The present invention relates generally to a method for interfering with various activities of blood cells in a mammal and thereby inhibiting, reducing or otherwise controlling an inflammatory response and/or reducing, preventing and/or inhibiting the adhesion of blood cells to endothelial cells. The present invention is also directed to pharmaceutical compositions useful in such a method comprising granule membrane protein (GMP) 140 and/or its derivatives and like proteins.

Granule membrane protein (GMP) 140 is a 140 Kd glycoprotein found in platelet alpha granules and in Wiebel-Palade bodies of endothelial cells (1,2,3) and is expressed on both platelet and endothelial cell membrane surfaces following cell activation (4,5,6). This property has been employed to image athersclerotic plaques in vivo using labelled monoclonal antibodies to GMP140 (Internation Patent Application No.

PCT/US90/01864). The structure of GMP140 has recently been defined (3) and appears to belong to the family of adhesive proteins that includes the human endothelial leukocyte adhesion molecule, ELAM-1, which is involved in the adhesion of blood cells to endothelium and the mouse lymphocyte homing receptor, the MEL-14 antigen (3,7,8). MEL-14, G P140 and ELA -1 contain lectin (L) and epidermal growth factor like (E) and complement binding like (C) domains. For this reason, ELAM has been termed a cell adhesion molecule (CAM) and GMP140 is, therefore, grouped with ELAM-1 in the class of LEC-CAM adhesion structures (9).

The capacity of some white blood cells, such as neutrophils, to be involved in inflammation and to damage tissues depends on a number of functions including the

production of toxic oxygen radicals such as superoxide anions (0 ₂ ^" ), degranulation of proteolytic enzymes, phagocytosis and intra- or extra-cellular killing of cells or pathogens. These processes are powerfully enhanced by cytokines such as TNF-α and GM-CSF (13,14).

In accordance with the present invention, it has been surprisingly discovered that soluble GMP140 affects the ability of some white blood cells such as neutrophils to assume an activated phenotype and that it reduces, prevents and/or inhibits adhesion of blood cells, such as activated neutrophils, to endothelium. Furthermore, it prevents the activation of these cells by exogenous stimuli. The present invention is useful, therefore, in controlling an' ^' inflammatory response and in reducing, preventing and/or inhibiting adhesion of blood cells to endothelium as is desired in the treatment of, but not necessarily limited to, vascular injury after reperfusion of arteries, uncontrolled inflammatory reactions, for example septicemia, and to aid recovery from frost bite or reattachment of severed limbs.

Accordingly, one aspect of the present invention contemplates a method for inhibiting, reducing or otherwise controlling an inflammatory response in a mammal comprising administering to said mammal an effective amount of GMP140 and/or GMP140-like protein and optionally in combination with an agonist for GMP140 and/or its like protein.

More particularly, the present invention is directed to a method for inhibiting, reducing or otherwise controlling an inflammatory response in a mammal comprising administering to said mammal an effective amount of GMP140 and/or GMP140-like protein and optionally in combination with an agonist for GMP140 and/or GMP140-like

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protein for a time and under conditions sufficient for the function of white blood cells involved in said inflammatory response to be inhibited and thereby controlling said inflammatory response.

Yet another aspect of the present invention relates to a method for reducing, preventing and/or inhibiting adhesion of blood cells to endothelial cells in a mammal which method comprises administering to said mammal a blood cell adhesion-reducing, -preventing and/or - inhibiting effective amount of GMP140 and/or GMP-like protein for a time and under conditions sufficient to reduce, prevent and/or inhibit adhesion of blood cells to endothelial cells.

Still yet another aspect of the present invention is directed to a pharmaceutical composition comprising GMP140 and/or GMP140-like protein and optionally agonistic and/or synergistic factors to GMP140 and/or its like protein and one or more pharmaceutically acceptable carriers and/or diluents.

A further aspect of the present invention relates to the use of GMP140 and/or GMP140-Iike protein in the manufacture of a medicament for the reduction, prevention and/or inhibition of adhesion of blood cells to endothelial cells and/or for the inhibiting, reducing or otherwise controlling of an inflammatory response in a mammal.

Preferably, the mammal is a human.

The present invention is described using neutrophils as the preferred white blood cells. This is done, however, with the understanding that the present invention extends to any white blood cell involved in an inflammatory

response and whose function may be affected by GMP140 and/or its like protein in such a way as to inhibit, reduce or otherwise control the inflammatory response. The present invention is also directed to the affect on more than one type of white blood cell.

The following non-limiting figures further describe the present invention:

Figure 1 is a graphical representation showing that increasing concentrations of GMP140 in solution inhibits adhesion of activated neutrophils to endothelial cells.

Figure 2 is a graphical representation showing the generation of 0 ₂ ^" anions of either unactivated neutrophils (NIL) or neutrophils activated by 100 U/ml of TNF-α (TNF). Cells are added to uncoated (plastic) microtitre plates or one coated with fibrinogen (FBN) or GMP140 (GMP). 0 ₂ ^* generation is measured after 30' and bars are arithmetic mean of 9 replicates of 3 experiments ± SEM. 0 ₂ ^" generation in GMP group differs significantly (p < 0.005) from FBN or plastic.

Figure 3 is a graphical representation showing the effect of increasing doses of GMP140 on 0 ₂ " generation and adhesion of TNF-α activated neutrophils. Increasing doses of GMP140 are used to coat plastic microtitre plates before addition of neutrophils. Adhesion to GMP140 and 0 ₂ " generation appear inversely related. Simi-Uar results are obtained with unactivated neutrophils.

The first aspect of the present invention is predicated, in part, on the surprising discovery that when neutrophils are added to plastic microtitre plates coated with GMP140, little 0 ₂ ^" generation is seen even when

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neutrophils are activated with TNF-α (Fig. 2). However, neutrophils added to plastic wells or fibrinogen coated microtitre wells produce significant levels of 0 ₂ " either when unactivated or activated with TNF-α (Fig. 2). The lack of 0 ₂ " production by neutrophils in GMP140 coated wells is observed even though neutrophils adhere to GMP140 vigorously (Fig. 3) - suggesting adhesion to GMP140 is qualitatively different from that to plastic or fibrinogen in not allowing 0 ₂ " production. Interestingly, neutrophils adherent to GMP140 also fail to spread and are maintained as a rounded phenotype.

Soluble GMP140 also prevents 0 ₂ ^~ production as shown in Table 4. Soluble GMP140 inhibits 0 ₂ " production of neutrophils added to plastic even when the cells are activated by TNF-α. This inhibition is prevented by affinity purified rabbit anti-GMP140 Fab' but not by control Fab' antibodies. It is also shown in Table 4 that GMP140 is effective even when added 15' after the experiment begins suggesting that it also inhibits the ongoing generation of 0 ₂ ~.

The results described herein indicate that GMP140 is a natural anti-inflammatory substance for neutrophils and that it moderates other aspects of neutrophil function such as degranulation and cytotoxicity. Thus, GMP140 or its like protein will be a useful anti-inflammatory agent and have use in acute or chronic inflammatory conditions. Furthermore, the depletion of GMP140 stores may be responsible for sequelae of acute stress, trauma or inflammatory reactions, such as the respiratory distress syndrome, and use of soluble forms of this protein may be beneficial.

Additionally, as the action of GMP140 extends to eosinophils and basophils it may also be useful to

prevent or moderate the activation of these cells which play a dominant role in the pathogenesis of allergic reactions and, in particular, asthma.

In further accordance with the present invention, the anti-inflammatory effect of GMP140 extends to monocyte function in general and in particular, to the role of monocyte 0 ₂ ^~ generating systems on the oxidation of lipids enhancing their uptake by the scavenger receptor and leading to accelerated atherogenesis.

Another aspect of the present invention is predicated in part on the surprising discovery that soluble GMP140 inhibits the adhesion of blood cells, and in particular neutrophils, to endothelial cells.

By "soluble GMP140" as used throughout the present specification is meant a GMP140 lacking a transmembrane domain and, therefore, secreted from platelets or endothelial cells. The term also extends to solubilized membrane associated GMP140 and to recombinant and synthetic forms of GMP140. The term "blood cells" herein is used in its broadest sense to mean any cell circulating in the blood and includes, but is not necessarily limited to, neutrophils and monocytes.

The present invention also extends to derivatives of' GMP140. By "derivatives" of GMP140 is meant any alteration to primary, secondary or tertiary structure of naturally or non-naturally occurring GMP140 or any alteration which affects the natural or non-natural activity or function of GMP140 or which enhances or delimits its anti-adhesive and/or its anti-inflammatory properties. Derivatives of GMP140 contemplated herein include, but are not limited to, those affecting the extent of glycosylation and/or association and/or

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conjugation of GMP140 or its derivatives with other molecules (eg. lipids or proteins or polypeptides), and to derivatives where the polypeptide portion of GMP140 has been altered. All such derivatives may also promote, stabilise and/or enhance the function of GMP140.

Accordingly, the present invention extends to naturally occurring soluble or solubilized GMP140 and to naturally and non-naturally occurring derivatives of GMP140 including those with or without glycosylation, association with other molecules and/or an altered amino acid sequence of the polypeptide portion of GMP140. By "non-natural" GMP140 is meant to include its recombinant and/or synthetic forms. All such derivatives and non- natural forms of GMP140 are encompassed herein by the term "GMP140-like proteins". In a preferred embodiment, the GMP140-like protein is a fragment of GMP140 having anti-inflammatory and/or anti-adhesion activity.

Another aspect of the present invention relates to a pharmaceutical composition comprising GMP140 and/or its like protein and one or more pharmaceutically acceptable carriers and/or diluents. The pharmaceutical compositions contemplated herein may comprise solely GMP140 or GMP140-like protein or may comprise combinations of GMP140 and its derivatives or combinations of different derivatives, the combination desired depending on the relevant anti-adhesive or anti- inflammatory activities and/or other properties of GMP140 and its derivatives. The pharmaceutical compositions may also contain agonistic and/or synergistic factors of GMP140 or its derivatives. One source of such agonists and/or synergists may be peptide, polypeptide or glycopeptide derivatives of GMP140 or peptide, polypeptide or glycopeptide derivatives of complementary molecules to GMP140 and/or its proteins. Such complementary molecules may be, but are not

necessarily limited to, polypeptides or glycopeptides which can associate with GMP140 or its derivatives.

The active ingredients of a pharmaceutical composition comprising GMP140 or the like are contemplated to exhibit excellent therapeutic activity in reducing, preventing and/or inhibiting adhesion of blood cells to endothelial cells or in inhibiting, reducing or otherwise controlling an inflammatory response when administered in amounts, although depending on the particular case, of about 0.001 ^• μg to about 200 mg per kilogram of body weight of the mammal per day. Dosage regima, however, may be adjusted to provide the optimum therapeutic response and more or less of the active component may need to be administered. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

The active compound may be administered in a convenient manner such as by the intravenous (where water soluble), intramuscular, subcutaneous intranasal, intradermal, pulmonary (eg via lungs) or suppository routes or implanting (eg using slow release molecules). Depending on the route of administration, the active ingredients which comprise GMP140 or the like may be required to be coated in a material to protect said ingredients from the action of enzymes, acids and other natural conditions which may inactivate said ingredients.

The pharmaceutical forms suitable for in ectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the

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contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilisation. Generally, dispersions are prepared by incorporating the various sterilised active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.

As used herein "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for-pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

The present invention is directed to the use of the subject pharmaceutical compositions in mammals, and in particular, humans.

A convenient reference source for pharmaceutical compositions can be found in Remington*s Pharmaceutical Sciences 16th ed., 1980, Mack Publishing Company, edited by Osol et al.

The present invention also extends to the use of GMP140 and/or its like proteins in the manufacture of a medicament for the reductions, prevention and/or inhibition of adhesion of blood cells to endothelial cells and/or the inhibiting or otherwise controlling of an inflammatory response in a mammal.

The present, invention is also described by reference to the following non-limiting examples.

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EXAMPLE 1 Materials and Methods

Endothelial cells are prepared by collagenase treatment of human umbilical vein. The cells are grown on gelatin coated plastic and used within 6 days following establishment of culture. Cells are harvested by trypsin-EDTA treatment replated at 2 x 10 ⁴ cells/well in microtitre wells and grown to confluence overnight. Before use in the assay, the monolayers are washed in

RPMI 1640 2.5% (v/v) fetal calf serum (FCS). Neutrophils are prepared from freshly drawn blood by destran sedimentation and ficol-hypaque separation. The erythrocytes are removed by hypotonic shock.

The GMP140 diluted in RPMI 1640 - 2.5% (v/v) FCS are added to the monolayers of endothelial cells either with or without tumour necrosis factor (TNF). Neutrophils are then added and incubated for 30 minutes at 37°C, 5% C0 ₂ . The supernatant is removed, the cells stained with Rose Bengal and the adherence ± SEM (standard error of the mean) is given either as optical density (OD) or a percent adherence for triplicate determinations performed for each group.

Adhesion assays are as defined in references 10 and 11. Pure GMP140 (12), as verified by N-terminal sequencing, was passed through a column to remove detergent and used immediately.

EXAMPLE 2

The results in Table 1 show that pure GMP140 (3) when coated onto plastic surfaces, mediates adhesion of neutrophils. Similar data apply to monocytes and eosinophils (but not lymphocytes).

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TABLE 1

" Arithmetic mean ± SEM (standard error of the mean) Triplicate determinations. ^b Differs from 0 control by p<0.01.

EXAMPLE 3

The results in Figure 1 show that soluble GMP140 inhibits the adhesion of neutrophils to endothelial cells. In Figure 1, GMP140, RNF (lOU/ml) and neutrophils are incubated together with the endothelial monolayers for 30 minutes at 37°C before the level of adherence determined as described.

The inability to inhibit adhesion to activated endothelium (Fig. lb) shows the effect of GMP140 is selective for one type of adhesion (i.e. that mediated by activated neutrophils, and perhaps other cells) activation is prominent. Such examples will include septicemia, thrombosis, or therapeutic administration of agents such as TNF or GM-CSF that will activate blood cells. (The solid symbols represent adherence in the absence of any stimulation).

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The anti-adhesive property is also observed both when GMP140 is added to the adhesion reaction as well as when neutrophils are pre-coated with this molecule as shown in Table 2. In Table 2, the neutrophils are incubated for 15 minutes at 37°C with GMP140, washed once and then added to the endothelial monolayers together with lOU/ml TNF, incubated for 20 minutes at 37°C and adherence determined.

TABLE 2 Effect of GMP140 pretreatment of PMN on adherence levels to endothelium

% Adherence

GMP140 GMP140 in Assay ¹ in Pretreatment ²

PMN only 7.8 (2.6) 10.4 (2.2) PMN + lOU/ml TNF-α 80.4 (3.7) 61.5 (2.9) PMN + lOU/ml TNF-α + 8ug/ml GMP140 50.4 (4.4) ^: 45.2 (4.O

¹ PMN together with TNF-α and GMP140 were added to monolayers of HUVES, incubated for 30 minutes at 37°C and adherence levels determined.

PMN were pretreated with GMP140 or control medium for 15 minutes at 37°C, immediately placed on ice, diluted in ice cold medium and centrifuged for 15 seconds at 12,000g. The cells were resuspended and added to HUVES together with TNF-α.

p < 0.05 compared to group receiving TNF-α but no GMP140.

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EXAMPLE 4

The results in Table 3 show the effect of antibodies to GMP140 on inhibition of neutrophil attachment.

TABLE 3 Effect of rabbit antibodies to GMP140 on activity of GMP140

GMP140 is preincubated with rabbit anti-GMP140 for 15 minutes 37 ^C C. The mixture is then added to endothelial monolayers together with neutrophils and TNF. Adherence levels are determined 20 minutes later.

EXAMPLE 5 Measurement of 0 ₂ ^~ generation

Superoxide production is measured by the reduction of cytochrome c as described (15) by using an extinction coefficient of 21.1 x 10 ³ M ^"1 cm ^_1 (16). The assay is adapted for use in microtitre wells and nmoles of reduction is calculated from standard curves. Superoxide dismutase (Sigma) completely abolishes the reduction of cytochrome c. The results are expressed as means of duplicate determinations in Tables 4 and 5 and in Figure 2 are the mean ± SEM of 9 determinations from 3 experiments, and Figure 3 is one representative experiment of three similar ones performed and points are mean of triplicates ± SEM.

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TABLE

Effect of soluble GMP140 on 0 ₂ ^" generation by neutrophils

Significantly p < 0.05 different from no treatment control Time of addition of GMP in relation to the beginning of the assay = O min. Affinity purifiedRabbit αGMP Fab' or Fab' from normal rabbit

IgG at 50 ug/ml.

S B T

EXAMPLE 6 Effect of GMP140 on activation of blood cells by exogenous Stimuli

GMP140 prevents or at least reduces activation of blood cells such as neutrophils by exogenous stimuli. The results are shown in Table 5.

TABLE 5

0 ₂ ^" generation (nMoles/10 ⁶ cells/30 min)

Stimulus Plastic Fibrinogen GMP140

Nil 7.5 ± 0.3 5.5 ± 0.3 2 ± 0.3

FMLP" (10 ^_7 M) 28 ± 0.5 26 ± 0.3 7.5 ± 0.3 TNF (lOU/ml) 21 ± 0.3 32.5 ± 0.0 9 ± 1.0 TNF + FMLP 36 ± 0.5 46 ± 0.5 21 ± 0.3

^• N-formyl-methionyl-leucyl-phenylalanine

This shows that FMLP (N-formyl-methionyl-leucyl-phenylalanine), a bacterial derived neutrophil activator, has a reduced 0 ₂ ^* stimulatory effect when neutrophils are in the presence of GMP140. The effect of FMLP is evident even in the presence of TNF-alpha, an agent that also activates neutrophils.

Those skilled in the art will appreciate that the invention described herein is suseptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

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REFERENCES

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