminoglucane or derivatives thereof.

11. Use as claimed in claim 9 or 10 wherein the polyanhydroglucuronic acid contains in their polymeric chain from 8 to 30 per cent by weight of carboxyl groups, at least 80 per cent by weight of these groups being of the uronic type, at most 5 per cent by weight of carbonyl groups, and at most 0.5 per cent by weight of bound nitrogen.

12. Use as claimed in any preceding claim in the preparation of a medicament for use as an immunosuppressor .

13. Use as claimed in any preceding claim in the preparation of a medicament for use as a immunostimulator.

14. Use as claimed in any preceding claim in the preparation of a medicament for use in stimulating the production of TNF-α .

15. Use as claimed in any preceding claim in the preparation of a medicament for use in stimulating bone marrow hematopoieses.

16. Use as claimed in any preceding claim in the preparation of a medicament for use in adjuvant therapy.

17. Use as claimed in any preceding claim in the preparation of a medicament for use in disorders including malignancies, immunodeficiencies such as AIDS, viral infections such as epidemic influenza or prion infections such as Creutzfeld-Jacob disease, and inflammatory diseases.

18. Use as claimed in any preceding claim in the preparation of a medicament for use in the proplylaxis of recurrent infections.

19. Use as claimed in any preceding claim in the preparation of a medicament for use in respiratory tract infections.

20. Use of a mixed salt containing more than one inorganic cation of polyanhydroglucuronic acid (PAGA) or derivative thereof in the preparation of a medicament for stimulating an immune response in an organism.

21. Use as claimed in any preceding claim comprising at least one other biocompatible biologically active substance.

22. Use as claimed in any preceding claim comprising at least one biologically acceptable adjuvant.

23. Use as claimed in any preceding claim comprising at least one pharmaceutically active adjuvant.

24. A biocompatible Ca/Na salt of polyanhydroglucuronic acid (PAGA) or derivative thereof.

25. A biocompatible salt as claimed in claim 24 wherein the polyanhydroglucuronic acid comprises a microdispersed oxidised cellulose or derivative thereof.

26. A composition comprising a biocompatible Ca/Na salt of polyanhydroglucuronic acid or derivative thereof.

27. A composition as claimed in claim 26 comprising at least one other biocompatible biologically active substance.

28. A composition as claimed in claim 26 or 27 comprising at least one biologically acceptable adjuvant.

29. A composition as claimed in claims 26 to 28 comprising and/or at least one pharmaceutically active adjuvant.

30. A composition as claimed in any of claims 26 to 29 suitable for peroral, intraperitoneal, intravenous, intraarterial, or inhalation administration.

31. A composition as claimed in claim 30 in the form of a tablet, pellet, capsule, granule, microsphere, solution, dispersion or aerosol.

32. A composition as claimed in any of claims 26 to 31 for the preparation of a medicament for use as an immunosuppressor.

33. A composition as claimed in any of claims 26 to 31 for the preparation of a medicament for use as an immunostimulator.

34. A composition as claimed in any of claims 26 to 31 for the preparation of a medicament for use in hematopoieses.

35. A composition as claimed in any of claims 26 to 31 for the preparation of a medicament for the prophylaxis and/or treatment of disorders including malignancies, immunodeficiencies such as AIDS, viral infections such as epidemic influenza or prion infections such as Creutzfeld-Jacob disease, and inflammatory diseases.

36. A composition as claimed in any of claims 26 to 35 wherein the PAGA comprises a microdispersed oxidised cellulose or derivative thereof.

37. A method for modulating an immune response in an organism comprising administering a biocompatible salt of polyanhydroglucuronic acid (PAGA) or derivative thereof wherein the PAGA comprises a microdispersed oxidised cellulose or derivative thereof.

38. A method as claimed in claim 37 wherein the derivative is an inorganic salt.

39. A method as claimed in claim 38 wherein the inorganic salt is selected from any one or more of K, Na, Ca, Mg, Zn, Al or Co.

40. A method as claimed in claim 38 or 39 wherein the derivative is a 'mixed' salt containing more than one inorganic cation.

41. A method as claimed in claim 38 wherein the derivative is a Ca/Na salt.

42. A method as claimed in claim 37 wherein the derivative is an organic derivative selected from any one or more of urea, gelatine or arginine.

43. A method as claimed in any of claims 37 to 42 wherein the PAGA comprises a microdispersed oxidised cellulose or salt or derivative thereof.

44. A method as claimed in any of claims 37 to 43 wherein the polyanhydroglucuronic acid and salts thereof comprises a biocompatible intermolecular polymer complex of

- polyanhydroglucuronic acid; and

a cationic component comprising a linear or branched natural, semi- synthetic or synthetic oligomer or polymer.

45. A method as claimed in claim 44 wherein the cationic component is selected from any one or more of derivatives of acrylamide, methacrylamide and copolymers thereof, cationised natural polysaccharide, a synthetic or semi- synthetic polyamino acid, a synthetic anti-fibrinolytic, a natural or semi- synthetic peptide, or an aminoglucane or derivatives thereof.

46. A method as claimed in claim 44 or 45 wherein the polyanhydroglucuronic acid contains in their polymeric chain from 8 to 30 per cent by weight of carboxyl groups, at least 80 per cent by weight of these groups being of the uronic type, at most 5 per cent by weight of carbonyl groups, and at most 0.5 per cent by weight of bound nitrogen.

47. A method for suppressing an immune response in an organism comprising administering a biocompatible salt of polyanhydroglucuronic acid (PAGA) or derivative thereof wherein the PAGA comprises a microdispersed oxidised cellulose or derivative thereof.

48. A method for stimulating an immune response in an organism comprising administering a biocompatible salt of polyanhydroglucuronic acid (PAGA) or derivative thereof wherein the PAGA comprises a microdispersed oxidised cellulose or derivative thereof.

49. A method for stimulating the production of TNF-α comprising administering a biocompatible salt of polyanhydroglucuronic acid (PAGA) or derivative thereof wherein the PAGA comprises a microdispersed oxidised cellulose or derivative thereof.

50. A method for stimulating bone marrow hematopoieses comprising administering a biocompatible salt of polyanhydroglucuronic acid (PAGA) or derivative thereof wherein the PAGA comprises a microdispersed oxidised cellulose or derivative thereof.

51. A method for the prophylaxis and/or treatment of disorders including malignancies, immunodeficiencies such as AIDS, viral infections such as epidemic influenza or prion infections such as Creutzfeld-Jacob disease, and inflammatory diseases, comprising administering a biocompatible salt of polyanhydroglucuronic acid (PAGA) or derivative thereof wherein the PAGA comprises a microdispersed oxidised cellulose or derivative thereof.

52. A method for the prophylaxis of recurrent infections comprising administering a biocompatible salt of polyanhydroglucuronic acid (PAGA) or derivative thereof wherein the PAGA comprises a microdispersed oxidised cellulose or derivative thereof.

53. A method for the prophylaxis of respiratory tract infections comprising administering a biocompatible salt of polyanhydroglucuronic acid (PAGA) or derivative thereof wherein the PAGA comprises a microdispersed oxidised cellulose or derivative thereof.

54. A method as claimed in any of claims 37 to 53 comprising at least one other biocompatible biologically active substance.

55. A method as claimed in any of claims 37 to 53 comprising at least one biologically acceptable adjuvant.

56. A method as claimed in any of claims 37 to 53 comprising at least one pharmaceutically active adjuvant.