MXPA97000037A - Modulators of accession of pneumococs to cellular objectives that involve the receptordel platelet activation factor, and uses of the mis - Google Patents

Abstract

The present invention relates to the use of an effective platelet activating factor receptor antagonist for inhibiting the binding of S. pneumoniae to host cells, in the preparation of compositions for preventing or treating an infection with Streptococcus pneumoni.

Description

f ADJUSTMENT OF PNEUMOCOCES TO CELLULAR 0B3ETIVQS INVOLVING THE RECEIVER OF THE PLATELET ACTIVATION FACTOR, AND USES OF THEM The research that led to the present invention was supported in part by AI-27913 of the National Institutes of Health. Therefore, the Government has certain rights in the invention.

FIELD OF THE INVENTION The present invention relates to compositions and methods for preventing infection by pneumococci. In particular, the invention relates to the identification of the main reception for Streptococcus pneumoniae on activated human cells, diagnostic and therapeutic compositions and methods based thereon.

BACKGROUND OF THE INVENTION Streptococcus pneumoniae is a highly positive pathogen that is the main cause of lobar pneumonia, sepcia and meningitis (Bur an et al., 1995, Rev. Infect. Dis 7: 133). This pathogenic bacterium is the most frequent cause of bacterial pneumonia in children of developed countries and represents up to 70% of cases in adults (Pennington, 1986, Am. Fa.

* Physician 33: 153; Dohnston, 1991, Revs. Infect. Dis. 13 (Suppl 6); S509; iusher, 1992, Clin. Infect. Dis. 14: 801). Pneumococci usually enter the host through the nasopharynx where they attach to the epithelial cells and in some cases persist for several months (Anderson et al., 1981, Infect, Irnmun, 32: 311). In experimental models, progression to pneumonia results from the desi ination of pneumococci by aerosol form and transport from the nasopharynx to the ^^^ lower respiratory tract. 10 The early neurnococcal lesion is characterized by fluid-filled alveoli that contain pneumococci, which are often seen to line the alveolar walls, a distribution that suggests a retention of specific interaction rates in the alveolar space (Wosd, 1941, 3.

Esp. Med. 73: 222). Pneumococci readily gain access to the bloodstream from the alveolar space, suggesting an aggressive ability to cross the vascular endothelial cells of the alveolar capillaries (Rake, 1936. 3. Esp. Med. 63: 191). 20 Bacterial adherence to eukaryotic cells commonly involves specific (adhesive) bactrian proteins that recognize glycoconjugates of host cells. The adhesion of pneumococci to human oral emptily is individual in the presence of the disaccharide N-acetyl-glucosamino-β-3-galactose suggesting that the carbohydrate can serve as a receptor, perhaps relevant to the nasopharyngeal passage (Anderson et al., 1983, 3. Esp. Med. 158: 559). It remains unknown precisely what type of cells support the neurnococic adhesion in the alveolar space. It has been reported that pneumococci that bind to purified glycoconjugates containing GalNAcßl-4Gal, terminal or internal, a structure in lung secretions and lung tissue (Dri an et al., 1988. Proc. Ntl. Acad. Sci. USA 85 : 6157). The structure differs signi icantly in the stereochemistry of that proposed for the nasopharyngeal receptor. The identity of neurnococcal adhesions capable of mediating adhesion to the nasopharynx, lung and vascular otelio is unknown. Consistent with the ability to easily produce experimental bacteremia, neu ococular adhesion to vascular endothelial cells has shown that it is dose-dependent, rapid, and independent of the capsular type (Teelen and Otos, 1993, Infect. I unol. 1538). The components of the cell wall and the protein components contribute almost equally to the association, but specific pneumococcal ligands remain to be specified (Tuomanen et al., 1985, 3. Infect. Dis. 151: 859). Invasive pneumococcal infection is characterized by particularly intense inflammation induced mainly by components of the cell wall of pneumococci (Tuomanen et al., 1985, 3. Infect. Dis. 151: 535; Tuomanen et al., 1985, cited above; Tuomanen et al., 1985, Am. Rev. Respir. Dis. 135: 869). The teicoic acid and lipoteichoic acid of pneumococci has the highest inflammatory capacity of all the components of the cell wall (Cabellos et al., 1992, 3. Clin.Research 90: 612) and it has been shown that its bioactivity depends critically on the presence of an uncommon component: phosphorylcholine (Tomasz and Saukkonen, 1989, Paediatr. Infect. Dis. 3. 8: 902). Phosphoricoline is also a critical determinant of the biological activity of the inflammatory measurer, platelet activation factor (FAP) (Wissner et al., 1986, 3. Med. Che. 29: 329), suggesting a unique pro-inflammatory relationship between pneumococci and FAP in the pathogenesis of meningitis and pneumonia (Hair and others, cited above). As indicated above, pneumococci adhere to glycoconjugates containing N-acetyl-D-galactosamino (31-4 galactose (GalNAcßl-4Gal) or GalNAcßl-3Gal either in purified form or presented on the surface of vascular endothelial and epithelial cells of Pulmonary type II (Krivan et al., 1988, Proc. Nati. Acad. Sci. USA 85: 6157) However, the local generation of inflammatory cytokines, such as tumor necrosis factor (TNF) and interleukin-1 ( IL-1), during the course of infection can drastically alter the presentation of potential receptors on cells (Rozdzinski et al., 1993, 3. Esp. Med. 178: 917, van de Kar et al., 1992, Blood 11: 2725) For example, the activation of vascular endothelial cells by TNF and by IL-1 stimulates the synthesis of FAP (Bussolino et al., 1988, Biochi, Biophys, Acta 927: 43, Chao and Olson, 1993, Biochem. 292: 617; Zirnrnerrnan et al., 1992, Im unol. Today 13: 93-100) and increases the expression from a variety of cell surface receptors including E-selectin (Rozdzinski et al., cited above) and globotriosilcerarnide (van de Kar et al., cited above). Thus, there is a need in the art to identify ligands for bacterial adhesion. There is a particular need in the art to identify said ligands on cells activated by inflammatory mediators. There is also a need to characterize the role of pneumococci in the inflammatory processes associated with pneumococcal infections. The citations of references in this should not considered as an admission that they are prior art of the present invention.

«* BRIEF DESCRIPTION OF THE INVENTION The present invention relates to the discovery that the platelet activating factor receptor (FAP) is a ligand for neurnococcal adhesion to activated lung epithelial cells (ie, hosts) and activated venous endothelial cells. It has also been found that the The binding of pneumococci to the PAF receptor activates the receptor, which in turn can increase inflammation. Accordingly, the present invention is directed to a method of preventing or treating an infection by Streptococcus pneurium which comprises administering to a subject believed to be in need of such treatment an amount of an antagonist. of platelet activation factor receptor effective to inhibit the binding of S. pneumoniae to host cells. And to inhibit the activation of the FAP receptor subsequent to said binding. Most preferably the antagonist FAP receptor is & amp; amp; effective to elute (ie, remove or detach) pneumococci adherents of host cells. In a specific embodiment, the platelet activating factor receptor antagonist is selected from the group consisting of L659,989 L652,731 WER 2086, kadsurenone and FR72112; Additional antagonists are described in Hwang and Lam, 1991, Lipids 26: 1148-1553, Hwang et al., 1989, Mol. Pharmacol. 35: 48-58, which are specifically incorporated herein by reference in their entirety. The inhibition of pneumococcal binding to FAP receptor has important implications for preventing the migration of pneumococci through epithelial tissues and endothelial. The migration of bacteria from the nasopharynx and lung into the blood can lead to a systemic bacterial infection, resulting in bacteremia, sepsis and meningitis. Activation through the PAF receptor further complicates these pathological pathways, in particular, generating or exacerbating inflammation leading to increased vascular permeability (see, eg, Tufnanen et al., 1985, 3. Infect. Dis. 151: 535-540). The invention further relates to the recognition that adhesion to activated cells also involves a carbohydrate ligand found on said cells activated. Therefore, a method for inhibiting pneumococcal adhesion may comprise administering an amount of carbohydrate containing an effective N-acetyl-D-glucose binding inhibitor to inhibit the binding of S. pneumoniae to Efa host cells. In a specific modality, the carbohydrate is N-acetyl-D-glucosamine. Preferably, said method is used in conjunction with the administration of a receptor antagonist of the FAP. It has been found that resting lung epithelial and endothelial cells have two kinds of receptors containing different carbohydrate main elements. Therefore, the invention further provides for administering an amount of a second carbohydrate selected from the group consisting of a carbohydrate containing a disaccharide-binding motif N-acetyl-D-20 galactose inaßl-4Gal, a disaccharide containing a disaccharide N-acetyl-D-galactosamine binding (31-3Gal, and mixture thereof, effective to inhibit the binding of S. pneumoniae to host cells.) In a specific embodiment, the second carbohydrate is selected from the group consisting of Forssman glycolipids, globoside ,. asialo-GMl and asialo-6M2. The method for inhibiting the binding of pneumococci to host cells may further comprise administering an amount of a carbohydrate selected from the group consisting of maasose, N-acetyl-glactose, rnanosa-D-mannose, and methyl-aD-anopyranosi or effective for inhibit the binding of S. pneumoniae to host cells. In a preferred aspect, wherein the carbohydrates are used in the methods of the invention, the carbohydrate or carbohydrates are multivalent. Mr In one modality, administration comprises the atomization and inhalation. For example, atomization can be done by nebulization. Alternatively, the invention provides parenteral administration, e.g., intravenous injection. In a further embodiment, a FAP antagonist can be administered orally. The invention further recognizes that expression of the PAF receptor increases in host cells activated with inflammatory mediators, such as the cytokine tumor necrosis factor (TNF) and interlucin-1 (IL-1). Pneumococci directly stimulate inflammatory responses, in particular for the activity of the wall components of the pneumococcus teichoic acid and lipoteichoic acid. Therefore, the invention further contemplates administering an effective inhibitor of inflammation to inhibit the expression of platelet activating factor receptor. In a specific aspect, The inflammation inhibitor is selected from the group consisting of a neutralizing antibody to JF tumor necrosis factor, a soluble tumor necrosis factor receptor in the neutralizer, a neutrophilizing antibody to interleukin-1, and an interleukin-1 receptor. soluble in the neutralizer. In addition to the therapeutic methods, the invention provides a pharmaceutical composition comprising an effective amount of a platelet activating factor receptor antagonist to inhibit the binding of S. pneumoniae to host cells, an amount of a carbohydrate containing an effective N-acetyl-D-glucosamine binding promoter for inhibiting the binding of S. pneumoniae to host cells, and a pharmaceutically acceptable carrier. The platelet activating factor receptor antagonist can be selected from the group consisting of L659,989, L652,731, kadsuresona, FR72112 and UEB 2086; the carbohydrate can be N-acetyl-D-glucosamine. In a further embodiment, the pharmaceutical composition comprises an amount of a second carbohydrate selected from the group consisting of a carbohydrate that contains a disaccharide-binding motif N-acety-D-galactosamine β-3Gal and a carbohydrate containing disaccharide-binding motif N-acetyl-D-galactosamine β-4Gal to inhibit the binding of S. pneumoniae to host cells.

The second carbohydrate can be selected from the group consisting of Forssman glycolipid, globoside, asialo-GMl and asialo-GM2. flf In a further embodiment, the pharmaceutical composition comprises an amount of a carbohydrate selected from the group consisting of mannose, N-acetyl-galactose, mannose-D-mannose and effective methyl-a-D-mannopyranose. to inhibit the binding of S. pneumoniae to host cells. Preferably, in the pharmaceutical compositions containing a carbohydrate or carbohydrates, the carbohydrate or carbohydrates are multivalent. mk In a particular aspect, the invention specifically provides a pharmaceutical composition in which the pharmaceutical composition is an aerosol formulation, said formulation containing a dispersant. For example, the dispersant can be ur / surfactant. In one embodiment, the pharmaceutical composition is an aerosol formulation of dry powder, in which the FAP receptor antagonist, and, if present, carbohydrate or carbohydrates are present in a finely divided powder. The dry powder aerosol formulation may further comprise a body forming agent. In another embodiment, the pharmaceutical composition is a The liquid aerosol formulation further comprising a pharmaceutically acceptable diluent. The diluent can be selected from the group consisting of sterile water, saline, saline regulated at its pH and dextrose solution. In a specific aspect, the invention provides a pharmaceutical composition in an aerosol formulation that comprises a FAP receptor antagonist. Said formulation may be an aerosol formulation of dry powder, or a liquid aerosol formulation. Said aerosol formulation may further comprise a carbohydrate containing a N-acetyl-D-glucosarnine binding motivator. In additional embodiments, the composition may comprise a second carbohydrate selected from the group consisting of a carbohydrate containing a disaccharide-binding motif N-acetyl-D-galactosamine β-3Gal and a carbohydrate containing a disaccharidic binding motif N-acetyl-D-galactosarnine ßl-4Gal effective to inhibit the binding of S. pneumoniae to host cells. A principal object of the invention is to provide a method and associated compositions for inhibiting adherence of pneumococci to activated host cells, in particular lung epithelial cells and venous endothelial cells. Another object of the invention is to inhibit the activation of pneumococci of the FAP receptor. These and other objects of the present invention will be understood more fully by reference to the drawings and the following detailed description.

BRIEF DESCRIPTION OF THE DIBU30S FIGURE 1. Adherence of pneumococci to cultured human type II neurnocytes stimulated with cytokine (A) and vascular endothelial cells (B). The monolayers in Terasaki boxes with 60 wells were stimulated with IL-la (pulmber cells of type II, 5 ng / ml, 4 hr at 37 ° C, Rozdsinski and others 1993, 3. Exp. Med, 178: 917) and TNFa (vascular endothelial cells, 10 ng / ml, 3 h at 37 ° C, Elias and others 1990, Am. 3. Respir Cell, Mol. Biol. 3:13). Pneumococci R6 and AII were cultured for 18 hours on trypticase soy agar containing 3% sheep blood, labeled with fluorescin isothiocyanate and adjusted to 105-107 cfu / ml (Geelen et al. 1993, Infect.Inmun. 61: 1538), and co-incubated with the monolayers. The control adherence in the absence of cytokine is indicated by a dashed line (). Bacteria adhering to monolayers stimulated with cytokine () and at rest (0) were determined as the number of bacteria fixed by 100 5 pulmonary or endothelial cells. The results are means ± SD for duplicate wells in at least six independent experiments. FIGURE 2. Effect of FAP receptor antagonists on pneumococcal adhesion to cultured human type II lung cells stimulated by IL-lc (A) and vascular endothelial cells stimulated by TNFa (B). Monolayers stimulated by cytokine were incubated (10 min, 37 ° C) either with albumin pH regulator (-) or concentrations in increments of FAP receptor antagonists L659,989 5 () V WEB 2086 I and R6 pneumococci (10? Cfu / ml) were allowed to adhere to the monolayers for 30 minutes at 37 ° C. Control adherence in the absence of cytokine stimulation is indicated by a horizontal dotted line (). Adherent bacteria were determined as the number of bacteria fixed per 100 lung or endothelial cells. The results are means ± SD for duplicate wells in at least six independent experiments. *? > 0.05 compared with cytokine stimulation in the absence of antagonists. FIGURE 3. Effect on the adherence of pneumococci to w FAP receptors for receptor glycosylation, antagonists WEB 2086 and L659.989 (lμ) and removal of phosphorylcholine from the pneumococcal cell wall (Tuomanen et al. 1985, 3. Infect. Dis. 151: 859). FIGURE 4. Effects of "simple" sugars on the adhesion of R6 to cultured human type II pneumocytes stimulated by cytokine and at rest (A) and endothelial cells (B). R6 (10 cmu / ml) was exposed to several simple sugars (15 minutes, room temperature) and then co-incubated with monolayers at rest or stimulated by cytokine i for 30 minutes. The adherence of pneumococci to a designated sugar was defined as bacterial adherence to 100 host cells in the absence of sugar-bacterial adherence to 100 host cells in the presence of sugar. The results are means ± SD for duplicate wells in at least six independent experiments. Glc = D-glucose. Gal = D-galactose, Fue = L-25 fuctose, GalNAc = N-acetyl-D-galactosamine A GlcNAc = N-acetyl-D-gl? Cosarnine, Man = D-mannose, NANA = sialic acid, Lac = lactose and 2-ADGG = 2- acetamido-2-deoxy-3-0-BD-galactopi ranosil-D-galactopirani. FIGURE 5. Glyconjugated pneumococcal receptors on monolayers stimulated by cytokine. The number of pneumococci adherent to the population of GalNAcßl-4Gal was defined by Asialo-GM2 I and those adherent to the population of GalNAcßl-3Gal by globoid i. To define whether carbohydrate speci fi cities were contained in one or more receptors, the capacity and mixture of two components of GlcNAc (o) plus either globoside and organelle -GM2 I were evaluated to alter the adherence of pneumococci to a greater degree than it was sugar alone. The final concentrations were composed of simple sugar or equally of two sugars tested. Control adhesion to monolayers stimulated by FNT-oc indicated by the discontinuous line (). * <0.05 in comparison with soybean-GM "or GlcNAc alone The adhesion of pneumococci to each designated sugar was determined from the bacterial adherence to 100 host cells in the absence of sugar-bacterial adherence to 100 host cells in the presence of sugar. results shown are means ± SD for duplicate wells in at least six independent experiments FIGURE 6. Pneumococci activate the FAP receptor COS cells in a monolayer were treated with control platelet activation factor (108 bacteria / rnl) , rnococci (108 bacteria / rnl), pneumococci cultured with ethanolamine (108 bacteria / l) FAP + L659,989 and pneumococci + L659,989 Activation was measured by detecting the 3H incorporation level in inositol triphosphate, which reports as the percentage above the basal level.

DETAILED DESCRIPTION OF THE INVENTION It is known that pneumococci adhere to and activate pulmonary epithelial cells and vascular endothelial cells during the course of pneumonia and bacterirnia. Cytokine activation of human vein endothelial cells and lung type II cells results in pneumococcal adhesion greatly increased. The present invention is based in part on the unexpected discovery that the binding of pneumococci to activated pulmonary epithelium and activated vascular endothelium was eliminated in the presence of platelet activating factor receptor (FAP) antagonists. In one example, under transfection with FAP receptor human kidney epithelial cells acquired the ability to withstand pneumococcal adherence consists of specific receptor activity. It has also been observed that the presence of FAP antagonists. FlcNAc, FAP receptor or specific site commutation to remove the individual glycosylation site on the transfected FAP receptor resulted in a drastic reduction of pneumococcal binding. Adherence to activated type II pneumocytes, vascular endothelial cells and transfected cells depended on phosphorylcholine on the bacterial surface indicating that this unique component of teichoic acid, like the phosphorylcholine portion in FAP, directs pneumococci to the receptor of the FAP. Accordingly, the present invention relates to compositions and methods for treating or preventing infection by S. pneumoniae. As briefly explained above, the present invention is based on the discovery that the pneumococcus recognizes, binds and activates the platelet activation factor receptor, which is expressed by pulmonary epithelial cells and venous endothelial cells activated with mediators. inflammatory, such as tumor necrosis factor (TNF) and interleukin-1 (IL-1). The invention is also based on the discovery that the activation of said cells is accompanied by the expression of a portion of carbohydrate GlcNAc, which also mediates the adherence of pneumococcus, a property of GlcNac not previously recognized before the present invention. The invention also relates to the recognition that pneumococci are associated with at least two other main elements of different carbohydrates, one of which has recently been recognized. This last aspect of the invention is more fully developed in copending application serial number 08 / 254,577, case of Attorney No. 600-1-084, filed on June 6, 1994, by Tuomanen and Cundell, entitled "MODULATORS OF ~ 'PNEUMOCOCCAL ADHERENCE TO PULMONARY AND VASCULAR CELLS AND DIAGNOSTIC AND THERAPEUTIC APPLICATIONS. ", Hereinafter referred to as" Tuomanen and Cundell ", which is specifically incorporated herein by reference in its entirety. In specific embodiments, the invention helps to inhibit the adhesion of pneumococci, or to elute adherent pneumococci, of pulmonary epithelial cells and of venous endothelial cells. á > The inventors of the present invention have discovered that the platelet activating factor (FAP) receptor interacts with a phosphorylcholine entity in pneumococci; the teichoic and lipoteichoic acid portions of the bacterial cell wall. The inventors have also discovered a role for carbohydrates containing GalNacßl-3Gal and the group of 5 carbohydrates in the FAP receptor in the adhesion of pneumococci to host cells, such as lung epithelial cells and vascular endothelial cells. Moreover, the inventors have discovered that the internalization of the bacteria is associated with binding to the PAF receptor. This internalization seems to give the bacterium a migration path in or through the endothelium and epithelium, resulting in a systemic infection, which can lead to bacteremia, sepsis and meningitis. In this way, the present invention provides a powerful tool, effective against antibiotic resistant bacteria, as well as against antibiotic-sensitive bacteria, to prevent Wf infection. pneumococci and pathogenesis. To better understand the invention, the definitions for certain terms are subsequently provided; aspects of the invention - FAP receptor antagonists, inhibitors of FAP receptor expression are also presented; carbohydrates; therapeutic compositions and methods; and examples. This invention relates in part to techniques : k conventional molecular biology, microbiology and DNA recombinant within the technique. Such techniques are fully explained in the literature. See, for example, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (in the present "Sambrook and others, 1989"); DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed., 1985); Qligonucleotide Synthesis (M.3.Gait ed., 1984); Nucleic Acid Hibridization CB.D. Hames S S.3. Higgins eds (1985) 3; Transcription And Translation CB.D. Hames & S.3. Higgins eds (1984) 3; Animal Cell Culture CR.I. Freshney, ed (1986) 3; Immobilized Cells And Enzymes CIRL Press, (1986) 3; B. Perbal, A Practical Guide to Molecular Cloning (1984). A composition consisting of "A" (where "A" is a single molecule, such as an antagonist, protein, carbohydrate, etc., of the FAP receptor), substantially lacks "B" (in where "B" consists of one or more contaminating molecules) when at least about 75% by weight of the? components of the composition (depending on the category of the species to which "A" and "B" belong) in the composition is "A". Preferably, "A" consists of at least about 90% by weight of species A + B in the composition, most preferably at least about 99% by weight. It is also preferred that a composition, which is substantially free of contamination, contains only species of only molecular weight that 'j? have the activity or characteristic of the species of interest. The phrase "pharmaceutically acceptable" refers to entities and molecular compositions that are physiologically tolerable and that typically do not produce an allergic or similar adverse reaction, such as gastric discomfort, dizziness and the like, when it is administered to the human being. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by the regulatory agency of the state or federal government or listed in the pharmacopoeia of E.U.A. or another pharmacopoeia generally recognized for use in animals, and very particularly in humans. The term "vehicle" refers to a diluent, adjuvant, excipient or vehicle together with which the compound is administered. Such pharmaceutical vehicles can be sterile liquids, such as water and oils, including those of petroleum, animal origin, Vegetable or synthetic, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The solutions wß. Water salt or water solution and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. As used herein, the term "host cell" refers to a cell capable of colonization by pneumococci, for example, lung epithelial cells and venous endothelial cells. As used herein, the term "pulmonary administration" refers to the administration of a formulation of the invention in the lungs by inhalation. As used herein, the term "inhalation" refers to the entry of air into the alveoli. In specific examples, the entry may occur by the self-administration of a formulation of the invention by inhaling, or by administration through a respirator, v. G., To a patient on a respirator. The term "inhalation" used with respect to the formulation of the invention is synonymous with "pulmonary administration". As used herein, the term "aerosol" refers to suspension in the air. In particular, aerosol refers to the particularization of a formulation of the invention and its suspension in the air. In accordance with the present invention, an aerosol formulation is a formulation consisting of a FAP receptor antagonist., and also possibly a carbohydrate, which is W aerosolized, that is, atomized and suspended in the air, for inhalation or pulmonary administration. As used herein, the term "parenteral" refers to the introduction of an antagonist of platelet activation factor and possibly carbohydrate, which inhibits the binding or adhesion of pneumococci in mammalian cells within the body, and in particular, oral, intravenous (iv), intraperitoneal (i) intraperitoneal routes jllf (ip), intramuscular (i.) intraventricular and subcutaneous routes (s.c.) Preferably, the route is intravenous or oral. As used herein, the term "systemic" refers to a disease or disorder, or site of original injury distant from the lung or that encompasses the entire body of the organism. The term "local" is therefore used in the present with respect to the lung. In this way, a systemic infection is one in which S. pneumonia is found in the blood and can lead to bacteremia, sepsis and meningitis. A local infection is one in which pneumococci have only run into the lung, and can lead to pneumonia. The compositions of the invention, or the administration of a composition, can be used to protect or treat an animal subject against S. pneumoniae infection. In this way, a composition of the invention can be used in birds, such as chickens, turkeys and pets; and in mammals, Preferably, a human being, although the compositions of the invention are contemplated for use in other species of mammals, including but not limited to domesticated animals, (canines and phenyls); farm animals (cattle, sheep, horses, goats, pigs and the like); rodents and non-domesticated mammals. 5 FAP receptor antagonists In its first aspect, the present invention relates to methods for inhibiting or preventing infection and activation of the recep > of FAP with pneumococci, which consists in inhibiting the binding of pneumococci to the FAP receptor, or the? ir pneumococci adherent to the FAP receptor, or both. In one aspect of the invention, FAP receptor antagonists can be used to inhibit such binding or eluting adherent bacteria. As used herein, the term "platelet activating factor receptor antagonist" refers to a molecule that competitively inhibits the binding of pneumococci to the platelet activation factor receptor. Preferably, the agonist does not activate itself However, these molecules are not excluded where they can effectively inhibit the adhesion of pneumococci to the FAP receptor. For example, FAP itself competitively inhibits the binding of pneumococci to recep > tor. Such antagonists, or competitive inhibitors of binding to the PAF receptor, include but are not limited to molecules containing phosphorylcholine (see, for example, Tence et al. 1983, in Platelet-Activating Factor and Structurally Related Ethei-Lipids, INSERM Sy posi? Rn No. 23. 3. Benveniste and B. Arno? X (eds.) Elsevier Science Publishers: Amsterdam, pp. 41-48). Most particularly, an FAP receptor antagonist can be selected from the group consisting of LS59,989, L652,731, WEB 2086, kads? Renona and FR72112, vr.g., Hwang and Lam, 1991 Lipids 26: 1148-53; Hwang et al., 1989, Mol. Pharrnacol. 35: 48-58, which are incorporated herein by reference. In specific examples, mentioned below, FAP receptor antagonists include, but are not limited to, L659,989 (available from Merck a Co., Rahway, N3) and WEB 2086 (available from Boehringer Ingleheirn, Indianapolis, IN) .

Inhibitors of FAP receptor expression In another embodiment, administration of an inhibitor of FAP receptor activation in a subject can be used to inhibit the binding and internalization of bacteria. As used herein, an inhibitor of FAP receptor activation can be any immunosuppressive agent, including but not limited to: non-steroidal anti-inflating agents (such as, but not limited to: aspirin, salicylic acid, ibuprofen, etc.) .), steroidal anti-inflammatory agents (co or cortisone, etc.), and inhibitors of inflammatory cytokines, such as TNF and IL-1.

As used herein, the term "inflammatory cytokine inhibitors" refers to molecules that neutralize the activity of inflammatory cytokines. Such molecules can be antibodies that bind to the cytokine, and inhibit the activity of the cytokine, while either directly (through steric interference with the binding of the cytokine to its receptor) or indirectly (providing rapid clarification of the cytokine through the cytokine). clarification trails mediated with antibody) (see, for example, Dinarello and Uolff 1993, New * 10 Engl. 3. Med. 328: 106-113). Such antibodies can be obtained from commercial sources or from other sources, or they can be prepared. Such antibodies include but are not limited to polyclonal, monoclonal, chimeric and single chain Fab fragments, and a Fab expression library. In consecuense, As used herein, the term "antibody" refers broadly to fragments of i munoglobin or antigen-binding thereof. As an alternative, soluble anti-inflammatory cytokine receptors can be used to inhibit the activity of cytokines (see, Dinarello and Uolff, mentioned above).

Main elements of carbohydrates that bind to pneumococci In another aspect, the invention relates to the identification of carbohydrate main elements that mediate the adherence of pneumococci to host cells, in? particular pulmonary epithelial cells (lung type II cells), and venous endothelial cells. In particular, the inventors of the present have discovered that the stimulation of the host cells to which the pneumococci bind with inflammatory cytokines, such as the turnoral necrosis factor (TNF) interleukin-1 (IL-1), etc., induces a previously known carbohydrate specificity: N-acetyl-D-glucosarnine (GlcNAc). It has also been discovered that this GlcNAc sugar specificity is associated with the recipient population * 10 GalNacßl-3Gal in this way, a composition consisting of a carbohydrate with the binding motif GalNAcßl-4Gal and the binding motivator GlcNAc (both major elements can be found in the same oligosaccharide, or in two different oligosaccharides) can be use to prevent Adhesion of pneumococci to, or elute adherent pneumococci from, host cells. Thus, as used herein, the term "pneumococcal carbohydrate binding motivator" refers to the structure of a carbohydrate that is recognized for the pneumococcal bacterial adhesion complex, or the minimum receptor unit for such binding or adhesion. It is well known that a carbohydrate (also called oligosaccharide and saccharide) in a glycoprotein can be a highly complex structure formed by a set of onosaccharide subunits (sugar) arranged in a variety of bonds, (see, v. G, "Darnel et al. Molecular Cell Biology, Scientific American ^ Books, 1986, pp. 957-964.) The binding driver of the present invention is the minimal receptor unit, that is, the minimum structure capable of binding with pneumococci, regardless of the "decoration" (additional saccharide subunits) found in the compound.Thus, the carbohydrate binding motivator to which pneumococci binds can it can be located in a complex oligosaccharide, or it can be the simplest possible oligosaccharide that has the structure of the binding engine.Moreover, a binding motivator can be a derivative of the • 10 group > specific saccharides shown herein to mediate pneumococcal binding. A functional definition of binding linker of the invention is that it can demonstrate pneumococcal binding and elution properties as described in the examples herein As used herein, the term "carbohydrate" is refers both to compounds containing saccharide subunits and to those compounds associated or conjugated with a polymer as described herein In a further aspect, the invention takes advantage of the identification of a carbohydrate binding motivator to which pneumococci binds that has not previously been known to mediate the binding of pneumococci. This binding promoter contains a disaccharide group N-acetyl-D-galactosamine β-3Gal (GalNAcßl-3Gal). This motivator of union can be, but does not need to be, linked to a trickster. Examples of carbohydrates that contain this binding motivator include, but are not limited to,, forssman glycolipid, globose, etc. It has been discovered that quantitative or quasi-quantitative elution of host pneumococci from host cells can be achieved by contacting the pneumococci with the GalNAcßl-3Gal binding motif and a second binding motif, which was previously known to be pneumococci. adhere This second binding promoter is a carbohydrate containing a disaccharide group N-acetyl-galactosamine β-4Gal (GalNAcßl-3Gal). This motivator of union can be, but does not need to be, ^^ P 10 linked to a trickster. Examples of carbohydrates that contain that second binding motivator include, but are not limited to, asialo-GMl, asialo-GM2, etc. Quantitative elution of pneurnococci from unstimulated pulmonary epithelial cells can be achieved (type II pulmonary cells) and venous endothelial cells contacting these cells with a composition containing | carbohydrates that have both main elements (Tuornanen and Cundell, cited above). In the most preferred mode, one can use a compound that consists of carbohydrates representing the three main elements, in conjunction with an antagonist of the PAF receptor. Such a composition is particularly desirable for treating an early or ongoing infection, in which the immune system has been activated and the inflammatory mediators have been released. Preferably, such a composition is administered prior to the internalisation of pneumococci and the migration of pneumococci into the blood. The invention contemplates including additional portions of carbohydrate that have been discovered to interfere with the binding or adhesion of pneumococci in host cells. Examples of such carbohydrates include, but are not limited to, mannose, N-acetyl-D-galactosarnine, rnanose-D-mannose and methyl-a-D-mannopyranoside. The present invention further contemplates the use of multivalent carbohydrates or structures containing carbohydrates to increase the potency of the drug. In one embodiment, a binding motivator is found in multiple copies in a compound for use in the invention. In another embodiment, more than one binding motivator is found in one or multiple copies in a compound for use in the invention. Multivalent carbohydrates can be prepared by preparing a complex branched carbohydrate, which conceptually resembles a tree or brush in which each branch or sow contains a major nanococcal binding element. Alternatively, monovalent carbohydrates can be covalently or non-covalently associated with a polymer (see, Langer et al., International Patent Publication No. UO 94/03184, published February 17, 1994, which is specifically incorporated herein by reference). Suitable polymers include, but are not limited to, a protein, polysine, dextran, β-glycosaminoglycan, cyclodextrin, agarose, SEPHAROSE and polyacrylamide. Carbohydrates with major elements of neurnococcal binding according to the invention can be obtained from any source. For example, such carbohydrates can be obtained from commercial sources. Alternatively, carbohydrates can be prepared synthetically, using known enzymatic or chemical methods. The glycosyltransferase glycosyltransferase enzymes for the synthesis of a carbohydrate (is Said, saccharide) containing a neomocoxic binding junction engine, can be prepared as described in International Patent Publication No. U093 / 13198 by Roth. (published on July 8, 1993), which is incorporated herein by reference. The preparation catalyzed with glycosyltransferase of saccharide compositions has also been described in (Roth, US Patent No. 5,180,674 issued January 1, 1993, and International Patent No. 91/16449, published October 31, 1991), which has an apparatus for preparing such compositions (Roth.

E.U.A. No. 5,288,637, issued February 22, 1994) (each of these references is specifically incorporated herein by reference in its entirety).

^ J Compositions and therapeutic methods The present invention contemplates formulations consisting of an antagonist of the FAP receptor, preferably with a carbohydrate containing a pneumococcal binding junction enhancer as described above for pulmonary or parenteral administration, especially i.v. or oral, for the prevention and treatment of ip infection) - pneumococci and resulting disease conditions that include, but are not limited to, bacteremia, meningitis and pneumonia. Subsequently, in the present and for convenience purposes, the term "carbohydrate" in the singular or plural form should be interpreted as a carbohydrate containing a pneumococcal binding partner, unless provide another specific term. Accordingly, the present invention provides pharmaceutical compositions which consist of an antagonist of the FAP receptor, preferably with one or more carbohydrates containing a pneumococcal binding partner and a pharmaceutically acceptable carrier or excipient, as defined above. Since certain FAP receptor antagonists, such as L659,989 are orally active, the invention contemplates oral administration of said compounds (see Hwang and Lam, 1991, Lipids 26: 1148-53). For the treatment of a nascent Pneumococcal infection or of a systemic infection, a composition of the therapeutic invention can be administered by inhalation, to prevent colonization of pulmonary epithelial cells leading to infection, or to elute adherent bacteria of 5 cells pulmonary epithelial. Pulmonary administration is an effective form of administration for the bloodstream as well, since the drugs pass from the alveoli to the capillaries easily. Moreover, since neuroscotic systemic infections generally begin with the colonization of pulmonary epithelial cells, the administration of a therapeutic agent through the lungs is a rational route to treat the infection. Similarly, it is also possible that a parenterally administered drug, v. G, iv., Crosses from the capillaries to the 5 alveoli. Accordingly, the present invention contemplates the parenteral administration of a pharmaceutical composition of the invention, in particular i.v. administration, for the treatment of both systemic and pulmonary (local) lung infections. Accordingly, a wide variety of devices that are designed for the delivery of pharmaceutical compositions and therapeutic formulations in the respiratory tract or parenterally can be used in this aspect of the invention. The preferred pulmonary administration route 5 in the present invention is in the aerosol or inhaled form. The FAP receptor antagonist preferably with a carbohydrate or carbohydrate of the present invention, combined with a dispersible or dispersing agent, can be administered in an aerosol formulation as a dry powder or in a solution or suspension with a diluent. However, as mentioned above, the composition of the invention can also be administered parenterally. As used herein, the term "dispersant" refers to an agent that aids in the aerosolization of the PAF receptor antagonist, preferably with a carbohydrate or the absorption of this agent by the lung tissue, or both. Preferably the dispersant is pharmaceutically acceptable. For example, surfactants that are generally used in the art to reduce surface-induced aggregation of FAP receptor antagonists, or FAP receptor antagonists and carbohydrates, caused by the atomization of the solution forming the liquid aerosol can be used Non-limiting examples of such surfactants are surfactants such as esters of polyoxyethylene fatty acid alcohols, and polyoxyethylene sorbitan fatty acid esters. The amounts of surfactant used will vary, being generally within the range or 0.001 and 4% by weight of the formulation. Suitable surfactants are well known in the art, and may be selected on the basis of desired properties, Sg depending on the specific formulation, FAP receptor concentration and, if present, carbohydrate or carbohydrate, diluent (in a liquid formulation). ) or in powder form (in a dry powder formulation), etc. In addition, depending on the choice of the PAF receptor antagonist, and, if present, carbohydrate or carbohydrates (e.g., disaccharide or oligosaccharide complex), the desired therapeutic effect, the quality of lung tissue (e.g., lungs) healthy or sick) and * 10 other numerous factors, the liquid or dry formulations may comprise additional components, as discussed below. Liquid aerosol formulations contain the PAF receptor antagonist, and, if present, carbohydrate or carbohydrates and a dispersing agent in a physiologically acceptable diluent. The dry powder aerosol formulations of the present invention consist of a finely divided solid form of the FAP receptor antagonist, and, if present, carbohydrate or carbohydrates, and an agent dispersant. Parenteral formulations contain the PAF receptor antagonist, and, if present, carbohydrate or carbohydrates, in a vehicle suitable for injection. With the liquid formulation or dry powder aerosol, the formulation must be aerosolized. That is, it must be divided into liquid or solid particles to ensure that the pf-aerosolized dose actually reaches the alveoli. In general, the dynamic diameter of the mass medium will be 5 microns or less to ensure that the drug particles reach the pulmonary alveoli (Wearley, LL, 1991, 1991, Crit. Rev. in 5 Ther. Drug Carrier Systems 8: 333 ). The term "aerosol particle" is used herein to describe the liquid or solid particle suitable for pulmonary administration, i.e., which reaches the alveoli. Other considerations such as the construction of the assortment device, components Additional 10 in the formulation and particle characteristics are important. These aspects of pulmonary administration of a drug are well known in the art, and the handling of formulations, aerosol forming means and construction of an assortment device require at most routine experimentation by one skilled in the art. As for the construction of the assortment device, any form of aerosolization known in the art, including but not limited to spraying, Atomization or aerosolization with pump of a liquid formulation, and aerosolization of a dry powder formulation, can be used in the practice of the invention. Often, aerosolization of a liquid or dry powder formulation will require a propellant. The propellant can be any propellant generally used in the art. Specific non-limiting examples of such useful propellants are a * chlorofluorocarbon, a hydrofluorocarbon, a hydrochlorofluorocarbon or a hydrocarbon, including trifluoromethane, dichlorodifluorornetan, dichlorotetrafluoroethanol, and 1,1,1, -tetrafluoroethane, or combinations thereof. In a particular aspect of the invention, the device for aerosolization is a dose measuring inhaler. A dose-measuring inhaler provides a specific dose when administered, instead of a variable dose U depending on the administration. A dose measuring inhaler As such it can be used with a liquid formulation or a dry powder aerosol. Metered dose inhalers are well known in the art. Once the FAP receptor antagonist, and, if present, carbohydrate or carbohydrates reach the lung by inhalation, a number of formulation-dependent factors perform the binding activity. It will be appreciated that when treating a pneumococcal infection, which requires circulating levels of the PAF receptor antagonist, and, if present, carbohydrate or carbohydrate, such factors as aerosol particle size, aerosol particle shape, the presence or absence of lung disease or embolism that may affect the absorption of the FAP receptor antagonist, and, if present, carbohydrates, the pH of the lungs or pharmaceutical vehicle, etc. For each of the In the case of the formulations described herein, certain lubricants, absorption enhancers, stabilizing or suspending agents may be appropriate. The choice of these additional agents will vary depending on the objective. It will be appreciated that in some cases where the local assortment of the PAF receptor antagonist, and, if present, carbohydrate or carbohydrate, is desired or sought, such variables as an absorption enhancer will be less critical. In a further embodiment, an aerosol or parenteral formulation of the present invention may include ^ Me other active ingredients in addition to the receptor antagonist of the FAP, and, if present, carbohydrate or carbohydrates. In a preferred embodiment, said active ingredients are those used for the treatment of lung diseases. For example, said additional active ingredients include, but are not limited to, bronchodilators, antihistamines, epinephrine, and the like, which are useful in the treatment of pulmonary conditions. "In a preferred t-modality, the additional active ingredient may be an antibiotic, for example, for the treatment of pneumonia In a preferred embodiment, the antibiotic is pentamidine While in a preferred aspect the compositions of the present invention are administered together with antibiotics, a particular advantage of the present invention is that it provides a strategy for treating antibiotic-resistant pneumococci. important in view of the recent increase in antibiotic resistance among virulent bacteria, a phenomenon that is of great concern in public health. The recognition that pneumococci activate the PAF receptor provides added incentive to use PAF receptor antagonists in the treatment of pneumococcal infection. The use of such inhibitors not only prevents colonization and internalization of the bacteria, but can also alleviate the inflammatory response associated with exposure to the bacteria. In general, the FAP receptor antagonist, and, if present, carbohydrate or carbohydrates of the present invention are introduced herein in the form of aerosol or parenteral in an amount between 0.001 mg per kg body weight of the mammal. to about 100 mg per kg body weight of said mammal. In a specific modality, the dose is dose per day. One skilled in the art can readily determine an aerosol volume or weight corresponding to his dose based on the concentration of the PAF receptor antagonist, and, if present, carbohydrate or carbohydrates, in an aerosol or parenteral formulation of the invention; alternatively, one can prepare an aerosol formulation with the appropriate dose of FAP receptor antagonist, and, if present, carbohydrate or carbohydrate, in the volume to be administered, as is readily appreciated by one skilled in the art. It is also clear that the dose will be higher in the case of inhalation therapy for a syngeneic pneumococcal infection, and may be lower to treat only the lung infection, because of the local concentration of the PAF receptor antagonist, and, if present, carbohydrate or carbohydrates, in the lung will be much greater with pulmonary administration. An advantage of the present invention is that administration of an antagonist of the FAP receptor, and, if present, carbohydrate or carbohydrates, directly to the lung allows target drug assortment, thus limiting the cost and unwanted side effects. The formulation can be administered in a single dose or in multiple doses depending on the severity of the infection or need for prophylaxis. For example, when administering a subject on a respirator as protection against pneumococcal infection (a common consequence of long-term treatment on a respirator), less than the composition may be effective. If an infection begins, more of the composition can be provided to facilitate elution of adherent bacteria as well as to prevent adhesion and colonization by resistant bacteria. It will be appreciated by one skilled in the art that the exact amount of prophylactic or therapeutic formulation to be used will depend on the state and severity of the disease, the physical condition of the subject, and a number of other factors, which can be readily determined by the patient. medical expert (see, for example, Langer et al., International Patent Publication No. UO 9403184, published February 17, 1994, which is specifically incorporated herein by reference). The aerosol assortment systems, such as the pressure dose inhaler and the dry powder inhaler are described in Newman, S.P., Aerosols and the Lung, Clarke, S.U. and Davia, D. editores, pp. 197-22 and can be used in connection with the present invention.

Liquid aerosol formulations The present invention provides liquid aerosol formulations and dosage forms for use in treatment subjects suffering from or in danger of acquiring a pneumococcal infection. In general, such dosage forms contain an antagonist of the PAF receptor, and, if present, one or more carbohydrates, in a pharmaceutically acceptable diluent.

Pharmaceutically acceptable diluents include but are not limited to sterile water, saline, buffered saline, dextrose solution, and the like. In a specific embodiment, a diluent that can be used in the present invention or the The pharmaceutical formulation of the present invention is phosphate buffered saline, or a saline solution generally regulated between the pH scale of 7.0-8.0, or water. The liquid aerosol formulation of the present invention may include, as optional ingredients vehicles, diluents ,. solubilizing agents or emulsifiers, surfactant and pharmaceutically acceptable excipients.

The liquid aerosol formulations of the present invention will typically be used with a sprayer. The sprayer can be driven to compressed or ultrasonic air. Any sprayer known in the art may be used in conjunction with the present invention such as but not limited to: Ultravent, Mallinckrodt, Inc. (St. Louis, MO); the Acorn II sprayer (Marquest Medical Products, Englewood CO). Other sprays useful in conjunction with the present invention are described in the U.S.A. Us. 4,624,251 issued November 25, 1986; 3,703,173 issued November 21, 1972; 3,561,444 issued February 9, 1971 and 4,635,627 issued January 13, 1971. The formulation may include a vehicle. The vehicle is a macromolecule that is soluble in the circulatory system and that it is physiologically acceptable that the means of physiological acceptance for those skilled in the art would accept injection of said vehicle into a patient as p > art of a therapeutic regimen. This vehicle is preferably relatively stable in the circulatory system with a plasma acceptable of half life for space. Said macromolecules are included but not limited to soy lecithin, oleic acid and sorbitan trioleate, with preferred sorbitan trioleate. Lae formulations of the present modality also may include other agents useful for stabilization or for regulation of osmotic pressure. Examples of the agents * include but are not limited to salts, such as sodium chloride, or potassium chloride, and carbohydrates, such as glucose, galactose or mannose, and the like.

Dry powder spray formulations It is also contemplated that the present pharmaceutical formulation will be used as a dry powder inhaler formulation comprising a finely divided powder form of the FAP receptor antagonist, and, if present, carbohydrate and carbohydrate and dispersant. The form of the PAF receptor antagonist, and, if present, carbohydrate, will generally be a lyophilized powder. Freeze-dried forms of the PAF receptor antagonist, and, if present, carbohydrates, can be obtained by standard techniques. In another embodiment, the dry powder formulation comprises a finely divided dry powder containing an antagonist of the FAP receptor, and, if present, one or more carbohydrates, a dispersing agent and also a binding agent. Binders useful in conjunction with the present formulation include such agents as lactose, sorbitol, sucrose, or mannitol, in amounts that facilitate dispersion of the powder from the device. The present invention will be better understood from one. review of the following illustrative description that presents the details of the constructions and procedures that were followed in their development and validation.

E3EMPL0 1 In this example, we describe the profound effects of TNF and IL-1 on the adhesion of pneumococci to vascular epithelial and white endothelial cells, and identify The receptor specificity higher in activated cells like the FAP receiver. The adhesion of the encapsulated AII chain of S. pneumoniae (serotype 2) and the non-encapsulated R6 chain isogenicity to human vein endothelial cells (EC) and lung epithelial cells type II (LC) measured before and after after stimulation of eukaryotic cells with IL-1 or TNF. The adherence to EC was increased approx. 70% by stimulation of TNFa (figure 1). Stimulation of IL-1 resulted in a more modest increase in pneumococcal adherence (increase from 240 + 10 to 363 + 13 bacteria / 100 CE; increase from 270 + 15 to 355 + _ 18 bacteria / 100 LC). The adhesion was not affected by the presence of a type II capsule. At an input concentration of 107 cfu / ml, the adherence of AII and R6 to 100 EC stimulated by TNFa was 405 + 14 and 400 + _ 10, respectively. 25 The identity of the new receptor activity was suggested by two observations. The purified pneumococcal JP pneumococcal cell wall (50 ug / ml Tuomanen et al., 1985, 3. Infect. Dis. 151: 859) competitively inhibited the improved pneumococcal adhesion to the activated EC (415 + 25 decreased to 289 +. 11 bacteria / 100 EC) and LC (355 + .18 decreased to 249 + 13 13 bacteria / 100 LC). In addition, improved adherence was profoundly attenuated (71 and 62% decrease, respectively) at the time of replacement of choline in the pneumococcal cell wall with ethanolamine by * MA substitution of the aminoalcohol in the culture medium (T? Onanen et al., 1985, cited above) (decrease from 415 + 25 to 121 + _ 11 bacteria / 100 EC, decrease from 355 +18 to 135 + _ 13 bacteria / 100 LC). Choline is a critical determinant of the bioactivity of platelet activating factor (FAP) and FAP has been shown to play a particularly pronounced role in the inflammatory response during pneumonia and pneumococcal meningitis (Cabellos et al., 1992, 3. Clin Invest. 90: 612). The direct participation of the PAF receptor in the adherence of pneumococci was suggested by the ability of two PAF receptor antagonists to virtually eliminate the Improved adhesion of pneumococci to activated cells (Figure 2). Antagonists of FAP L659,989 (Hair and others, cited above) and UEB 2086 (Kunz et al., 1992, 3. Biol. Chem. 267: 9101) inhibited adhesion to EDso of 5 x 10-8M and 1 x 10 ~ 8M, respectively. Similar values of EDso have been recorded in animal models for L659,989 (Hair and others, cited above) and in transfected cells of the in vitro FAP receptor for UEB 2086 (Kunz et al., Cited above).

No receptor antagonist affected the adherence of pneumococcus to the resistant DC (240 + .15 against 224 + 12 and 237 + _ 9 bacteria / EC in the presence of 1 uM UEB 2086 and L659,989, 5 respectively) or LC (270 +.10 bacteria against 264 i 8 and 272 +. 8, respectively). Specific receptors for FAP have been identified in numerous tissues and cells including rt-lung and brain membranes, platelets, neutrophils, eosinophils and rnacrophages (Chao and Olson, 1993, Biochern 3. 292: 617). The PAF receptor is a protein of approximately 39,000 Da that "consists of an extracytoplasmic aminoterrninal region, seven putative membrane spanning domains, and a carboxy terminal cytoplasm.

(Chao and Olson, cited above). These aspects are characteristic of the seven members of the rhodopsin receptor family (Kunz et al., Cited above). FAP receptors appear to be well conserved between species. The comparison of human and guinea pig sequences Indians indicate that both consist of 342 amino acids, which are organized into seven domains that rotate the putative membrane and possess approximately 83% sequence similarity at the amino acid level (Kunz et al., Cited above). Unique for the human FAP receptor among this class of molecules, without However, it is the presence of an individual glycosylation site in the putative extracellular loop between JP amino acids 169 and 171 (Kunz et al., Cited above). The glycosylation sites of FAP differ in p > osition and number of other species. For example, the FAP receptors of a guinea pig possess two glycosylation sites; one between amino acids 5 169 and 171 and the second in the extracellular loop of N-terrninal "The high affinity binding of FAP to the FAP receptor results in the signaling of transmembrane, which is thought to involve G proteins and is coupled to phospholipase A2 and phospholipase C (Chao and Olson, cited above). Recently, the FAP receptor was cloned from human leukocytes and successfully transfected into kidney epithelial cells (Kunz et al., Cited above). To determine if pneumococci can be directly added to the FAP receptors, human U937 cells (ATCC CRL1593; embryonic kidney epithelial cells stably transfected with the SV40 T antigen) and COS-7 cells (ATCC CRL 1651, monkey kidney) were transinfected with the human FAP receptor cDNA (Gerard and Gerard, 1994, 3. Immunol 152: 793) or the cDNA that contains a flag construction (Kunz et al., Cited above) by electroporation (Kunz et al., Cited above) or the adhesion of cationic liposomes (Gerard and Gerard, cited above). The binding of ligands to these receptors is temperature dependent, sensitive to FAP receptor antagonists, and results in internalization The receptor is fast, comparable to those of cells that have native receptors (Gerard and Gerard, cited above). The P control cells were transinfected with only with vector. The transfected and control cell monolayers were prepared in 60-well Terasaki boxes. Monolayers of COS-7 and U937 cells are prepared in culture dishes of excavation 48 using the previously described methods for EC and LC (Geelen et al., 1993, Infect.Immun 61: 1538). The expression of FAP receptors was confirmed by means of staining im unohistoquímica when using a monoclonal antibody . ^ against the flag gene FAP receptor product (K? nz and others, cited above). The adherence of pneumococci to control COS-7 and U937 cells was low, probably because the kidney is not normally a target for pneumococcal infection (figure 3). At the time of transfection As the FAP receptor in these cells, the adherence increased even more than 6 times (Figure 3). The adherence was not affected by the • E presence of a type II capsule. At an input concentration of 107 cfu / ml, adhesion to U937 cells by R6 and AII cells was 295 +.30 and 227 + 38, respectively. The The adherence of pneumococci cultured in meta olamine was approximately 10 fold less than the bacteria cultured in a medium containing choline. At an input concentration of 10? cfu / ml, adhesion. to U937 cells was 25 ± 10 for R6 bacteria cultured in ethanolamine, compared to 296 + 30 for R6 bacteria grown in choline. Further confirmation that the pneumococci were directing the pP the PAF receptor came from the ability of FAP and FAP antagonists WEB 2085 and 1-659,989 to inhibit the adherence of pneumococci (figure 3). It may be of interest to determine if pneumococci recognized FAP strictly in a choline-dependent model or if the individual glycosyl determinant of the FAP receptor contributed to receptor recognition. As noted above, the human FAP receptor differs from the ^^ recep > FAP of other species in the absence of a site of N-linked glycosylation in the putative extracellular sequence of H2 -terminal and the presence of only one extracellular glycosylation site at positions 169-171 (Kunz et al., Cited above). This site of individual glycosylation was removed by means of direct mutagenesis instead of triple amino acid glycosylation. The non-glycosylated PAF receptor reduced pneumococcal adhesion for J | J »COS-7 and U937 cells by approximately 75% (Figure 3). At an input concentration of 107 cfu / ml, the adhesion of R6 to U937 cells expressing the non-glycosylated PAF receptor was decreased from 296 ± 30 to 74 ± 20. The contribution of carbohydrate recognition to the adherence of pneumococci for EC and LC stimulated by cytokine was tested using competition tests performed in the presence of monosaccharides (1-50 nm) or glycoconjugates (0.003-2mM) the role of carbohydrate recognition in the adhesion of pneumococci is described Mk completely in copending application Serial No. 08 / 254,577, Proxy Case No. 600-1-084, filed on 6 June 1994 by Tuomanen and Cundell, entitled "MODULATORS OF PNEUMOCOCCAL ADHERENCE TO PULMONARY AND VASCULAR CELLS AND 5 DIAGNOSTIC AND THERAPEUTIC APPLICATIONS" (MODULATORS OF PNEUMOCOCCAL ADHESION TO PULMONARY AND VASCULAR CELLS AND DIAGNOSIS AND THEIR THERAPEUTIC APPLICATIONS); hereafter Tuornanen and Cundell. Bacteria were preincubated for 15 minutes at room temperature with final concentrations of * 10 sugars were established, centrifuged (3,000 rprn, 3 min) to remove unbound sugar, resuspended at 1 x 107 cfu / ml in albumin buffer and added to the adhesion test. The comparison of the capacity of simple sugars (Tuomanen et al., 3. Exp. Med. 168: 267), for inhibiting adherence indicated that while GalNAc and D-rnanosa were effective in inhibiting adherence to cells Oj at rest, GlcNAc became effective in activated cells (figure 4). GlcNAc showed no effect on the adhesion of pneumococci without phosphorylcholine, a finding consistent with the function proposal of the hill in the direction of pneumococci to the PAF receptor. The adherence of bacteria without choline to the activated cells in the presence and absence of GlcNAc, ue of 121 ± 11 and 119 ± 5/100 EC and 137 ± 7 and 135 ± 13/100 LC, respectively. Pneumococcal adhesion to COS-7 cells and U937 transinfected also decreased by approximately 40% in the presence of GlcNac (Figure 3). * rJm The resting and LC ECs express two classes of pneumococcal receptors: GalNAcßl-4Gal or GalNAcßl-3Gal in a nucleus of mañosa (Tuomanen and Cundell, cited above). These two specificities of recep > They are best defined by the 5 inhibitory activities of the glycocid played asialo-GM2 for the β-4 receptor and globose for the β-3 receptor. The contrast to the results with GlcNAc, however, neither the globósido nor asialo-GM2 decreased the adherence of pneumococci to | k transfected transplants. At an input concentration 0 of 107 cfu / rnl, the adherence of R6 to U937 cells in the presence of globose and asialo-GM2 was 318 ± 30 and 327 + 39, compared to 296 ± 30 in the absence of the glycoconjugates. Notwithstanding the foregoing, the cytokine stimulation of EC and LC results in a greater number of both these resting cell receptors (Table 1), as well as the appearance of new specificity of GlcNAc.The latter specificity was dependent on the 6alNAcßl-4Gal specificity since the inhibitors were additive, GlcNAc was related to the population of the GalNAcßl-3Gal receptor since these inhibitors are not additive (Figure 5) .If the effects of the sugars were not additive then they are considered to occupy the mimos receptors or linked receptors, if they are additives, they are interpreted as being present in separate receptors.

TABLE 1 Neococeptor populations in resting cells and human type II lung cells stimulated with cytokine (LC) and vascular endothelial cells (EC) Type of Cell Number of pneumococci adherent to the designated receptor in host cell 6alNAcßl-46al GalNAcßl-36al Control EC 140 + 12 110 ± 8 240 ± 10 EC + TNFa 180 ± 13 200 ± 9 415 + 25 LC 140 ± 10 140 ± 10 270 ± 15 LC + IL-la 205 ± 9 200 ± 11 355 ± 18 R6 (107 cfu / nl) was exposed to increasing concentrations (0.003-2 millon) of asylum-GM2 or globose and then co-incubated with resting cells or monolayers stimulated with cytokine for 30 minutes at 37 ° C. Asialo-GM2 was used to define the number of pneumococci attached to the GalNAßl-46al population of the receptors and globósidos that adhere to the GalNAßl-3Gal population. Neurococcal adherence to each designated sugar was determined from bacterial adherence to 100 host cells in the absence of sugar (control values) bacterial adherence to 100 host cells in the presence of sugar. The results are shown for the maximum (2 rnM) of each sugar tested and are the mean ± SD for duplicate vessels in at least 6 independent experiments. Previous studies have indicated that although PAF receptor antagonists markedly reduce inflammation during experimental pneumococcal pneumonia, the components of pneumococcal teicoicates do not activate the receptor PAF in model in vitro cell culture systems and do not prevent PAF activation of the receptor (Hair and others, cited above). However, as shown in the following example PP, these studies were incorrect. In fact, pneumococci appear to be potent activators of the PAF receptor. These results suggest that the PAF receptor binds pneumococci to the target cell in a carbohydrate-choline-dependent manner. This union seems to lead to a shot of a translation cascade signal. In addition, it is known that the PAF receptor is rapidly internalized after expression on the surface of the cell (Gerard and Gerard, cited above), PP suggesting that this binding can provide the bacterium with a migration route to endothelial or epithelial cells or through them. Pneumococci have been visualized within vascular endothelial cells in vitro. The ability of the cells to carry the PAF receptor to capture pneumococci was tested directly in a bacterial protection test. In In this experiment, U037 and COS-7 cells transfected with PAF receptor by non-glycosylated PAF receptor were incubated with 1Q7 cfu / ml of R6 for 2 hours at 37 ° C. Then the cells were washed twice with medium 199 and tested with 0.5 rnl of 50μg / ml gentamicin for 90 minutes at 37 ° C. He Treatment with gentamicin destroys bacteria that are not protected by internalization in mammalian cells. The cells were washed twice with medium 199 and lysed by resuspension in 0.1% Triton X-100. Cell suspensions were seeded in 100-fold serial dilutions on plates of blood and cfu / ml was determined after 24 hours of incubation at 37 ° C. Of the 1-2 x 10 * bacteria initially adherent to K? In a single layer, 300 were recovered from the lysates of the cells treated with gentamicin, indicating that approximately 3% of the adherent bacteria entered the cells over time and protected themselves from the antibiotic extrecelular. The non-glycosylation of the PAF receptor resulted in a 10-fold reduction in the number of pneumococci interned. It is concluded that the pneumococcal adhesion to human type II neurotrocytes and vascular endothelial cells is qualitatively and quantitatively affected by # 10 cytokine and pneumococcal activation of the target cell. This is consistent with observations of increased adherence of pneumococci to virally infected cells and increased susceptibility to pneumococcal disease during viral infection (Plotowski et al., Am. Rwev. Respir. Dis. 134: 1040). The resting cells LC and EC carry two classes of receptors containing GalNAcßl-4Gal and GalNAcßl-3Gal. Cytokine stimulation results in the expansion of the receptor with ^ * Improved pneumococcal adhesion being directed toward the recep > PAF tor. This example reports that it has been found by The first time a pathogen binds to its receptor. It is also the first identification of a receptor for an adhesive ligand present in a suitable bacterial cell wall, in this case, the teichoicates containing choline unique to pneumococci. The presence of choline as a determinant bioactive for PAF and teichoic acid / neurotococcal lipoteichoic acid suggests imitation of ligand as a basis for the direction of pneumococci to the PAF receptor. The profound decrease in pneumococcal inflammation in the presence of PAF receptor antagonists suggests the physiological importance of the association of pneumococci with the PAF receptor during pneumonia and introduces a novel use of PAF receptor antagonists to interfere with pneumococcal binding to eukaryotic cells.

E3EMPLQ 2: PAF Receptor Activated by Pneumococcus 10 Activation of C0S-7 cells transfected with the PAF receptor was tested. The cells were exposed to medium (control), platelet activation factor (100 rnM), pneumococci (108 bacteria / rnl), and pneumococci cultured in ethanolamine (108 bacteria / ml). The inhibition of activation of PAF and pneumococci was also measured with the Wjte receptor antagonist PAF L659.989 (2 μg / ml). Cell activation was measured detecting incorporation of 3H in ositoltriphosphate (IP3). The results of this test are shown in the Figure 6. Very little 3 3-labeled IP3 was detected in the control cells, or after exposure to pneumococci cultured in ethanolamine. As noted above, pneumococci cultured in ethanolarnin lacking choline can not appreciably bind to the PAF receptor. As expected, the PAF treatment results in significant incorporation of 3H into "IP3." However, surprisingly, contacting the transfected cells with pneumococci produced a very strong response, with much higher 3H-IP3 than the observed with 100 rnM PAF, both activations, mediated by pneumococci and PAF could be almost completely inhibited with the PAF receptor antagonist L659, 989. These results indicate that the activation of COS cells by pneumococci proceeds through the PAF receptor. of the bacteria cultured in ethanolamine to activate the receptor also indicates a function of the acid teicoico in the union to, and the activation of, the PAF receptor. This invention may be embodied in other forms or carried out in other ways without departing from the spirit or essential characteristics thereof. Therefore the present description is considered in all aspects illustrative and non-restrictive, the scope of the invention is indicated by the appended claims and all changes that come within the meaning and scale of equivalences are intended to be encompassed by it. Different references are cited throughout this specification, each of which is incorporated as a reference in its entirety.

Claims (2)

1. NOVELTY OF THE INVENTION CLAIMS 1. The use of an effective platelet activating factor receptor antagonist to inhibit the binding of S. pneumoniae to host cells, in the preparation of compositions for preventing or treating an infection with Streptococcus pneumoniae.
2. 2. The use of a platelet activating factor receptor antagonist further characterized in that said platelet activating factor receptor antagonist is a carbohydrate containing an effective N-acetyl-D-glucosarnin binding promoter to inhibit the Binding of S. pneumoniae to 15 host cells. % 3. The use of a platelet activating factor receptor antagonist according to claim 2, further characterized in that said platelet activation factor receptor antagonist is a second carbohydrate Selected from the group consisting of a carbohydrate containing a disaccharide of β-4Gal N-acetyl-D-galactosamine binding motif disaccharide, a disaccharide of β-3Gal binding motivator N-acetyl-D-galactosamine, and a mixture of same, effective to inhibit the binding of S. pne? moniae to host cells. 4. The use of a platelet activating factor receptor antagonist according to claim 1, further characterized in that said platelet activating factor receptor antagonist is selected from the group consisting of L559,989, L652, 731, UEB 2086, kadsurenone, and FR72112. 5. The use of a platelet activating factor receptor antagonist in accordance with the claim 2, further characterized in that the carbohydrate is N-acetyl-D-gl? Cosarnine. -, _ 6.- The use of a factor receptor antagonist 10 activation of platelets in accordance with the claim 3, further characterized in that the second carbohydrate is selected from the group consisting of glycolipid forssrnan, globoside, asialo-GMl and asialo-GM2. 7.- The use of a factor receptor antagonist 15 platelet activation according to claims 1, 2 or 3, further characterized because together With the platelet activating factor antagonist, a carbohydrate selected from the group consisting of mannose is used, N ~ acetyl-galactose, mannose-D-mannose, and methyl-a-D-mannopyranoside 20 effective to inhibit the binding of S. pneumoniae to host cells. 8. The use of a platelet activating factor receptor antagonist according to claim 2 or 3, further characterized in that the carbohydrate or carbohydrates are multivalent. 9. The use of a platelet activating factor receptor antagonist further characterized in that the composition obtained is for administration p > or atomization and inhalation. 10. The use of a platelet activation factor 5 receptor antagonist according to claim 9, further characterized in that the atomization is nebulization. 11. The use of a platelet activating factor receptor antagonist in accordance with the claim .A, 1 2 or 3, further characterized by the composition obtained 10 is for administration by intravenous injection. 12. The use of a platelet activating factor receptor antagonist according to claim 1, further characterized in that a inhibitor is used together with the platelet activating factor antagonist. 15 effective inflammation to inhibit the expression of the platelet activation receptor. •? Tjg 13.- The use of a platelet activating factor receptor antagonist according to claim 12, further characterized in that the inhibitor of inflammation is 20 selects from the group consisting of a neutralizing antibody for tumor necrosis factor, a neutralizing soluble tunoral necrosis factor receptor, a neutralizing antibody for interleukin-1 and a soluble neutralizing interleukin-1 receptor. 14. A pharmaceutical composition comprising an amount of a platelet activating factor receptor antagonist effective to inhibit the binding of S. pneumoniae to host cells, an amount of a carbohydrate containing an N-acetyl-binding partner. D-glucosarnine effective to inhibit the binding of S. pneumoniae to 5 host cells, and a pharmaceutically acceptable carrier. 15. The pharmaceutical composition according to claim 14, further characterized in that it comprises an amount of a carbohydrate selected from the group consisting of (from a carbohydrate containing a disaccharide of rnotivador de 10 β-3Gal binding N-acetyl-D-galactosarnine, and a carbohydrate containing a disaccharide of β-4Gal N-acetyl-D-galactosamine binding motivator, effective to inhibit the binding of S. pneumoniae to host cells. 16. The pharmaceutical composition according to claim 14, further characterized in that the platelet activating factor receptor antagonist is selected from the group consisting of L659,989, L652,731, UEB 2086, kadsurenone, and FR72112. 17. The pharmaceutical composition according to claim 14, characterized in that the carbohydrate is N-acetyl-D-glucosamine. 18. The pharmaceutical composition according to claim 15, further characterized in that the second carbohydrate is selected from the group consisting of glycoside forssman, globoside, asialo-GMl and asialo-GM2. 19. The pharmaceutical composition according to claims 14 or 15, further characterized in that it comprises an amount of a carbohydrate selected from the group consisting of mannose, N-acetyl-galactose, mannose-D-rnannose, and methyl-aD-mannopyranoside. effective to inhibit the binding of S. pneurnoniae to host cells. 20. The pharmaceutical composition according to claim 14 or 15, further characterized in that the carbohydrate or carbohydrates are multivalent. ^ 21.- The pharmaceutical composition in accordance with 10 claim 14 or 15, further characterized in that the pharmaceutical composition is an aerosol formulation, which formulation contains a dispersant. 22. The pharmaceutical composition according to claim 21, further characterized in that the dispersant is a surfactant. 23. The pharmaceutical composition according to claim 21, further characterized in that it is a dry powder aerosol formulation, in which the carbohydrate or carbohydrates are present in finely divided powder. 24. The pharmaceutical composition according to claim 23, further characterized in that it comprises a filler. 25. The pharmaceutical composition according to claim 21, further characterized in that it is a liquid aerosol formulation comprising a pharmaceutically acceptable diluent. 26. The pharmaceutical composition according to claim 25, further characterized p > or the diluent is selected from the group consisting of sterile water, saline, regulatory saline, and dextrose solution. 27. The pharmaceutical composition according to claim 14 or 15, further characterized in that the host cell is selected from the group consisting of lung epithelial cells and vascular endothelial cells. SUMMARY OF THE INVENTION The present invention relates to compositions and methods for preventing infection by pneumococci; in particular, the invention relates to the identification of the main reception of Streptococcus pneumoniae on activated human cells, and diagnosis and therapeutic compositions and methods based on the same; in particular, the invention relates to ^ discovery that the platelet activating factor (PAF) receptor is an adhesive ligand for adhesion of pneumococci to activated venous endothelial and lung epithelial cells (ie, host cells); therefore, the present invention is directed to a method for preventing or treating an infection by Streptococcus pneumoniae by 15 administration of a platelet activating factor receptor antagonist; this invention further relates to the recognition that adhesion to activated cells also includes a carbohydrate ligand found in these activated cells; in this way, a method to inhibit The pneumococcal adhesion may further comprise the administration of an amount of carbohydrate containing a N-acetyl-D-glucosarnin binding motif; it has been found that resting venous and epithelial lung endothelial cells carry two classes of receptors that contain different 25 carbohydrate functions; thus, the invention further provides for administration an amount of a second carbohydrate selected from the group consisting of a carbohydrate containing a disaccharide with a β-4Gal N-acetyl-D-galactosamine binding partner, a disaccharide of binding factor ßl ~ 3Gal N-acetyl-D-galactosarnine, and mixtures of 5 same; In addition, the invention provides pharmaceutical compositions comprised of these agents that inhibit the binding of pneumococci to human cells; activator factor receptor antagonists are shown in a specific example ^ g ^ of platelets and saccharides to inhibit the binding of uf pneumococci to venous endothelial cells and activated lung epithelial cells as well as cells transinfected with the recep > of the platelet activation factor in vitro. 15 33 / 3N / BS / EA / ls * ycl * cgt * ieoh * casv P96 / 985F