Application of heparin-binding antagonists in inhibiting bradykinin releasing

FIELD: medicine, pharmaceutics, pharmacology.

SUBSTANCE: one should apply mammalian anti-HBP-antibodies. The ways are being suggested to identify monoclonal antibody bound, at least, with one epitope upon native HBP (heparin-binding protein) and methods to detect whether a mammal produces HBR being bound with a monoclonal antibody and, also, the kits for the above-mentioned purpose. The present innovation provides the opportunity to apply the mentioned antibodies in preventing and treating disorders associated with bradykinin releasing.

EFFECT: higher efficiency of application.

25 cl, 11 dwg, 3 ex, 1 tbl

 

Description

The scope of the invention

The invention relates to a method of treating or preventing diseases resulting from the release of bradykinin in a mammal, particularly a mammal, which produces a heparin-binding protein, wherein the specified heparin-binding protein binds to antagonist heparin-binding protein, which includes the introduction of the specified mammal antagonist heparin-binding protein in an amount effective to reduce the release of bradykinin in a mammal. Moreover, the invention relates to methods and kits for identifying, producing, whether the mammal is a heparin-binding protein that binds to antagonist heparin-binding protein, and a method for detecting antagonist heparin-binding protein.

Prior art

Inflammation

Acute inflammatory response associated with several phenomena, including changes in size and vascular tone, as well as increased vascular permeability, which leads to the formation of enriched protein exudate (Lewis in Mediators of Inflammation, Wright, Bristol, U.K., 1986).

Because neutrophils (PMA) receive a chemotactic signals, they are located on the periphery of vessels and adhere to vascular endothelial cells through specifices is their adhesion molecules, synthesized on the surface of endothelial cells and on the surface of neutrophils. After attachment of neutrophils to endothelial cells in the endothelium are formed gaps, inducing vascular permeability and creates the opportunity for migration of neutrophils in the interstitial region of tissue.

Phase-contact system covers three enzymatic factor, factor XI (F (XI), factor XII (F XII) and prekallikrein (pre-kallickrein) plasma (RK)and non-enzymatic cofactor, N-kininogen (NC), which forms equimolar complexes with F XI and RK, respectively. The contact phase is present on monocytes, fibroblasts and neutrophils. Specific binding sites exhibited endothelial cells and noncellular negatively charged surfaces, such as kaolin, generally referred to here as the "contact phase"create local clusters of critical components. Turning Imogene F XII in an active enzyme, F He activates such as phase-contact system (Colman et al., 1986, Grit. Rev. Oncol. Hematol. 5: 57-85). Reciprocal activation is associated with the surface F XII and RK, anchored on the surface by means of NC, promotes the development F Ha and Pka, amplificare thereby signal initiation. Then factor He can activate factor XI, and activates the intrinsic coagulation pathway. You know, Thu the ROK also hydrolyzes NC with the formation of strong nonapeptide, bradykinin (Kaplan and Silverberg, 1987, Blood 70:1-15). Kinini are the primary mediators of inflammation, which causes pain, vasodilatation and increased vascular permeability resulting from direct actions on endothelial cells, causing them to fall and allowing at the same time and migration of neutrophils, and the formation of transudate content of plasma (Oyvin et al., 1970, Experentia 26:843-844). May be involved the process of local production of prostaglandins and nitric oxide, NO (Hall, 1992, Pharmacol. Ther. 56:131-190). Therefore, activation of the phase-contact system can lead to a number of damaging effects, such as inflammation, septic shock, respiratory distress syndrome of adults, disseminated intravascular coagulation, post-operative bleeding in cardiovascular surgery.

Phase-contact system can be activated, in particular, when neutrophils are associated with endothelial cells, the presence of endotoxins and bacterial infection (review Colman et al., 1997, Blood 90:3819-3843). For example, it was found that sepsis is the activation of factor XII and prekallikrein at which they split and as a result turn into enzymes that react quickly with C1-inhibitor with the formation of complexes of factor XIIa-C1-inhibitor and kallickrein-C1-inhibitor. Znachitelno.soderzhanie education complex kallickrein-C1-inhibitor is observed when stimulated in vitro (artificial) the circulation.

Found that Aprotinin, an inhibitor at the same time and plasmin, and plasma kallikrein, reduces blood loss after cardiac surgery and reduces high postoperative clotting time. In particular, it was shown that Aprotinin in model-driven extracorporeal circulation helps reduce the formation of complexes kallickrein-C1-inhibitor and C1-C1-inhibitor (Wachtfogel et al., 1993, J. Thorac. Cardiovasc Surg. 106:1). Adding Aprotinin to the artificial circle of blood circulation, perfuziruemami whole blood volume, anticoagulative heparin, Aprotinin actually complement the action of heparin (Bannan et al., 1998, Brit. J. Haem. 101:455-461). Therefore, Aprotinin has an additional gemostaticski a beneficial effect compared to the individual effect of heparin in the specified model.

Aprotinin also increases the viability of endothelial cells under hypoxic conditions during cooling, when these cells are stored in solutions for the preservation of organs, and increases the safety of the lungs and myocardium in models that use the bodies as a whole (Sunamori et al., 1991, Ann. Thor. Surg. 52:971-978 and Roberts et al., 1998, Ann. Thorac. Surg. 66:225-230). Moreover, while bradykinin, as shown, increases vascular permeability, Aprotinin reduces the permeability of blood vessels and the number of neutrophils (O'brien et al., 1997, an. J. Physiol. Pharmacol. 75:741-749 and Dwenger et al., 1995, Eur. J. Clin. Chem. Clin. Biochem. 34:207-214).

Heparin-binding protein

Currently found covalent structure of two closely related proteins isolated from polymorphonuclear leukocytes in human peripheral blood and swine (cf. H. Flodgaard et al., 1991, Eur. J. Biochem. 197: 535-547; J. Pohl et al., 1990, FEBS Lett. 272: 200 ff.). Both proteins have great similarity with elastases neutrophils, but as a result of selective mutations of the active serine 195 and histidine 57 (chymotrypsin numbering (B.S. Hartley, "Homologies in Serine Proteinases", Phil. Trans. Roy. Soc. Series 257,1970, p. 77 ff.)) these proteins are deprived by activity. These proteins were called heparin-binding protein human (hHBP) and heparin-binding protein pigs (rnvr), respectively, due to their high affinity to heparin.

Schafer et al. (Schafer et al., 1984, Infec. Immun. 53:651) called this protein cationic antimicrobial protein (SAR) due to its antimicrobial activity. NVR tightly binds to the lipid a component of LPS and endotoxin (Kass=0,8×109M-1). It has been suggested that the bactericidal effect of HBP due to the binding with lipid A (Petersen et al., 1993, Eur. J. Biochem. B214: 271-279, Flodgaard et al., 1994, J. Cell. Biochem. Suppl. 18A: Abstr. E505; Pereira et al., 1993, Proc. Natl. Acad. Sci. USA 90: 4733-4737). The proposed binding site of native HBP with lipid A/LPS localized in the form of uncharged area between the main and kislotno the m patches on the NVR and contains residues 20-26 and 41-43. In the binding site of the lipid A/LPS Phe25, Cys26, Cys42, and Phe43 form a hydrophobic pocket that is suitable for linking or chain fatty acids, or glucosaminidase sugar rings lipid A. Near this pocket is ionic and hydrophilic pocket (Asn20, Gln21, and AGD), which should very well be suitable for binding glucosaminyl-linked phosphate groups of lipid A (Iversen et al., 1997, Nature Struct Biology 4: 265-268).

Moreover, in animal models of fecal peritonitis was shown that the treatment of HBP rescues mice from lethal damage (Mercer-Jones et al., 1996, In Surgical Forum, pp. 105-108 and Wickel et al., 1997, In 4mInternational Congress on the Immune Consequences of Trauma, Shock and Sepsis, Munich, Germany, pp.413-416). It was suggested that heparin-binding protein or LPS-binding fragments can be used to treat septic shock (WO 95/28949, US Pat. Nos. 5458874, 5607916 and 5650392).

Originally NVR studied in connection with the fact that he has bactericidal and LPS-binding properties (Gabay et al. 1989, Proc. Natl. Acad. Sci. USA. 86:5610-5614 and Pereira et al., 1993, Proc. Natl. Acad. Sci. USA 90: 4733-7). However, accumulating data now support the concept that HBP, in addition to its bactericidal role involved in the development of inflammation through its effects on the recruitment and activation of monocytes (Pereira et al., 1990, J. Clin. Invest. 85:1468-1476 and Rasmussen et al., 1996, FEBS Lett. 390:109-112), the recruitment of T cells (Chertov et al., 196, J. Biol. Chem. 271: 2935-2940).

It was found that HBP induces contraction of endothelial cells and fibroblasts (Ostergaard and Flodgaard, 1992, J. Leuk.Biol. 51: 316-323). In this connection, WO 93/05396 describes a method of screening inhibitors of HBP by incubation NVR or cells that produce HBP, in the presence of the substance, which was assumed properties of the inhibitor HBP, and in the presence of tissue or cells that are able to interact with HBP; weak interaction (for example, reduction of endothelial cells) indicates that the substance is an inhibitor of HBP.

In the application WO 99/26647 described the use of a heparin-binding protein in modulating or preventing apoptosis of mammalian cells. In this application also described that HBP rescues cells from rat insulinoma from IL-1-induced apoptosis.

It was also found that the heparin-binding protein human, unlike heparin-binding protein pigs, associated with Aprotinin (BPTI) (Petersen et al., Eur. J. Biochem. 214:271-279). In particular, BPTI has the ability to bind NVR with Kd=0,1×10-6M (Petersen et al., 1993, Eur. J. Biochem. B214: 271-279). P1-specificity NVR been shown to be primarily associated with Lys or Leu (Kiczak et al., 1999, Biol. Chem. 380: 101-105). It was postulated that the most characteristic remains in the structure of the HBP, which are responsible for the binding of BPTI are Gly169, Gly175 Ser192, and Asp201, in accordance with Asp189, Ser195, Gly216, and Asp226 in trypsin (Petersen et al., 1993, Eur. J. Biochem. B214: 271-279). Kiczak et al., 1999, Biol. Chem. 380:101-106, using the techniques of phage distribution, constructed a library of mutants of the side chain of P1 Aprotinin. These researchers found that the NVR has a great affinity in relation to the P1 Lys, as well as with uncharged amino acids of P1 - Leu, Thr, Met, Gin.

Summary of invention

By chance it was discovered that the heparin binding protein (HBP) is signalism link neutrophils in induced vascular permeability and activation of the phase-contact system with simultaneous formation of bradykinin and that this, in particular, affects mediated RK splitting NC with getting bradykininase sequence. In addition, it has been shown that antagonists of HBP reduce the permeability of endothelial cells. As shown here, the "HBP antagonist" is a substance that binds to the heparin-binding protein and inhibits the action of the heparin-binding protein.

This invention is directed to a method of treatment or prevention of diseases resulting from the release of bradykinin in a mammal, particularly in a patient person, in particular in a mammal, which is produced by the heparin-binding protein that binds to anti nistam NVR, includes introduction to the specified mammal, if necessary, antagonist heparin-binding protein in an amount effective to modulate or reduce the release of bradykinin in the specified mammal. Such diseases include, but are not limited to, a syndrome of systemic inflammatory response, ischemic reperfusion injury, anaphylaxis and allograft rejection. Such diseases can include respiratory distress syndrome of adults as a side effect syndrome systemic inflammatory response. Anaphylaxis may occur on a background of undesirable activation PMA during artificial blood circulation, pulmonary surgery, head injuries and total injuries to the entire body. Modulation or reduction of the degree of liberation of bradykinin occurs as a result prevent contact NVR with endothelial cells and/or phase-contact system. In a particular aspect of the HBP antagonist modulates or reduces mediated kallicrein cleavage of N-kininogen obtaining bradykininase sequence. Further, the invention is directed to the use of HBP antagonist for the manufacture of a medicinal product, applicable to the treatment of the syndrome of systemic inflammatory response, ischemic reperfusion injury, anaphylaxis and allograft rejection is the patient, with HBP, which is associated with HBP antagonist. Antagonists NVR can also be used to treat or manufacture of the medicinal product, applicable to the treatment of the complications of the syndrome of systemic inflammatory reactions, respiratory distress syndrome in adults.

In a specific aspect the invention is directed to a method of preventing diseases resulting from the release of bradykinin in a mammal, particularly in a patient person, in particular in a mammal, which is produced by the heparin-binding protein that is associated with HBP antagonist, which includes the introduction of a given mammal, if necessary, domain, inhibitor of serine proteinase-like serine proteases of Konitza or analog or derivative that binds to HBP, in an amount effective to modulate or reduce the release of bradykinin in the specified mammal.

In another particular aspect this invention is directed to a method of preventing diseases resulting from the release of bradykinin in a mammal, particularly in a patient person, in particular in a mammal, which is produced by the heparin-binding protein that binds to a monoclonal antibody that binds at least one the m epitope on the NVR, moreover, the specified epitope associated with prekallikrein-N-kininogen complex and activates the release of bradykinin, which includes the introduction of a given mammal, if necessary, a monoclonal antibody that binds at least one epitope on the NVR, and the specified epitope associated with prekallikrein-N-kininogen complex and activates the release of bradykinin, in a quantity effective to modulate or reduce the release of bradykinin in the specified mammal.

This invention further is directed to the detection of HBP antagonist. In one aspect the method comprises (a) culturing endothelial cells in the presence of HBP and in the presence and in the absence of the substance, which presumably is specified antagonist, and (b) detecting any of the actions specified substances on the permeability of endothelial cells, and reduced permeability of these endothelial cells compared with the permeability of these cells when they are incubated in the presence of HBP, but in the absence of this substance indicates that the substance is an antagonist. In another aspect, the method includes (a) incubation of HBP in the presence of the first substance which interacts with the NVR, and the second substance is, which by assumption is an antagonist of HBP, and (b) detecting any of the actions specified second substance, which by assumption is an antagonist of HBP, interaction NVR with these first substance.

In a specific aspect this invention is directed to methods of identifying a monoclonal antibody that binds at least one epitope on HBP, in particular, NVR, with the specified epitope associated with prekallikrein-N-kininogen complex and activates the release of bradykinin. In one aspect the method comprises (a) culturing endothelial cells in the presence of HBP and in the presence and in the absence of monoclonal antibodies, which presumably binds at least one epitope HBP, in particular, NVR, with the specified epitope associated with prekallikrein-N-kininogen complex and activates the release of bradykinin, and (b) detecting any of the actions specified substances on the permeability of endothelial cells, and reduced permeability of these endothelial cells compared with the permeability of these cells when they are incubated in the presence of HBP, but in the absence of the indicated antibodies indicates that the antibody is a monoclonal antibody that binds, what about the least with one epitope HBP, and the specified epitope associated with prekallikrein-N-kininogen complex and activates the release of bradykinin. In another aspect, the method comprises (a) incubating prekallikrein-N-kininogenase complex in the presence of HBP and in the presence and in the absence of monoclonal antibodies, which presumably binds at least one epitope on HBP, in particular, NVR, with the specified epitope associated with prekallikrein-N-kininogen complex and activates the release of bradykinin, and (b) detecting any of the actions specified antibodies to the release of bradykinin, and reduced the release of bradykinin indicates that the antibody binds at least with the specified epitope on HBP.

This invention is also directed to methods and kits for determining, produce whether mammals HBP, which is associated with HBP antagonist. The method includes the following stages: (a) the allocation of HBP or cells or tissues, producing HBP, from a mammal, in particular a sick person; (b) incubating the specified NVR or cells or tissues, producing HBP, with substance, tissue, cells or their components, which interact with HBP and specified HBP antagonist, and (C) detecting the impact of the decree of the aqueous heparin-binding antagonist interaction NVR with the specified substance tissue, cells or their components, and reduced interaction indicates that the HBP is associated with the specified HBP antagonist. The set of tests includes (a) HBP antagonist; (b) native HBP and (C) a substance, tissue, cells or their components, which interact with HBP.

In a specific aspect this invention is also directed to methods and kits for identifying, producing, whether mammal, in particular a sick man, HBP, which binds to a monoclonal antibody that binds at least one epitope on native HBP, in particular, NVR, with the specified epitope associated with prekallikrein-N-kininogen complex and activates the release of bradykinin, which includes (a) the allocation of HBP or cells or tissues, producing HBP, from the specified mammal; (b) culturing the specified NVR or cells or tissues, producing HBP, with endothelial cells in the presence or absence of the indicated antibodies, and (C) detecting any effects of the indicated antibodies on the permeability of endothelial cells, and reduced permeability indicates that HBP is associated with the indicated antibody. In another aspect, the method includes (a) the allocation of HBP or cells or tissues, producing HBP, from the specified mammal; (b) inkberrow is of the specified NVR or cells or tissues, producing HBP, with prekallikrein-N-kininogenase complex in the presence or absence of the indicated antibodies, and (C) detecting any effects of the specified NVR on the release of bradykinin from prekallikrein-N-kininogenase complex, and reduced the release of bradykinin indicates that the HBP is associated with the indicated antibody. Set according to the invention includes (a) a monoclonal antibody that binds at least one epitope on native HBP, in particular, with NVR man, with a specified epitope associated with prekallikrein-N-kininogen complex and activates the release of bradykinin, (b) native HBP person, and (C) prekallikrein-N-kininogenase complex attached to the solid carrier.

Brief description of drawings

Figure 1 shows the effect of cross-linking CD 18 with secondary goat antibody against mouse F{AB}2.

Figure 2 shows dose-dependent effect of HBP (25-75 µg/ml) on the permeability of monolayers of endothelial cells.

Figure 3 shows the inhibition of HBP-induced increase in permeability of endothelial cells (EC) polyclonal anticorodal against HBP.

Figure 4 shows the influence of antibodies against NVR on the function of endothelial barrier under the action of the secretions of polymorphonuclear is aconito (PMA).

Figure 5 shows the inhibition caused by bradykinin and NVR increase the permeability of the EC under the action of monoclonal antibodies MWC against bradykinin. Bradykinin (100 nm; Fig. 5A) or NVR (75 µg/ml; fig.5b) was administered during the time corresponding to zero, in the side plate of monolayers of endothelial cells, preincubating in the presence of mAb MVC (40 µg/ml).

Figure 6 shows the inhibition caused by HBP increase the permeability of the EC under the action of monoclonal antibodies RCN against plasma kallikrein. NVR (75 µg/ml; filled circles) or bradykinin (100 nm; hollow circles).

7 shows the inhibition caused by HBP increase the permeability of the EC under the action of processing the peptide NCN.

On Fig shows the inhibition caused by HBP increases EC permeability in the processing of peptide SDD31.

Figure 9 shows the inhibition caused by HBP increase the permeability of the EC's handling Aprotinin.

Figure 10: the release of Il-6 from human monocytes. Monocytes were cultured in 1 ml serum-free medium for 24 hours in the presence of LPS and/or native HBP /[R23S, F25E]HBP/ [G175Q] HBP in quantities indicated on the figure.

11: Saturation native HBP, [R23S,F25E]HBP [G175Q]HBP (A)3H-LPS and (B) competition for binding125I-BPTI fixed, increasing concentrations of unlabeled BPTI. (C) the Concentration of the 125I-BPTI shown at 0 nm % unlabeled BPTI. The apparent difference in binding between HBP and [R23S,F25E]HBP discussed in the section "Results". The arrows indicate the standard deviation.

Detailed description of the invention

Heparin-binding protein

In this context, the term "heparin-binding protein" ("HBP") means a protein that (i) proteoliticeski inactive; (ii) contained in azurophilic granules of polymorphonuclear leukocytes; and (iii) is a chemoattractant for monocytes and/or activates monocytes. HBP can, respectively, be the protein of a mammal, in particular humans. In particular, NVR is a Mature NVR person having at least about 80% identity with the amino acid sequence presented in SEQ ID NO:1, more preferably at least about 90%, even more preferably at least about 95%, and most preferably at least about 97% (hereinafter in the description of "homologous polypeptides"), which are qualitatively retain the activity of HBP.

SEQ ID NO:1:

Amino acid sequences homologous polypeptides differ from the amino acid sequence represented in SEQ ID NO:1, inclusion or deletion of one or more amino acid residues and/or replacing one or more amino acid is different balances of the different amino acid residues. Preferably, the amino acid changes were of a minor nature, i.e. that there is such conservative amino acid substitutions that do not have a significant impact on the spatial packing and/or activity of the protein; small deletions, typically of the order of 30 amino acids; small amino - or carboxy-terminal pendant, such as amino-terminal meinenemy residue; a small linker peptide consisting of about 20-25 residues; or a small pendants that facilitates purification by changing neutral charge or perform another function, such as polyhistidine tract, an antigenic epitope or binding domain.

Examples of conservative substitutions made within the group of basic amino acids (such as arginine, lysine and histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine and asparagine), hydrophobic amino acids (such as leucine, isoleucine and valine), aromatic amino acids (such as phenylalanine, tryptophan and tyrosine), and small amino acids (such as glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not normally alter the specific activity are known in this field and are described, for example, at N. Neurath and R.L. Hill, 1979, in, The Proteins, Academic Press, New York. Most often occur following substitutions: Ala/Ser, al/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly, and Vice versa.

This term is specifically introduced in order to enable peptide fragments HBP, in particular, fragments having a chemotactic effect similar to that of the NVR. Moreover, NVR used in the methods according to the present invention, contact antagonists NVR, such as Aprotinin and/or monoclonal antibody generated against native HBP person. Native HBP person in its Mature form has an amino acid sequence represented in SEQ ID NO:1, and in addition, is (i) proteoliticeski inactive; (ii) contained in azurophilic granules polymorph nuclear leukocytes; and (iii) is a chemoattractant for monocytes and/or activates monocytes.

NVR can be isolated from blood platelets, obtained from human blood using the methods described in U.S. patent No. 5814602. More specifically, a protein obtained by fractionation of the extract of blood platelets. For this purpose, applied column chromatography using heparin-sepharose. This method of chromatography, including elution from a column in a NaCI gradient from 0.5 M to 3 M, through which the first was skipped extract of blood platelets, resulting in such two elution peak. The first peak in the area, 1.2 M m NaCI who should be registered at 280 nm in the form of broad protein peak, which is known in itself platelet factor (PF<). In the field of 1.8 M NaCI number of protein below the registration limit for the system, but the fraction in this area have angiogenic activity. The active fraction was further purified using microprobe, using HPLC with a reversible phase column With a4and at 214 nm was recorded completely purified protein peak; this protein was identical to the present NVR, whether NVR pig or human types, depending on the type platelets.

If HBP is used to detect antagonists NVR, it is preferable that it has been produced by recombinant DNA as described below. The sequence of the nucleic acid encoding HBP can be obtained artificially by using established standard methods, for example, phospholidine method described S.L. Beaucage and M.H. Caruthers, 1981, Tetrahedron Letters 22:1859-1869, or the method described by Matthes et al., 1984, EMBO Journal 3: 801-805. According potamididae method, oligonucleotides were synthesized, for example, in an automatic DNA synthesizer, purified, and hybridized, legirovanyh and cloned in appropriate vectors.

The methods used for selection or cloning nucleic acid sequence that encodes a heparin-binding protein, and is used in the method according to the present invention, well known in this field and include isolation from genomic DNA, from cDNA, or a combination of both. Cloning of nucleic acid sequences according to the present invention from such genomic DNA can be implemented, for example, using the well known polymerase-cableway reaction (PCR) or by screening expression libraries of antibodies for the registration of cloned DNA fragments with partial structural properties (see, for example, Innis et al., 1990, A Guide to Methods and Application, Academic Press, New York). You can use other methods of nucleic acid amplification, such as ligase-zalewa reaction (LCR), legirovannye activated transcription (LAT) and amplification-based nucleic acid sequence (first NASBA).

Then the sequence of the nucleic acid is included in a recombinant expression vector which may be any vector, which will be convenient for carrying out the methods of recombinant DNA. The choice of vector will often depend on the host cell into which it is included. Thus, the vector may be a stand-alone can replicate vector, i.e. a vector which exists in the form of extrachromosomal substances, and replication which does not depend on chromosomal replication, e.g. a plasmid. Alternatively, as a vector may be selected such vector, which is when introducing it into the cell of the host integrates into the host genome and replicated together with the chromosome(s), in which it is integrated.

The nucleic acid sequence SEQ ID NO:2, the coding sequence of SEQ ID NO:1, can be operatively linked to a nucleic acid that encodes a heterologous Pro - and/or signal sequence. Alternatively, a nucleic acid encoding SEQ ID NOS: 4 (signal sequence + Mature HBF) and 6 (signal sequence + Pro-sequence + Mature NVR), SEQ ID NOS: 3 and 5, respectively, can be included in a recombinant vector.

The sequence of the nucleic acid encoding HBP, may also be operatively associated with the respective terminator, such as the terminator of human growth hormone (Palmiter et al., op. cit.) The vector may additionally contain other elements. The latter include polyadenylation signals (for example, from SV 40 or from the area 5 adenovirus Elb), transcriptional enhancer sequences (e.g., enhancer SV 40) and translational enhancer sequences (e.g., sequences encoding the RNA of adenovirus VA). Recombinant expressing the vector may also contain a DNA sequence enabling the vector to replicate in the cell of the relevant owner. The vector may also what to include selective marker, for example, a gene the product of which complements a defect in the cell host, such as the gene encoding dihydrotetrazolo (DHFR), or such gene, which imparts resistance to the drug, for example, neomycin, geneticin, ampicillin or hygromycin.

In a particular aspect of the NVR can be produced cultured host cells containing a DNA sequence encoding a Mature NVR, preceded by N-terminal additive in an appropriate culture medium under conditions dopustimih expression of HBP, in this case, the NVR is obtained from the culture medium in the form of HBP, containing additional insertion material on the N-end.

N-terminal supplementation may represent a sequence of about 5 to 25 amino acid residues, in particular from about 8 to 15 amino acid residues. The nature of the amino acid residues in the N-terminal sequence is not considered critical.

In order to facilitate Mature NVR is generally preferred that the DNA sequence encoding the N-terminal additive included a DNA sequence encoding a site subject to proteolytic cleavage, localized between the DNA sequence that encodes a N-terminal additive, and a DNA sequence that encodes a Mature HBP. Examples of relevant sites about politicheskogo splitting are the site of cleavage by enterokinase with the amino acid sequence, the site of cleavage by factor XA with the amino acid sequence.

Alternatively, the host cell containing the DNA encoding Mature NVR, preceded by a signal sequence may be cultured in an appropriate culture medium under conditions permitting the expression of HBP, and the resulting NVR get from the culture medium in the form of Mature HBP.

The procedure used for ligating nucleic acids sequences encoding HBP or NVR with N-terminal addition, Dpro-HBP (signal + Mature NVR), promoter and terminator, respectively, and to include them into the appropriate vectors containing the information necessary for replication, are well known to specialists in this field (see, for example, Sambrook et al., city).

The host cell(s)into which the expression vector may be any cell(s), which are capable of producing HBP and is preferably a eukaryotic cell(s), such as the cell(s) invertebrates (insects) or cell(s) of vertebrates, for example, the cell(s) mammals, in particular, the cell(s) and insect cell and mammalian. In a preferred aspect, the cell of a mammal is a cell that can be grown under anaerobic conditions, after transfection with nucleic acid encoding HBP mammal. In bol is e preferred aspect, the cell of a mammal is a transformed by adenovirus cell(s) or cell(s)derived from an embryo. In this context, the cell derived from embryonic stem cells is a cell(s)derived from a primary culture of embryonic cells, or cell(s)obtained from the cell line, originally perasaanku from a primary culture of embryonic cells. An example of such transformed by adenovirus cells or cells derived from an embryo, is a cell and embryonal human kidney (SOME), in particular, the cell SOME 293. Among insect cells such cells can be, for example, cells of Lepidoptera or Drosophila.

Methods for transfection of mammalian cells and expression of DNA sequences introduced into these cells, as described, for example, Kaufman and Sharp, 1982, J. Mol. Biol. 159:601-621; Southern and Berg, 1982, J. Mol. Appl. Genet. 1:327-341; Loyter et al., 1982, Proc. Natl. Acad. Sci. USA 79:422-426; Wigler et al., 1978, Cell 14:725; Corsaro and Pearson, 1981, Somatic Cell Genetics 7:603, Graham and van der Eb, 1973, Virology 52:456; Fraley et al., 1980, JBC 225:10431; Capecchi, 1980, Cell 22:479; Wiberg et al., 1983, NAR 11:7287; and Neumann et al., 1982, EMBO J. 1:841-845. The cells of the insect can be appropriately transliterowany baculovirus vector, as described in U.S. patent No. 4745051.

Used for cell culture medium can be any of the commonly used media suitable for growth of mammalian cells, such as the environment with the obsession serum or serum-free medium, containing the appropriate supplements, or environment suitable for growth of mammalian cells. The necessary protection can be purchased through dealers or to prepare them in accordance with the published writings (e.g., in catalogues of the American type culture Collection). The cells are then subjected to screening for resistance to antibiotics. After that, the selected clones are subjected to analysis to determine the activity of HBP using known in the field of methods, such as determining chemotactic activity and the analysis of the release of cytokines from monocytes (see, e.g., Rasmussen et al., 1996, FEBS Lett. 390:109-112).

Produced by cells of the NVR can then be obtained from the culture medium using conventional methods, including separating the host cells from the medium by centrifugation or filtration, deposition of proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, purification of various chromatographic methods, for example, by the method of ion exchange chromatography, affinity chromatography, etc.

If the NVR has the N-terminal additive, after receiving it from the culture medium of the N-terminal additive NVR can be successfully derived under the action of a suitable proteases; and the result is a Mature (and active) HBP. Examples of relevant which can be which include, not limited to, enterokinase and factor XA.

ANTAGONISTS HBP

Antagonists NVR can be either a polyclonal antibody against HBP, or monoclonal antibodies against HBP. To obtain polyclonal antibodies against NVR can be used in the following procedures. To obtain antibodies it is possible to immunize animals of different types, using them as a host, by injection they NVR, preferably NVR person, including, but not limited to, rabbits, mice, rats, sheep, goats, etc. In one aspect, the NVR can be anywhereman with immunogenic carrier, e.g., bovine serum albumin (BSA) or hemocyanin mollusk keyhole limpet (KLH). To enhance the immune response can be used in a variety of adjuvants, depending on the species of the host, including, but not limited to, beta-blockers (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, plutonomies a polyalcohol, polyanion, peptides, oil emulsions, hemocyanine mollusk keyhole limpet, dinitrophenol, and potentially useful adjuvants, such as BCG (Bacillus Calmette-Guerin) and Corynebacterium parvum.

To obtain monoclonal antibodies that bind at least one epitope on HBP, in particular, NVR man, W is specified epitope associated with prekallikrein-N-kininogen complex and activates the release of bradykinin, can be used the following method. In a preferred aspect, the monoclonal antibody is a monoclonal antibody man. Can be used any technology that enables the production of antibody molecules in the culture in the long-term cultivation of cell lines. Such methods include, but are not limited to, hybridoma technology, originally developed Kohler and Milstein (1975, Nature 256:495-497), as well as teamnow technology, technique b-cell hybridomas person (Kozbor et al., 1983, Immunology Today 4:72) and technology EBV-hybridomas for production of monoclonal antibodies (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). In an additional aspect of the present invention, with the help of modern technology it is possible to achieve the production of monoclonal antibodies in transgenic animals (PCT/US90/02545; Green, 1999, J. Immunol. Methods 231:11-23 and US. Pat. Nos. 5625126 and 5633425). According to the invention, human antibodies can be used and can be obtained by using human hybridomas (Cote et al., 1983, Proc. Natl. Acad. Sci. USA 80:2026-2030) or by transforming b cells human virus EBV in vitro (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96).

According to the invention available technology to obtain single-chain antibodies (U.S. Pat. No. 4946778) can be adapted to establish getting HBP-specific odnosima echnik antibodies. In an additional aspect of the present invention uses the technology in which the described method of constructing Fab-expressing libraries (Huse et al., 1989, Science 246:1275-1281)to be able to quickly and easily identify the Fab-fragments of the monoclonal antibodies of the desired specificity in relation to the NVR.

Antibody fragments which contain the idiotype of the antibody molecules, can be obtained by using known technologies. For example, such fragments include, but are not limited to, the fragment F(ab').sub.2, which can be obtained by cleavage of the antibody molecules under the action of pepsin; the Fab'fragments which can be obtained by recovery of the disulfide bridges of F(ab').sub.2 fragment, and Fab fragments, which can be obtained by processing the antibody molecules with papain and regenerating agent.

Upon receipt of antibodies, screening for the desired antibody can be produced using known in the field of technology, such as radioimmunoassay analysis, ELISA (enzyme-linked immunosorbent assay), "sandwich"-immune analysis, Immunoradiometric analysis, reactions diffuse precipitation in the gel, immunodiffusion analysis, immunological analysis in situ (e.g., using colloidal gold, enzyme or radioisotope labels), Western blot analysis, precipitation reactions, analysis of agglutination(for example, analysis of the gel agglutination, analysis of haemagglutination), analysis of complement fixation, immunofluorescence analysis, the protein and the analysis of immunoelectrophoresis etc. In one aspect of the binding of an antibody is determined by registering the label on the primary antibody. In another aspect, the primary antibody is determined by registering the binding of a secondary antibody or reagent to the primary antibody. In another aspect, the secondary antibody is associated with a label. In this area there are many known methods of registration binding with immune analysis, and they are all within the scope of this invention.

Alternatively, a library of anti-HBP-antibodies can be obtained using phage technology distribution (see, for example, Koscielska et al., 1998, Acta Biochimica Polinica 45:705-720). By screening this library it is possible to select antibodies that are most strongly associated with HBP, and use them as antagonists HBP.

The HBP antagonist can be a polypeptide containing the domain inhibitor serine protease type inhibitor Konitza that is associated with HBP. The peptide containing the domain inhibitor serine protease type inhibitor Konitza usually contains 60 residues and six specific spatially arranged cysteines, which form disulfide the ligature. In a particular aspect of the present invention, if the NVR is human HBP, type inhibitor inhibitor Konitza can be Aprotinin, also known as BPTI, or equivalent, which is associated with HBP. In a preferred aspect, similar Aprotinin contains mutations at positions 12-19 and 34-39, especially preferred are mutations at positions 15-19. Other analogues, which can also be used, as described in U.S. patents№№5162498, 5316923, 5395922, 5514585, 5510249, 5591603, 5618915, 5621074, 5673090, 5618696 and 5576294. In its most specific aspect, mutations are IN, CA, I18M, I19S, R17T.

Large libraries (107-109) polypeptides, including domain type domain Konitza, can be obtained using different approaches. For example, the method of error-prone PCR (Saiki et al., 1988, Science 293:487-491) can be applied to each domain type domain Konitza. As a result of this procedure will be randomly included erroneous nucleotides in each gene corresponding domain type domain Konitza during PCR reaction under conditions which aq polymerase under amplification includes erroneous nucleotides. The library can be divided into phage particles to select polypeptides, the most strongly binding with HBP.

Alternatively, when using degenerate oligonucleotide mutagenesis can be specific is etli (Schier et al., 1996, Gene 169:147-153). Artificial random oligonucleotides included in the domain type domain Konitza during PCR reaction, and the produced library options for individual loops.

In another aspect, libraries can be generated through DNA shuffling (Stemmer et al., 1994, Nature 370:389-391). This method can be used for recombination between homologous domains of the type of domains Konitza. Briefly, DNA encoding each of the options, mixed and fragmented to small pieces. Then these fragments come together again in the process of regular PCR reaction. During this reaction, small fragments will be combined with each other, because they are complementary to each other, and the result is the recombination of genes included.

REGISTRATION METHODS

Antagonists heparin-binding protein (HBP) can be registered by determining whether associated with HBP substance, which by assumption is an antagonist of HBP, as a result of (a) incubation NVR with the first substance that interacts with HBP, and with a second substance, which, by assumption, is an antagonist of HBP, and (b) detecting any effect of the specified second substance, which by assumption is an antagonist of the interaction with HBP, with a first substance, and a weakened interaction of HB is with the first substance indicates, that the second substance is an antagonist of HBP.

HBP or the first substance may be associated with the label. The label is preferably selected from the group consisting of enzymes, colored or fluorescent substances, radioactive isotopes and complexometric agents.

Examples of enzymes used as aiming substances are peroxidase, such as horseradish peroxidase, phosphatase (such as acidic or alkaline phosphatase), B-galactosidase, urease, glucose oxidase, carbonic anhydrase, acetylcholinesterase, glucoamylase, lysozyme, malatdegidrogenaza, glucose-6-phosphatedehydrogenase, b-glucosidase, proteases, piruvatcarboksilazy, esterases, luciferase, etc.

The enzymes themselves are not defined, but they must be combined with a substrate to catalyze the reaction, the final product in which the result is determined. Examples of substrates that can be used in the method according to the invention include hydroperoxide/tetramethylbenzidine or khlornaftil or o-phenylenediamine or 3-(para-hydroxyphenyl) propionic acid or luminal, intoxicat, para-nitrophenylphosphate, nitrophenylacetate, 4-methylumbelliferyl-0 galactopyranoside or luciferin.

Alternatively, the labeled substance may include colored or fluorescent substance, including particles angry is that, colored or fluorescent latex particles, particles of the dye, fluorescein, phycoerythrin or phycocyanin.

Radioactive isotopes, which can be used for the purposes of this invention, can be selected from125I131I111In3H,32P,14C and35S. Radioactivity emitted by these isotopes can be measured by Gemma-counter or scintillation counter method known in this field.

Kompleksoobrazuyushchie agents that can be used for the purposes of this invention, can be selected from Biotin (which forms complexes with Avidya or streptavidin), avidin (which forms a complex with Biotin), protein A (which forms a complex with immunoglobulins) and lectins (which form a complex with the receptor carbohydrates). Because the property is not defined, you must mark the substance with which the complex-forming agents to form a complex. The tagging may be performed by any of the labeled substances listed above for labelling enzyme.

In a particular aspect, the binding of HBP antagonist with NVR can be registered, for example, using technologies SPA, borrowed from Amersham Pharmacia Biotech. Briefly, biotinylated plasma membrane derived from endothelial cells of the umbilical vein (HUVEC), linked to avidin-conjugated granules PVT SPA. These granules can be obtained from Amersham Pharmacia Biotech. Labeled kininogen person (for example,3H,125I) add to the membranes up until the binding sites of kininogen will not be saturated. Add NVR in increasing concentrations to move labeled kininogen, and measure the decrease included the label. Obtain the standard curve for this decline. Test antagonist NVR on its ability to inhibit HBP-mediated reduction of radioactive kininogen on plasmatic membranes. In particular, NVR incubated in the presence of various concentrations of up to molar excess antagonists HBP, and determine their ability to inhibit HBP-mediated reduction of radioactive kininogen on plasmatic membranes.

Alternatively, antagonists NVR can be detected by culturing endothelial cells in the presence of HBP and in the presence or in the absence of the substance, which is supposed to be an antagonist. In a specific aspect, the antagonist is a monoclonal antibody that binds at least one epitope NVR, preferably NVR man, with a specified epitope associated with prekallikrein-N-kininogen complex and activates the release of bradykinin.

In a preferred aspect of monoclonalantibody is a monoclonal antibody man. In one aspect the antibody and NVR preincubated with each other before incubation with endothelial cells for 10 min at 37°C. the Impact of substances on the permeability of endothelial cells is determined by measuring transendothelial electrical resistance or by determining the clearance of albumin, as described in the examples below. Permeability reduction will indicate that the substance acts as an antagonist of HBP.

In a particular aspect, the binding of monoclonal antibodies to at least one epitope HBP, where the specified epitope associated with prekallikrein-N-kininogen complex and activates the release of bradykinin, can be determined by incubation of the antibody with the NVR and prekallikrein-N-kininogenase complex. As will be described in detail hereinafter in the examples, HBP is associated with prekallikrein-N-kininogenase complex, thus stimulating the release of bradykinin. Monoclonal antibody that binds at least one epitope NVR, should reduce or prevent the release of bradykinin. The release of bradykinin can be measured using methods known in this field, for example, using immunological analysis.

In one aspect, covering the buffer contains 0.05% Tween, 15,9 mm sodium bicarbonate, 35 mm sodium carbonate,pH of 9.6. Added N-kininogen and incubation is carried out overnight at 4°C. Covering the buffer is removed, and the plate is washed with Tris-buffer, pH 7.4, containing about 50 μm zinc. Then add prekallikrein, and the mixture is incubated for 1 hour at room temperature. After that add NVR in Tris-buffer, and sequentially, with approximately 10-minute intervals for approximately 1 hour starting after about 5 min after addition of HBP, samples are extracted and determined by the release of bradykinin. In one aspect the release of bradykinin may be determined using a commercially available kit ELISA method (MARKIT-M Bradykin, Dainippon Pharmaceutical Co., Ltd.). In this method, the bradykinin in the sample and labeled with peroxidase to bradykinin (BK) provide an opportunity to competitively react with anti-HC antibody (rabbit)associated with the antibody against rabbit IgG (goat), which is covered with striped wells of the microplate. The concentration of bradykinin is determined by the enzymatic activity of peroxidase labeled bradykinin associated with antibradykinin antibody.

Alternatively, prekallikrein and N-kininogen can be added in equimolar amounts to the monolayer of endothelial cells and preincubator for 1 hour at 37°C. After that add NVR, and consistently, about 10 minuts the mi intervals for approximately 1 hour, starting after about 5 min after addition of HBP, samples are extracted and determined by the release of bradykinin using the method described above.

In another aspect of prekallikrein and N-kininogen added in equimolar amounts to the solution of plasma containing ACE inhibitor (angiotensinase enzyme), and incubated for 1 hour at 37°C. After that add NVR, and consistently, with approximately 10-minute intervals for approximately 1 hour starting after about 5 min after addition of HBP, samples are extracted and determine the release of bradykinin using the method described above.

METHODS of TREATMENT

The composition according to the invention contains an aqueous solution. Preferably, this solution was physiologically acceptable, so that when it is added for delivery to the patient desired design this solution did not cause any adverse effect on the balance of electrolytes and volumetric balance of the patient. Thus, the aqueous medium for the composition may include, for example, normal saline solution (0.9% NaCI, 0.15 M), pH 7-7,4, or other pharmaceutically acceptable salt. Useful solutions can be prepared by any methods well known in the pharmaceutical field, described, for example, in "Remington's harmaceutical Sciences", (Gennaro, A., ed.). Mack Pub., 1990.

The concentration of HBP antagonist may vary widely, i.e. less than about 0.5%, for example, from 1%to 15-20% by weight. A single dose of the composition can typically contain from about 10 mg to 1 g of HBP antagonist. A therapeutically effective dose can be determined by either in vitro techniques or methods in vivo.

The HBP antagonist is assigned topically, subcutaneously or by intravenous injection. Dose prescribed by the attending physician in accordance with a specific state and consideration of specific patient being treated. Carefully selected dosage and frequency of administration and are in accordance with the parameters determined by the attending physician during the examination. The preferred route of administration may be, for example, intraperitoneal injection. Intravenous intraperitoneal injection of HBP antagonist can be administered every 24 h in the range from 0.1 to 100 mg, in particular from 0.5 to 50 mg, in particular from 1 to 25 mg per kg of body weight. This dose can be administered in 1-4 reception 24 hour or to enter the continuously through the catheter. Alternatively, the injection can be produced by continuous intravenous infusion during surgery, and then within 1-4 days after the operation.

The HBP antagonist will be administered only to those patients who produce NVR that is associated with what antagonista HBP. To determine is produced if the patient such NVR, platelets peripheral blood of the patient is necessary to select NVR using the methods described above. The binding of HBP with HBP antagonist (e.g., monoclonal antibody) or the effect of HBP on the permeability of endothelial cells can be estimated using the methods described above and compared with the effect of native HBP. Alternatively, the effect of HBP on airway endothelial cells or fibroblasts or on the aggregation of monocytes in the presence or in the absence of HBP antagonist can also be determined using the methods described in WO 93/05396. Reduced the extent of cell shortening or aggregation points to the interaction between HBP and HBP antagonist. The test set, which can be used to determine, is produced whether the patient such NVR, includes (a) HBP antagonist; (b) HBP or cells producing HBP, and (C) a substance, tissue, cells or their components, which interact with HBP.

In a particular aspect of the NVR or cells or tissues, producing HBP, are from the body of the patient. NVR or cells or tissues, producing the specified HBP, are incubated with endothelial cells, and the effect on the permeability determined in the presence or in the absence of monoclonal antibody, which binds on ENISA least one epitope NVR, moreover, the specified epitope associated with prekallikrein-N-kininogen complex and activates the release of bradykinin. This effect is comparable with the effect of native HBP person. In a specific aspect of native HBP person may be recombinant HBP, which is associated with such a monoclonal antibody. Alternatively, NVR or cells or tissues, producing HBP, are incubated in the presence or in the absence of prekallikrein-N-kininogenase complex and in the presence of monoclonal antibodies. Then determine the effect of monoclonal antibodies on the release of bradykinin. Reduced release of bradykinin in the presence of antibodies indicates products patient HBP, which binds with the antibody. In such case, the test set will contain (a) a monoclonal antibody that binds at least one epitope on the NVR, and the specified epitope associated with prekallikrein-N-kininogen complex and activates the release of bradykinin; (b) native human HBP and (C) prekallikrein-N-kininogenase complex, associated with a solid carrier.

EXAMPLES

Example 1

Activated neutrophils trigger changes in the function of endothelial barrier by signalizovania through B2-integrins

MATERIALS AND METHODS

Reagents

Medium 199, RPMI-1640 (containing L-glutamine), AMB the optional bovine serum (FBS), trypsin-EDTA, phosphate buffered saline (PBS) and balanced solution Hanks saline solution (HBSS) were purchased from Life Technologies (Gaithersburg, MD, USA). Gelatin, collagenase, penicillin, streptomycin, catalase, bovine serum albumin (BSA), N-formyl-methionine-leucyl-phenylalanine (fMLP), a blue dye Evans, hexadecyl-trimethylammonium bromide and tetramethylbenzidine were purchased at Sigma Chemical Co. (St. Louis/ MO/ USA). N in E. Merck (Darmstadt, Germany). Biomatrix I was purchased in Biomedical Technologies Inc. (Stoughton, MA, USA), a Dextran 70 (Macrodex) and Ficoll-Paque in Pharmacia Biotech AB (Uppsala, Sweden). The main culture medium consisted of a mixture of 1:1 M-199 and RPMI-1640 enriched with 20% V / V heat inactivated FBS, penicillin (100 u/ml) and streptomycin (100 µg/ml). Monoclonal antibody IB4 against the General β-chain (CD18) β2-integrins was provided by Dr. Claes Lundberg, Pharmacia &Up John (Uppsala, Sweden) with kind permission of Dr. Samuel D. Wright, Rockefeller University, mAb 60.1, recognizing αm-chain integrin (CD11b), was provided by Dr. Manuel Patarroyo, Karolinska Institutet, mAb DREG 200 (anti-L-selectin) gift of Dr. Eugene Butcher, Stanford University, mAb SK11 (anti-L-selectin) was obtained in a Becton Dickinson (San Jose, CA) and mAb G44-26 (aHTH-CD44) was obtained in PharMingen (San Diego, CA). F(ab')2-fragment of goat antibodies against mouse IgG was obtained from Jackson measurement Research Laboratories, Inc. (West Grove, PA), FITC-conjugated F(ab')2-fragment of rabbit antibodies against the murine IgG was obtained from Dako A/S (Glostrup, Denmark).

Endothelial cells

Endothelial cells of the umbilical vein of a person (HUVEC) and endothelial cells of the aorta bull (VAS) were isolated and cultured as described above (Gautam et al. 1998, Brit. J. Pharm. 125:1109-1114). HUVEC or WAES in the first to fifth passages were removed by brief (2 min) treatment with trypsin-EDTA (0.25% trypsin/0.01 % of EDTA) and transferred or inorganic Anopore membrane with pore size of 0.2 μm or polycarbonate filters with a pore size of 3.0 μm (Tissue Culture inserts, 10 mm; NUNC, Roskilde, Denmark). To accelerate cell differentiation and gain of attachment of endothelial cells (EC) filters pre-treated with 50 μl of Biomatrix I (167 μg/ml) and was dried in the air. EC were sown at a density of 2×105cells on the filter and incubated in culture medium at 37°C in humidified atmosphere of 5% CO2in the air. EC were cultured to education confluently of monolayers that were monitored daily by microscopic observation and measurement of the electrical resistance of the monolayer (Gautam et al. 1998, Brit. J. Pharm. 125:1109-1114).

Measurement of barrier function EK

To establish induced stimulation of permeability changes EK used two different approaches. To measure transendothelial electrical resistance (TEER) of the filter insert with the EC transferred into the chamber for measuring the electricity is resistance (see Gautam et al. 1998, Brit. J. Pharm. 125:1109-1114). Camera (bottom/lit compartment) with inserted filter (upper/lit compartment was filled, respectively, 2 ml and 400 µl of culture medium, placed in the incubator for cell culture. Transendothelial electrical resistance of the EC monolayer was measured at 37°With each compartment with electrodes, are clearly defined in relation to each other and to the monolayer. The electrical resistance of EC monolayers was obtained by subtracting the resistance of the corresponding intact filter coated with a layer of Biomatrix, measured before sowing its endothelial cells.

In independent experiments we used albumin conjugated with blue dye Evans (EVA), as a marker of macromolecular permeability of monolayers of EC. Before stimulation of EC culture medium in the upper compartment was changed to medium containing EVA (culture medium containing 4% BSA, mixed with blue dye Evans at a final concentration of 0.67 mg/ml). The concentration of EVE in liquid samples of the upper and lower compartments was determined spectrophotometrically by measuring the absorption at 620 nm (Titertek Multiskan MCC; Flow Laboratories, Solna, Sweden). Clearance of albumin was calculated in accordance with the relation: V1=A2×V2×1/A1, where V1 is the volume of the clearance (i.e. theoretical volume lit the th environment, purified from serum albumin by diffusion to abdominalgia the compartment), V2 - abdominally volume, and A1 and A2 - absorption lighted and unlighted environment, respectively. Base clearance in the absence of any stimulus, the average of 0.08±of 0.03 ál/min, subtracted from the amount of clearance received in response to the corresponding specific stimulus.

Obtaining and quantifying PMA

PMA person were isolated as described (Gautam et al., 1998, Brit. J. Pharm. 125:1109-1114), enriched in leukocytes, plasma, and resuspendable in culture medium at a concentration of 2-5×107cells/ml the purity PMA is > 98%, and the viability determined by the exception Trypanosoma blue is >95%. Before adding to the EC monolayers, PMA incubated for 30 min in the presence of monoclonal antibodies against cell surface molecules for subsequent cross-linking with secondary mAb (see experimental methods).

In some experiments the expression of CD11b/CD18 on the cell surface PMA before using exacerbated by changes in the temperature of incubation. PMA incubated in the presence of anti-GD18-mb IB4 at 4°C for 10 min, then at 37°C for 10 min, and then again at 4°C for another 10 min, after which cells were twice washed. PMA that are not incubated in presets the following antibodies subjected to the processing of filler instead of antibodies, in full accordance with the same method.

Adhesion PMA EC and transendothelial migration quantitatively evaluated by analyzing PMA-specific enzyme myeloperoxidase (MPO). Briefly, PMA was literally 0.5% of hexadecyltrimethylammonium bromide and centrifuged, then the supernatant was determined spectrophotometrically enzymatic activity as the change in absorbance at 650 nm, which occurs in redox reactions H2O2-tetramethylbenzidine peroxidase-catalyzed under the action of MPO (Suzuki et al., 1983, Anal. Biochem. 132:345-352). The MPO activity of adherent PMA and migrated through the EC layer, respectively, associated with the same total number PMA added to the monolayer of EC.

Experimental methods

Before transfer, the filter was introduced into the chamber to measure resistance (supported at 37°C)the environment in the filter box and the camera was replaced with fresh culture medium (37° (C)enriched with 10 mM Hepes. In some experiments, the EC monolayer before stimulation 10 μg/ml herbimycin And incubated for 15 min at 37°C and washed twice. PMA (2×106containing and not containing monoclonal antibody-related molecules on the cell surface, were added to the upper compartment (PMA:EC=10:1) is within 10 min gave them the opportunity to settle on the EC monolayer. Activation PMA induced either with fMLP (10-7M)added to the lower compartment, or by cross-linking associated with molecules on the cell surface antibodies and F(ab')2-fragments of secondary goat antibodies against mouse IgG. Transendothelial electrical resistance was measured before and every minute after the start of stimulation, until then, until he reached a phase of a plateau value of resistance changes, and then with 5-minute intervals. In those experiments, where he studied the permeability to albumin and migration PMA through the EC monolayer, the medium inserted in the filter contained EVE. After you define the filter insert with a 5-10 minute intervals was transferred into new wells containing fresh medium. All stages of the incubation was carried out at 37°C. At the end of the experiment, the bottom of the hole was centrifuged at HD and 4°C for 20 min, and the supernatant was determined by the content of EVE. The number PMA that migrated through the monolayer was determined by analyzing the activity of MPO in the sediment remaining in the hole. The environment in the filter box was subjected to quantitative determination in her reprecipitate PMA, and the membrane filter with EK removed for quantification of adherent fraction PMA.

In separate experiments, washed PMA, pre-treated anti-the D18-mb IB4 (6 µg 2× 106cells) for 30 min, incubated in culture medium in the presence and in the absence of goat anti-mouse F(ab)2for 10 min at 37°C. PMA besieged by centrifugation at HD for 15 min at room temperature, and cell-free supernatant containing secretions PMA, decantation for use in vivo (see below) or with EC monolayers. In the latter case, the environment in filter with a built-EC was replaced by medium containing secretions PMA, and, as described above, was determined by its effect on TEER and permeability to albumin. In some cases, the medium containing secretions PMA before using subjected to heat incubation (80°C for 15 min).

Determination of intracellular [CA2+] and formation of f-actin in EK

To determine changes in the content of intracellular free CA2+used CA2+-sensitive fluorescent probe, fluo-3/DM (Molecular Probes Europe BV, Leiden, the Netherlands). Confluently EC monolayers on filters were incubated for 30 min at 37°in the presence of fluo-3/DM (3 μm in HBSS containing 2% FCS and 10 mM HEPES), added on the apical and basolateral surface. The monolayers washed three times and incubated in the presence of fresh HBSS in the dark for 20 min at room temperature to complete the hydrolise essential dye. PMA treated with anti-CD18 or anti-L-selective mAb was added to the EC and, as mentioned above, the induced cross-linking of the receptor with secondary mAb. In separate experiments to stimulate EC in the absence of PMA used a medium containing secretions PMA. Changes in the content of intracellular [CA2+]iEC in the presence of these stimuli was measured in continuous registration of the intensity of fluorescence using laser scanning confocal system image (Insight Plus; Meridian Instruments Inc., Okemon, Michigan).

To analyze the formation of f-actin in EC in response to the activation PMA confluently EC monolayers grown on Biomatrix coated striped surfaces were incubated for 15 min at 37°or in the presence of PMA subjected to cross-linking with CD18 antibody or in the presence of medium containing secretions PMA, in full accordance with the procedures described above. The control were EC, inkubirovanie in the presence PMA processed IB4. The monolayers were fixed in 3.7% formaldehyde in PBS for 10 min at room temperature, washed twice and treated with 0.2% Triton X-100 in PBS (1 min at 4°). Cells are washed twice and stained to visualize actin filaments FITC-conjugated phalloidin for 20 min at 37°C. After three additional the leaching endothelial cells were viewed with a laser scanning confocal system image (Insight Plus; Meridian Instruments Inc., Okemon, Michigan).

Experiments in vivo

To study the influence of secretions PMA on the permeability of microvessels in vivo studies were performed study of the microcirculation using intravital microscopy intracellular in the cheek pouch of the hamster. As has been described in detail previously (Raud & Lindbom, 1994, Immunology of the Microcirculation, Academic Press, 1994, chapter 7), the left cheek pouch shot Golden Syrian hamsters were removed and prepared for microscopic observation under conditions of constant hyperfuse bicarbonate buffer while maintaining physiological levels of temperature, pH and pressure. FITC-conjugated dextran (150 Mm 000), injected intravenously (250 mg/kg body weight)was used to visualize changes in vascular permeability in respect of macromolecules. Microvascular system in the cheek pouch of the hamster observed in fluorescent light at low resolution in a microscope (Leitz Orthoplan, and video recorded. After controlling for the videos hyperfuse stopped, and the cheek pouch was placed 1 ml of medium containing secretions from PMA associated with CD18, at 37°C. In parallel experiments used the environment PMA treated with anti-CDlS, not subjected to cross-linking.

RESULTS

PMA (2×105), pre-treated anti-D18-mb (IB4)was added to the filter with built-EK (light surface EC/upper compartment of the chamber for measuring the resistance) and left for 10 minutes Cross-linking of CD18 with secondary goat F(ab)2antimelanoma antibodies caused a significant reduction in TEER, which was shown for 1 min, reaching its maximum (36+13 from control) after 15 min and remained at this level during the whole observation period (figure 1). Neither in the processing of polymorphonuclear leukocytes only one IB4 or when they are processed secondary mAb in the absence of IB4 no change in TEER not occurred.

In additional experiments studied the effect of the ability of cross-linking CD18 on strengthening macromolecular flow and migration PMA through the EC monolayer. PMA, pre-treated saturating concentration IB4, was added to the filter with integrated EC and left for 10 min in a medium containing EVA. Cross-linking of CD18 in a similar manner were induced goat F(ab)2antimelanoma antibodies, and the filter insert containing PMA/EVE, at regular intervals were transferred to new wells containing fresh medium. In the absence of secondary antibody is momentary and constant clearance of albumin, 0,08±μl of 0.03·min-1(average ± SD, n=13), which does not change with time and which exactly matches the rate of spontaneous clearance found in the filters inserts containing the raw PMA. Conversely, activation PMA under the action of the plumage is mestnogo CD18 binding caused an increase in the clearance of albumin, which eventually progressed (figure 1). The net increase in the clearance of albumin was increased from 0.5±0.1 µl during the first five-minute period and reached 20,0±4,5 µl (mean ± SD, n=10) after 60 minutes In contrast to the increasing clearance of albumin, cross-linking of CD18 did not cause the registrable adhesion PMA on EK or transendothelial migration (figure 1). Analysis of the activity of MPO in different compartments within 60 min after cross-linking CD18 showed that 91+6% (average ± SD, n=9) added PMA detected in the medium collected from the upper compartment, whereas no significant activity of MPO was not detected in the cell EC/filter or in the environment of the lower compartment. Direct microscopic observation of transparent filters, washed at the end of the experiment confirmed the absence PMA, adherent to the monolayer of EC.

Monolayers of endothelial cells were labeled CA2+-sensitive fluorophore fluo-3, and induced incentive to change content [CA2+]iin EC was observed by laser scanning microscopy. Adding to the EC monolayer of polymorphonuclear leukocytes treated with anti-CD18, did not cause changes in [CA2+]iin EC. However, cross-linking of CD18 with secondary mAb led to a rapid increase in the content of free [Ca2+]i in the cytosol EC, which reached its peak after about 100 seconds, and then slowly recovered. When cross-linking in the same way, antibodies to L-selectin was not able to induce any changes in [CA2+]iin EC, whereas after stimulation of raw PMA under the effect of fMLP observed CA2+-response is similar to that found in cross-linking of CD18 (cf. Gautam et al., 1998, Brit. J. Pharmacol. 125:1109-1114). A causal relationship between the activity changes of intracellular CA2+and functional response of EC activation PMA evident from experiments in which endothelial cells were pre-treated calcium-chelating agent VARTA AM. This treatment completely inhibited both the increase in the concentration of intracellular free CA2+in EC, and changes in TEER in response to cross-linking of CD18.

In further experiments, the EC monolayers were incubated with PMA, and then staining was performed with FITC-conjugated phalloidin to detect actin filaments. Using confocal laser scanning microscopy showed that, in endothelial cells, inkubiruemykh with IB4-processed PMA in the absence of secondary antibodies, there were a few fibers voltage and thin actin band along the marginal zone cells in the immediate SOP is kosnovenie with the neighboring cell. On the other hand, endothelial cells, inkubirovanie with PMA subjected to cross-linking with CD18, found a significant increase in the content of f-actin, as well as the number and density of fibers voltage.

To study the factor(s)secreted PMA, which regulates the answer, necessarily leading to increased permeability of the EC, PMA (2×106), pre-treated anti-D18-mb IB4 and washed to remove unbound antibodies were incubated for 30 min in the presence of goat F(ab')2slice antimelanoma antibodies, and then centrifuged in a soft mode. Adding cell-free supernatant to the EC monolayer induced a decrease in TEER and increased clearance of albumin, both of these changes in magnitude and time dependence coincided with those in the experiment in which EC was added PMA after cross-linking CD8. The supernatant was also caused changes in [CA2+]iand the content and distribution of f-actin in EC. Moreover, local application of supernatant containing secretions PMA on the cheek pouch of the hamster in vivo caused a rapid leakage of plasma from the postcapillary and small veins. The leakage was observed after 1.5 min after application, and the maximum response was observed after about 5 minutes After laundering fabric bufera the leak gradually abated. Such action on the permeability of the microvessels is lost after heat treatment secret PMA that indicates the sensitivity factor(s)responsible for the increase in their permeability to thermal effects. The overlay on the cheek pouch of the hamster supernatant IB4-processed PMA which is not subjected to cross-syazwani, did not cause any appreciable leakage of plasma.

DISCUSSION

The adhesion of polymorphonuclear leukocytes to the endothelial lining and the recruiting extravascular tissue in acute inflammation depends on the function β2-integrins. Increased vascular permeability for macromolecules is called in close connection with the response of the cells, leading to the emergence of exudation of plasma and the formation of edema. The results from the above experiments prove a causal link between the transmembrane (outside, inside) signalizovania β2-integrins on activated PMA and the ability of these cells to induce barrier dysfunction EC. It is shown that induced by antibody ligation and clustering β2-integrins initiates the process of increasing the permeability of the EC, similar to that observed after stimulation PMA chemoattractant. Such intercellular dialogue takes place in the absence of physical contact PMA with EC, suggesting that the phenomenon of adhesion PMA and structural the ligature as such is not directly related to the signal EC, which mediated increase in permeability. Rather, the binding of the ligand β2-integrin initiates through intracellular signalizovania cascade secretory phenomena in PMA and the release of a chemical mediator that contributes PMA-dependent change in the permeability of EC. Further, it is shown that the vascular permeability changes caused by this path is the result of active dependent tyrosine kinase, answers EK involved in the reorganization of the cytoskeleton and in the reversible formation of intercellular gaps, indicating neoliticheskoy in this respect, the function PMA-dependent factor(s).

For a more detailed study of the mechanisms by which activated PMA can in this process to transmit signals endothelial cells interaction PMA/EC and dependent adhesion engagement (engage) β2-integrins stimulated by cross-linking CD11/CD18. By cross-linking β2-integrins, bypassing induced chemoattractant activation of cells, showed that engage and clustering as such receptors β2-integrins by intracellular signaling starts the process of increasing the permeability of EC. Because this response occurred independently of adhesion PMA on the EQ, the signal transmission between PMA and EK can not is t to be attributed to the interaction of CD11/CD18 with protivorechiyami on the surface of EC, but rather should be achieved by the release of molecules intermediaries from PMA. This mechanism is further supported by the ability to induce hyperproliferate EC of the cell-free supernatant obtained after cross-linking CD18 on PMA in suspension.

Example 2

Identification obtained from neutrophils NVR (NVR) in the signal quality associated with induced neutrophil leakage of plasma from blood vessels

In example 1, adhesion PMA through P2-integrins was preceded by PMA-induced effects on the permeability of endothelial cells, and CA2+dependent transmembrane signaling by activated as a result of adhesion β2-integrin-induced secretion of soluble factor(s)that is directly responsible for the launch of changes in the barrier function of EC.

As described herein in example 2 was determined that a soluble factor is human HBP. In example 2, characterized by the interaction of HBP with phase-contact system.

MATERIALS AND METHODS

Reagents

Medium 199, RPMI-1640 (containing L-glutamine), fetal bovine serum (FBS), trypsin-EDTA, phosphate buffered saline (PBS) and balanced solution Hanks saline solution (HBSS) were purchased from Life Technologies (Gaithersburg, MD, USA). Gelatin, collagenase, penalties who Illin, streptomycin, bovine serum albumin (BSA), a blue dye Evans and FITC-conjugated phalloidin were purchased at Sigma Chemical Co. (St. Louis, MO, USA). Fluo-3/AM in Molecular Probes (Eugene, OR, USA). Biomatrix I was purchased in Biomedical Technologies Inc. (Stoughton, MA, USA), a Dextran 70 (Macrodex) and Ficoll-Paque in Pharmacia Biotech AB (Uppsala, Sweden). The main culture medium consisted of a mixture of 1:1 M-199 and RPMI-1640 enriched with 20% V / V heat inactivated FBS, penicillin (100 u/ml) and streptomycin (100 µg/ml). Monoclonal antibody IB4 against total (5-chain (CD18) (- integrins was provided by Dr. Claes Lundberg/ Pharmacia &Up John (Uppsala, Sweden) with kind permission of Dr. Samuel D. Wright, Rockefeller University. F(ab') 2 fragment goat antibodies against mouse IgG was obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA).

Polyclonal HBP-antibodies were obtained in accordance with the following procedure. New Zealand white rabbits were subcutaneously injected with 50 μg of human heparin-binding protein (HBP), emulsified in complete Freund's adjuvant (FCA), then three booster injections containing 50 μg of human HBP, emulsified in complete Freund's adjuvant (FIA), once in two weeks. Ten days after the last injection the animals were killed by exsanguination, and the serum was collected.

Monoclonal HBP-antibodies were obtained in accordance with the following procedure. The RBF mice were immunized by subcutaneous and is heterogene 20 μg of purified human HBP in FCA, then two injections containing 20 µg of human HBP in FIA. Mouse, have observed a strong response received intravenous booster injection containing 20 mg NVR, and three days later, their spleens were removed. Cells of the spleen was merged with Fox myeloma cell line. Supernatant were subjected to screening for the presence of products anti-HBP-antibodies using HBP-specific ELISA method. Positive cell lines were subjected to screening for binding to the intracellular part of the macrophage line using standard FACS method. Positive lines were cloned and used in both analyses. Monoclonal antibodies were purified using affinity chromatography using protein A.

Endothelial cells

Endothelial cells of the umbilical vein of a person (HUVEC) were isolated from fresh umbilical cords as described in the publication Jaffe et al., 1973, J. Clin. Invest. 52:2745-2756, and endothelial cells of the aorta bull (WAES) allocated in accordance with the method Booyse et al., 1975, Thromb. Diath. Haemmorrh. 24:825-839, with some modifications. Briefly, the lumen of the umbilical vein or aorta bull incubated in the presence of 0.25% solution of collagenase for 10 min, and ukrepivshis endothelial cells were washed with PBS solution. The collected cells were besieged and resuspendable in culture medium, were sown in bottles for culturing tissues and incubi is ovali under standard conditions. The medium was changed every 48 h until the culture had reached the stage of confluentes (3-5 days). In our experiments we used HUVEC or VES from the first to fifth passages. Identification and purity determination of EC was carried out on the basis of morphology, resembling cobblestone, and factor VIII/vWf-antigen (Booyse et al., 1975, Thromb. Diath. Haemmorrh. 24:825-839). Staining for factor VIII/vWf-antigen wore in the cells of homogeneous character.

Sowing on permeable membranes

Cultured EC were detached by brief (2 min) processing trypsin-EDTA (0.25% trypsin/0.01% of EDTA) and placed either on inorganic Anopore membrane with pore size of 0.2 μm or polycarbonate filters with a pore size of 3.0 μm (Tissue Culture inserts, 10 mm; NUNC, Roskilde, Denmark). To accelerate cell differentiation and gain of attachment EK filters were treated with 50 μl of Biomatrix I (167 μg/ml) and left to air dry. EC were sown with a density of 2×105cells on the filter and incubated in culture medium at 37°C in humidified atmosphere of 5% CO2in the air. EC were grown to education confluently of monolayers that were monitored daily by microscopic observation and measurement of the electrical resistance of the monolayer.

Measurement transendothelial electrical resistance (TEER)

To measure the electrical resistance of the monolayer EC filter box(with built-cells) was transferred into the chamber for measuring resistance (ENDOHM-12; World Precision Instruments, Sarasota, FL, USA), modified so that in the process of repeated measurements the electrodes were positioned in relation to each other and to the built-in filter. Camera (lower compartment) and filter insert (upper compartment) filled with, respectively, 2 ml and 400 µl of culture medium, placed in the incubator for cell culture. All measurements were carried out at 37°using equipment installed in the incubator for cell culture. The direct determination of electrical resistance were performed with an ohmmeter (EVOM; World Precision Instruments, Sarasota, FL, USA)connected to the electrodes of the chamber for measuring the electrical resistance. The electrical resistance of each of the individual EC monolayers was obtained by subtracting the resistance of the corresponding intact filter coated with a layer of Biomatrix, measured before sowing its endothelial cells.

Determining the clearance of albumin

Albumin conjugated with blue dye Evans (EVA)was used as a marker of macromolecular permeability of monolayers of EC. Blue dye Evans at a final concentration of 0.67 mg/ml was mixed with culture medium containing 4% BSA. In the experiments, which studied the permeability for albumin, the culture medium from the upper chamber was replaced with the food, containing EVA. The concentration of EVE in liquid samples of the upper and lower compartments was determined spectrophotometrically by measuring the absorption at 620 nm (Titertek Multiskan MCC; Flow Laboratories, Solna, Sweden). Clearance of albumin was calculated in accordance with the relation: V1=A2×V2×1/A1, where V1 is the volume of the clearance (i.e. theoretical volume of the apical medium, purified from serum albumin by diffusion to basolateral the compartment), V2 - basolateral volume, and A1 and A2 absorption apical and basolateral environment, respectively. Base clearance in the absence of any stimulus, the average of 0.08±of 0.01 μl/min, were read from the volume of the clearance received in response to the corresponding specific stimulus.

The selection and activation PMA

Enriched leukocytes plasma obtained from whole human blood by sedimentation in dextran, was carefully layered on Ficoll-Paque and centrifuged at HD within 30 minutes Containing PMA sediment resuspendable and washed twice (HD, 7 min) in ice-cold PBS. PMA resuspendable in culture medium at a concentration of 2-5×106cells/ml the purity PMA is > 98%, and the viability determined by the exception Trypanosoma blue is >95%.

Cross-linking antibody molecules CD18 on the surface of neutrophils was induced as described above. Briefly, purified the TWO (5× 106ml) were incubated in the presence of anti-CD18-mAb IB4, 10 μg/ml in HBSS for 30 minutes During the incubation period, cells were subjected to temperature changes from 4 to 37°to enhance the expression of CD11b/CD18 on the cell surface. Cells were twice washed and subjected to cross-linking of CD18 by adding F(ab')2fragments of goat antibodies against mouse IgG (diluted in the ratio 1:20). PMA besieged by centrifugation at HD for 15 min at room temperature, and cell-free supernatant was collected. In some experiments the cross-linking antibodies to CD18 caused by preincubating PMA in the presence of mAb IB4 added to the monolayers of EC (see below).

Experimental methods

Before transferring the filter inserts into the chamber for measuring resistance (supported at 37°C)culture medium in the insert and in the chamber was replaced with fresh culture medium (37° (C)enriched with 10 mM Hepes. HBP (25-100 μg/ml) or product secretion PMA added either to the upper or to the lower compartment, and every minute, until then, until he reached the phase plateau, measured value changes transendothelial electrical resistance, and then with 5-minute intervals. In separate experiments PMA (2×106), proinsurance in the presence of mAb IB4 (10 μg/ml), was added cornea the compartment (PMA:EC=10:1) and for 10 min gave them the opportunity to settle. Activation PMA induced by cross-linking antibodies to CD18 on the surface of leukocytes by adding F(ab')2fragments of goat antibodies against mouse IgG, diluted in a ratio of 1:20. In those experiments, where he studied the permeability of the monolayer of EC for albumin, the environment in the filter box contained EVE. In the course of the experiment, the filter insert bore with 5-10 minute intervals into new wells containing fresh medium. All stages of the incubation was carried out at 37°C. At the end of the experiment environment from the bottom of the wells were taken and it was determined the content of the EVA.

Determination of intracellular CA2+and distribution of F-actin in EK

To determine changes in the content of intracellular free CA2+used CA2+-sensitive fluorescent probe, fluo-3/AM (Molecular Probes Europe BV, Leiden, the Netherlands). Confluently HUVEC monolayers or BAEC, cultured on filters coated with Biomatrix I, incubated for 30 min at 37°in the presence of a solution of fluo-3/AM (3 μm in HBSS containing 2% PCS and 10 μm HEPES), added on the apical and basolateral surface. Before using the monolayers washed three times and incubated in fresh HBSS in the dark for 20 min at room temperature for complete hydrolysis of its ester dye to its calcium-sensitive free acid is army. EK was stimulated by addition of HBP to their apical surface. Changes in the contents of [CA2+]iin EC in response to stimulation HBP was measured by continuous registration of the intensity of fluorescence using laser scanning confocal system image (Insight Plus; Meridian Instruments Inc., Okemon, Michigan).

For analysis of the distribution of F-actin in EC in response to activation under the action of HBP confluently EC monolayers grown on surfaces coated strips Biomatrix, incubated for 15 min at 37°or in the presence of HBP, or in the presence of only one filler. The monolayers twice washed with culture medium and fixed in 3.7% formaldehyde in PBS for 10 min at room temperature.

EC for permeabilization were treated with 0.2% Triton X-100 in PBS (1 min at 4°C), washed twice and stained to visualize actin filaments FITC-conjugated phalloidin for 20 min at 37°C. After three additional washes with PBS endothelial cells were viewed with a laser scanning confocal microscope.

Western blot analysis

The sample HBP or secret PMA was added to the aliquot of the LTO-buffer and boiled for 5 min Electrophoresis was carried out in gradient (3-10%) of the LTO-polyacrylamide gel (LTOs-SDS page). For analysis of proteins Western blot turns electrophoretic RA who drove nitrocellulose membrane (Bio-Rad Laboratories, Richmond, CA). Additional binding sites were blocked by incubating the nitrocellulose membrane in milk for 2 hours. HBP was determined by the method of Western blotting.

RESULTS

Activated PMA release HBP

PMA in suspension activated by cross-linking of antigen CD18. PMA centrifuged, and the supernatant was collected and ran on LTO-PAG. Method Western blotting using antibodies against NVR recognize the visible band secret PMA corresponding to the 27 KD. In the control IgG rabbit no specific bands were detected.

HBP increases the permeability of monolayers of EC

Add NVR (75 µg/ml) to the upper compartment caused a significant reduction transendothelial resistance, which was shown for 1 min, reaching its maximum (30±3% of control) after 15 min and remained at this level during the whole observation period (figure 2). In response to HBP was also increased EC permeability for macromolecules, resulting in a progressive increase in the clearance of albumin, when the net amount of clearance through the 60-minute period was reached 20,4±and 3.4 μl (figure 2). The answers of EC to stimulation under the action of HBP was strictly dose-dependent, as is apparent from the less pronounced decrease in TEER of continuous exposure at 25 and 50 µg/ml of the BP (figure 2). However, the rate of occurrence of responses is the same for all doses of HBP.

Stimulation of EC under the action of HBP basolateral part of the monolayer (HBP) was added to the lower compartment) caused changes in permeability EC, similar to those invoked by the action NVR from the apical part, but at higher concentrations.

Neutralization NVR weakens induced PMA increased permeability EK

Purified IgG rabbit antisera against NVR used to neutralize the activity of the protein. Add NVR antibodies (50 μg/ml) to the EC monolayer before stimulation NVR (75 µg/ml) significantly reduced the induced changes in TEER and permeability to macromolecules. The change in TEER occurred after a latent period and reached 75±5% of control values (figure 3), indicating approximately 65% inhibition of HBP-induced response. Similarly, leakage of albumin inhibited by about 60% and has achieved a net clearance of the order 8,64±1,74 ál 60 min (figure 3). Treatment of nonspecific rabbit IgG (50 μg/ml) had no effect on HBP-induced decrease in TEER, reaching 31%±5% of control values after 15 minutes

Conducted to determine if antibodies against NVR inhibition and changes in the barrier function of the EC launched activated PMA. With this aim, PM the suspension is activated by cross-linking of antibodies to CD18 leukocyte. The supernatant, obtained after centrifugation, containing secretions PMA (see Materials and methods), were applied to the EC monolayer. In the presence of antibodies against NVR observed a significant suppression of the decrease in TEER and permeability increase of EC in respect of macromolecules, compared with responses to stimulation secretions PMA in the absence of anti-HBP (figure 4). Almost identical effects occurred after induced cross-linking of antibodies to CD18 after adding PMA to the EC monolayer, and therefore secretions PMA directly act on the EC monolayer.

Stimulation HBP increases the level of intracellular free Ca2+in EK

With the aim of determining whether the EC level rise of free CA2+in the cytosol in response to stimulation NVR, used the method of laser scanning confocal microscopy. Add NVR (75 µg/ml) to confluently the HUVEC monolayers or WAAS loaded CA2+-sensitive fluorophore fluo-3/AM, caused a rapid increase in fluorescence intensity, which reached its maximum value after 1 min, and then declined to baseline levels. This mobilization of intracellular CA2+strictly refers to the active EC response to stimulation under the action of HBP.

Stimulation HBP causes reorganization of ICRI the filaments EK

Confluently EC monolayers, inkubirovanie in the presence of HBP (75 µg/ml) or culture medium as such for 15 minutes, stained with FITC-conjugated phalloidin to visualize the fibers was observed in the laser scanning confocal microscope. Compared to untreated EC, which had a low density of F-actin fibers voltage and wherein the density of the peripheral strips along the marginal zone cells, HBP-stimulated EC were found to have significant changes in the content and distribution of F-actin fibers voltage. Increased density covering the cell F-actin fibers voltage and a discontinuity in the peripheral bands indicate the reorganization of the cytoskeleton EC due to stimulation under the action of HBP.

HBP increases vascular permeability in vivo

Local application of HBP (75 µg/ml) in the microvessels of the cheek pouch of the hamster after 2 min led to a significant leakage of plasma from both post and large veins. The maximum response leakage was observed after 7-10 min after application. Then leak gradually abated and completely stopped within a 30-minute period.

Inhibition of bradykinin - and HBP-induced increase in permeability of the EC under the action of monoclonal antibodies MVCS against bradykinin

Bradykinin (100 nm; f is GA) or NVR (75 μg/ml; fig.5b) were introduced in the zero time on the bright surface of EC monolayers, preincubation in the presence of monoclonal antibodies against bradykinin, MVCS (40 μg/ml) (Haasemann et al., 1991, J. Iniiounol. 147:3882-3892). MVC completely prevented the increase in EC permeability caused by bradykinin, and HBP. The specificity of action of mAb was determined on the basis of a lack of inhibition changes in permeability caused by histamine (histamine was injected 15 min after stimulation with bradykinin or NVR), N=6.

Inhibition of HBP-induced increase in permeability of the EC under the action of monoclonal antibodies RCN against plasma kallikrein

NVR (75 µg/ml; filled symbols) or bradykinin (100 nm; hollow symbols) were introduced in the zero time on the bright surface of EC monolayers, preincubation in the presence of monoclonal antibodies mAb PKH4 (40 µg/ml). The results are shown in Fig.6. PKH4 prevented the increase in EC permeability caused by HBP, but not bradykinin. Processing PKH4 not acted on the change in permeability caused by histamine (histamine was injected 15 min after stimulation NVR), N=6.

Inhibition of HBP-induced increase in EC permeability in the processing of peptide NKN

The EC monolayers were incubated in the presence NKN (3 μg/ml) for 30 min, and this effect was caused by the redistribution of svjazanno the surface of the N-kininogen. After washing, in zero time, on the bright surface of the EC monolayer was introduced NVR (75 µg/ml), and then at 15 minute bradykinin (100 nm). NC was attached to the surface of the EC with the help of domain 5 and 6, mainly with the help of domain 5. The minimum amino acid sequence in domain 5, which is required for binding of NC man with the EC presented a 20-amino acid peptide (ICH 20)having the following sequence: KKHGHGHHKKNKGKKNGKH (SEQ ID NO:7) (N-kininogen person 479-498). This peptide inhibits the attachment of the TC to EC (Hasan et al., 1995, J. Biol. Chem. 270:19256-19261 and Herwald et al., 1996, J. Biol. Chem. 271:13040-13047). The results are presented in Fig. 7. Processing EK peptide NCN prevented the increase in EC permeability caused by HBP, but not changes in permeability caused by the subsequent introduction of bradykinin, N=6.

Inhibition of HBP-induced increase in EC permeability in the processing of peptide SDD31

The EC monolayers were incubated in the presence SDD31 (3 μg/ml) for 30 minutes SDD31, also denoted NK, is a peptide derived from domain 6 of the human N-kininogen, NC), which blocks the binding of NC with kallicrein plasma and factor XII (Tait et al, 1986, J. Biol. Chem. 261:15396-15401 and Vogel et al., 1990, J. Biol. Chem. 265:12494-12502). He has the sequence SDDDWIPDIQTDPNGLSFNPISDFPDTTSPK (SEQ ID NO:8) (N-kininogen person 565-595). After washing, in zero time, to the illuminated surface monolayer introduced NVR (75 µg/ml), and then at 15 minutes bradykinin (100 nm). The results are presented on Fig. Processing EK peptide SDD31 largely prevents induced HBP increased permeability of EC. The EC response to direct stimulation by bradykinin after treatment with peptide SDD31 persisted, N=6.

Inhibition of HBP-induced increase in permeability of the EC's handling Aprotinin

NVR (75 µg/ml) was injected at the zero time on the lighted surface of EC monolayers, preincubating in the presence of Aprotinin (100 μg/ml; 30 min). The results are presented in Fig. 9. Treatment with Aprotinin to a large extent prevents induced HBP increased permeability of EC, but did not cause permeability changes induced following injection of bradykinin (100 nm), N=6.

Mutant NVR (G175N) mutation does not possess the ability to bind Aprotinin, although its spatial structure and characteristic distribution of the surface charge is not changed. When testing the model leak this mutant induced the release of bradykinin and increased permeability of EC. However, the addition of Aprotinin did not cause inhibition effect. A similar result was obtained when using NVR pigs with a high degree of homology with human HBP, but not capable of binding Aprotinin due to the presence of R at the bottom of the degenerate ka is eliticism pocket.

Example 3

Two mutant heparin-binding protein

For studies of structure-function relationships residues at the proposed site of binding of the lipid A/LPS and the binding site BPTI (inhibitor of trypsin in the pancreas of the ox) were obtained two mutant NVR person, [R23S,F25E]HBP [G175Q]HBP. Mutation G175Q does not change the protein structure, however, deprives him of the ability to bind BPTI, and mutant mediates only a very limited stimulation of lipopolysaccharide (LPS)-induced release of cytokines from human monocytes. The binding ability of lipid A/LPS comparable to that of native HBP. Mutations R23S.F25E not affect the binding of the lipid A/LPS and BPTI or lipopolysaccharide (LPS)-induced release of cytokines from human monocytes.

MATERIALS AND METHODS

Construction of mutant proteins

DNA polymerase Pwo acquired Boehringer Mannheim, DNA ligase BamH1 and T4 in New England Biolabs, cells spodoptera frugiperda (sf9) and carrying plasmid pVL1393 in Invitrogen, TC100 and SF900-II produced by the company Novo Nordisk A/S, fetal calf serum (FCS) at the company GibcoBRL. All other chemical reagents of high purity are widely available.

Mutant [R23S, F25E]HBP

Mutant [R23S, F25E]HBP constructed using the technique of overlapping extension PCR segments (But, S.N., Hunt, H.D., Horton, R., Pullen, J.K., and Pease, L.R., (1989) Gene (Amst) 77: 51-59). Two partially overlapping mu the new fragment obtained by PCR using oligonucleotides 1+2 and 3+6, and the resulting fragments are connected using oligonucleotides 1+6 (table 1). Amplified cDNA NVR person used as template in the PCR reactions. The resulting cDNA encodes propeptide, consisting of 19 amino acids, part of the Mature protein of 222 amino acids and C-terminal insertion material of the three amino acids. The BamH1 sites included on each side of the mutated cDNA for cloning. The mutated fragment Legerova in the transfer plasmid pVL1393 (Invitrogen). DNA for transfection was obtained with the use of kits Qiagen midiprep. After cleaning the insert sequenced.

Mutant [G175Q]HBP

In General, this mutant is constructed as described above. Two partially overlapping fragment obtained by PCR using oligonucleotides 1+4 and 5+6, and the resulting fragments are connected using oligonucleotides 1+6 (table 1).

Expression and purification

Cells sf9 insect supported in the environment TS, enriched with 10% FCS. Before infection, the medium was replaced with serum-free medium SF900-II. The expression of both mutants in serum-free medium and purification of mutants from the culture supernatant produced by the method described above for native HBP person (Rasmussen, P.W., Bjorn, S., Hastrup, S., Nielsen, P.F., Norris, K., Thim, L., Wiberg, F.C., and Flodgaard, H., (1996) FEBS Lett. 390:109-112).

Analysis indutsirovanno what about the LPS release of IL-6 from monocytes

The monocytes were isolated and processed in accordance with methods (Rasmussen, P.W., Bjorn, S., Hastrup, S., Nielsen, P.F., Norris, K., Thim, L., Wiberg, F.C., and Flodgaard, H., (1996) FEBS Lett. 390:109-112, except that the medium was removed with 1% non-essential amino acids.

The analysis of binding3H-LPS with HBP

Affinity for lipopolysaccharide (3H-LPS) was measured using the technique of scintillation analysis of micro gaps (scintillation proximity assay technology) (SPA)described in the literature (Linde, V., Bjorn, S., Kastrup, J.S., and Flodgaard, H., 2000, Biotechniques 28:218-222). All samples were presented in three parallel samples.

The analysis of binding125I-BPTI with HBP

The affinity in respect of BPTI were determined using the technique of SPA, basically as described for analysis of LPS (Linde, V., Bjorn, S., Kastrup, J.S., and Fiodgaard, H., 2000, Biotechniques 28:218-222), with several modifications. So,3H-LPS was replaced by125I-BPTI (obtained at the company Novo Nordisk A/S), and the buffer was replaced with PBS buffer (20 mm phosphate, pH 7.4, and 150 mm NaCl)containing 0.05% of BSA and 0.05% Triton X-100. The relative affinity of HBP and mutants was determined by the method of competitive analysis using a fixed, increasing concentrations of unlabeled BPTI.

RESULTS

Biological properties

The biological activity of the two mutants was compared with native HBP in relation to their ability to enhance, dose-dependent way, LPS-induced release of I1-6 of monocytopenia in vitro. The affinity against two LPS mutants and HBP was measured by the method of scintillation analysis of micro gaps (SPA) (Linde, V., Bjorn, S., Kastrup, J.S., and Flodgaard, H., 2000, Biotechniques 28:218-222). Both specimens were associated3H-LPS satisfying way, with similar apparent Kand(approximately 0.7 μg/ml), as in the case of native HBP (Fig. 11a). Moreover, for both mutants and native HBP was measured affinity in respect of BPTI similar method of scintillation analysis of micro gaps, where as a ligand for NVR used125I-BPTI. Mutant [R23S,F25E]HBP, as well as native HBP, bound BPTI, whereas no binding of the mutant [G175Q]HBP were not detected (Fig. 11b). Using competitive analysis using a fixed, increasing concentrations of unlabeled BPTI showed that125I-BPTI associated with HBP and [R23S, F25E]NVR with seemingly similar values IC50approximately components of 7 nm. As shown in figs, [R23S, F25E]HBP is more apparent ability of binding compared to the NVR. Perhaps this difference is due to the ability [R23S, F25E]NVR to link various forms used iodized BPTI. However, this increased the ability of binding does not affect the affinity of125I-BPTI. In addition, in the experiments of competitive analysis linking studied the ability of3H-LPS to oust125I-BPTI associated with native HBP and Vice versa: competition for binding 125I-BPTI was determined by adding up to 5 µg/ml of unlabeled LPS, and competition for binding3H-LPS by adding up to 50 mm unlabeled BPTI. In both situations, the effects of inhibition were not observed, which indicates that the binding sites LPS and BPTI localized in different parts of the molecule HBP.

Data analysis of biological activity in vitro showed that [G175Q1HBP, unlike [R23S, F25E]HBP and native HBP, is the mediator only very limited stimulation induced by lipopolysaccharide (LPS) release of cytokines from human monocytes (Fig. 10).

Volume described above and claimed invention is not limited to the listed specific aspects, since these aspects are presented only to illustrate some applications of the present invention. Any equivalent application are included in the scope of this invention. In fact, experts in this area it will be easy to make some modifications to this invention, in addition to the described and shown here in the foregoing description. Such modifications are also included in the scope of the attached claims.

In the description of a number of different links to publications, which are in full are included in this invention.

The list of sequences is given in the context of the e description.

1. The use of anti-HBP-antibody of a mammal for the preparation of a medicinal product for preventing or treating disorders associated with the release of bradykinin in a mammal, where the specified mammal produces HBP, which binds with anti-HBP-antibody, and the behavior is selected from the group consisting of a syndrome of systemic inflammatory response, ischemic reperfusion injury, respiratory distress syndrome in adults, anaphylaxis and allograft rejection.

2. The use according to claim 1, where anti-HBP-antibody present in an amount of about 0.1 - 100 mg/kg of body weight.

3. The use according to claim 1, where anti-HBP-antibody present in an amount of about 0.5 - 50 mg/kg of body weight.

4. The use according to claim 1, where anti-HBP-antibody present in an amount of about 1 to 25 mg/kg of body weight.

5. The use according to claim 1, where the aforementioned antibody is a monoclonal antibody, preferably a monoclonal antibody of the person.

6. The use according to claim 5, where the specified mammal is a sick man, and the specified NVR is a NVR person.

7 a Method for the identification of monoclonal antibodies which binds at least one epitope on native HBP, where the specified epitope associated with prekallikrein-N-kininogen complex and activates the release of bradykinin, and this method comprises (a) culturing endothelial cells in the presence of HBP and in the presence or in the absence of the substance, which is supposed to be an antagonist, and (b) identification of any action specified substances on the permeability of endothelial cells, with reduced permeability of these endothelial cells compared with the permeability of these cells when they are incubated in the presence of HBP, but in the absence of specified substances, suggests that said monoclonal antibody binds at least one epitope on the specified HBP.

8. The method according to claim 7, where the specified native HBP is a NVR person.

9. A method of identifying a monoclonal antibody that binds at least one epitope on native HBP, where the specified epitope associated with prekallikrein-N-kininogen complex and activates the release of bradykinin, while this method involves (a) incubating prekallikrein-N-kininogenase complex in the presence of HBP and in the presence or in the absence of monoclonal antibodies, which may be the positive binds at least one epitope on the specified NVR, and (b) identification of any action specified antibodies to the release of bradykinin, and reduced the release of bradykinin suggests that this antibody binds at least one epitope on the specified HBP.

10. The method according to claim 9, where prekallikrein-N-kininogenase complex attached to the solid substrate.

11. The method according to claim 9, which further includes incubating the mixture of stage (a) in the presence of endothelial cells.

12. The method according to claim 9, which further includes incubating the mixture of stage (a) in the presence of platelets.

13. The method according to claim 9, where the specified native HBP is a native HBP person.

14. The method according to claim 9, where the release of bradykinin detected using immunological analysis.

15. The test set also produces whether the mammal HBP, which is associated with HBP antagonist comprising (a) an antibody against HBP; (b) HBP or cells producing HBP; and (C) a substance, tissue, cells or component that interacts with the NVR.

16. The test set indicated in paragraph 15, where the NVR is labeled.

17. The test set indicated in paragraph 15 contains a substance which interacts with the NVR.

18. The test set at 17, where the substance is N-kininogen.

19. The test set at 17, where the substance is labeled./p>

20. The test set at 17, which further comprises granules.

21. The way to determine is produced by the mammal HBP, which binds to a monoclonal antibody that binds at least one epitope on native HBP, where the specified epitope associated with prekallikrein-N-kininogen complex and activates the release of bradykinin, thus this method includes (a) the allocation of HBP or cells or tissues, producing HBP, the mammal; (b) culturing the specified NVR, or cells or tissues, producing HBP, with endothelial cells in the presence or absence of indicated antibodies; and (C) identify any the actions specified antibodies on the permeability of endothelial cells, and reduced permeability suggests that the HBP is associated with the indicated antibody.

22. The method according to item 21, where the mammal is a patient man.

23. The method according to item 21, where native HBP is a NVR person.

24. The method of determining produces whether the mammal HBP, which binds to a monoclonal antibody that binds at least one epitope on native HBP, where the specified epitope associated with prekallikrein-N-kininogen complex and activates the release of bradykinin, enabling the th to (a) the allocation of HBP, or cells, or tissues, producing HBP, the mammal; (b) incubating the specified NVR, or cells or tissues, producing HBP, with prekallikrein-N-kininogenase complex; and (C) identification of any action specified NVR on the release of bradykinin from prekallikrein-N-kininogenase complex, and reduced the release of bradykinin suggests that the HBP is associated with the indicated antibody.

25. The test set also produces whether a mammal, in particular a sick man, HBP, which binds to a monoclonal antibody that binds at least one epitope on native HBP, where the specified set includes (a) a monoclonal antibody which binds to the epitope HBP; (b) native HBP person; and (C) prekallikrein-N-kininogenase complex attached to the solid substrate.

Priority items:

29.04.1999 - PP. 1-7;

01.10.1999 - PP. 8-25.



 

Same patents:

FIELD: veterinary science.

SUBSTANCE: the suggested preparation for parturient activity, prophylaxis and therapy of afterbirth delay and puerperal endometritis in cows includes 0.05 g carbacholine, 70 g ichthyol, 5 g crystalline glucose and 1 g phenol/1000 ml distilled water. The method for treating acute puerperal endometritis in cows deals with injection of the above-mentioned preparation at the quantity of 20 ml preheated up to animal's body temperature deeply into gluteal muscles 4-8 times at interval of 24-28 h. The preparation suggested is considered to be of high curative and prophylactic efficiency.

EFFECT: higher efficiency.

2 cl

FIELD: organic chemistry and drugs.

SUBSTANCE: New class of compounds of general formula 1, where R has formula 2 or 3; other residues are as described in claim of invention is disclosed. Said compounds are interleikyn-1β converting enzyme (ICE) inhibitors and have specific structural and physicochemical properties. Invention also relates to pharmaceutical composition containing said compounds. Compounds and composition of present invention are particularly useful in ICE activity inhibition and thereby can be used as drug for treating of diseases mediated by IL-1, apoptosis, IGIF and IFN-γ, as well as inflammations, autoimmune diseases, bone-destructive disorder, infections, disorder associated with cell proliferation, degenerative and necrotic disorders. Uses of claimed compounds and compositions as well as methods for production of N-acylamino compounds also are disclosed.

EFFECT: effective interleikyn-1beta converting enzyme inhibitors.

64 cl, 35 ex, 35 tbl, 21 dwg

FIELD: organic chemistry, pharmaceutical compositions.

SUBSTANCE: 5-aryl-1H-1,2,4-triazole derivatives of general formula I

, pharmaceutically acceptable salts thereof or pharmaceutical composition containing the same are described. In formula R1 is C1-C6-alkyl, C1-C6-haloalkyl or phenyl; R2 is C3-C8-cycloalkyl; phenyl optionally substituted with one or more substituents selected from C1-C4-alkyl; halogen, hydroxyl, C1-C4-alkoxy, nitro, di-(C1-C4)-alkylamino, C1-C4-alkylsulphonyl, C1-C4- alkylsulphonylamino, and methylenedioxy; phenyl-(C1-C4)-alkyl, wherein phenyl is substituted with C1-C4-alkoxy; or pyridil. New compounds are effective and selective cyclooxygenase-2 (COX-2) inhibitors and useful in treatment of inflammations.

EFFECT: new compounds for inflammation treatment.

10 cl, 36 ex, 1 tbl

FIELD: organic chemistry, heterocyclic compounds, biochemistry.

SUBSTANCE: invention relates to new compounds - purine derivatives of the general formula (I): in free form or salt wherein X means oxygen or sulfur atom or group NR5; R1 means alkyl, alkenyl, cycloalkyl, benzocycloalkyl, cycloalkylalkyl or aralkyl group that can be substituted optionally with hydroxy-, carboxy-group or alkoxycarbonyl; or if X means NR5 then R1 can mean alternatively heterocyclic group taken among benzylpiperidyl or the formula: ; or group of the formula (II): ; R2 means hydrogen atom, alkyl or alkoxy-group; R3 means hydrogen atom, alkoxy-, carboxy-group, carboxyalkyl, alkoxycarbonyl, -N(R9)R10, (C1-C4)-alkylene-SO2N(R11)R12 or -CON(R13)R14; or if two substitutes R2 and R3 are joined to adjacent carbon atoms in indicated benzene ring then in common with carbon atoms to which they are joined they mean heterocyclic group comprising 5-10 ring atoms among them one or two atoms mean heteroatoms taken among nitrogen, oxygen and sulfur atom; R4 means hydrogen atom, alkoxy-, carboxy-group, carboxyalkyl, -SO2N(R11)R12, -N(R9)R10 or -CON(R13)R14; or if two substitutes R3 and R4 are joined to adjacent carbon atoms in indicated benzene ring then in common with carbon atoms to which they are joined they mean heterocyclic group comprising 5-6 ring atoms among them one or two atoms mean heteroatoms taken among nitrogen, oxygen or sulfur atom; R5 means hydrogen atom or alkyl; R6, R7 and R8 mean hydrogen atom, or one of these radicals means -SO2NH2, -N(CH3)COCH3, -CONH2 and two others mean hydrogen atom; R9 means hydrogen atom or alkyl; R10 means hydrogen atom, -COR15 wherein R15 means alkyl, alkoxy-group; or R9 and R10 in common with nitrogen atom to which they are joined mean heterocyclic group comprising 5 or 6 ring atoms among them one or two atoms mean heteroatoms taken among nitrogen and oxygen atom; R11 means hydrogen atom or alkyl; R12 means hydrogen atom, alkyl, hydroxyalkyl, carboxyalkyl or alkoxycarbonylalkyl; or R11 and R12 in common with nitrogen atom to which they are joined mean heterocyclic group comprising 5 or 6 ring atoms among them one or two atoms mean heteroatoms taken among nitrogen and oxygen atom; R13 and R14 each and independently of one another means hydrogen atom or alkyl with exception of 2-(para-n-butylanilino)-6-methoxypurine, 2-(para-n-butylanilino)-6-(methylthio)purine, 2,6-di-(phenylamino)-purine, 2,6-di-(para-tolylamino)-purine and 2-(para-tolylamino)-6-(phenylamino)-purine.

EFFECT: valuable biochemical properties of compounds.

11 cl, 4 tbl, 221 ex

FIELD: organic chemistry, medicine, pharmacy.

SUBSTANCE: invention relates to a method for preparing a pharmaceutical composition eliciting anti-inflammatory and analgetic activity for oral administration and containing compound of the formula (I): A-X1-NO2 wherein A and X1 are given in cl. 1 of the invention claim and representing in the amorphous state or partially amorphous state. Method involves the following steps: stirring compound of the formula (I) with at least one filling agent that is able to confer the amorphous state to obtained mixture wherein this filling agent is taken among group consisting of (C5-C6)-polyalcohols, mono- and disaccharides and their derivatives, oligosaccharides comprising from 3 to 10 monosaccharide units, polysaccharides, their derivatives involving their salts, cyclodextrins and their derivatives, noncyclic derivatives of β-cyclodextrin, polymers and copolymers based on vinyl monomeric links, and/or comprising the carboxyl function, or methacrylic monomers wherein the mass ratio between amount of compound of the formula (I) and the amount of at least one filling agent is in the range = (1:20)-(1:0.5), and providing the amorphous state of obtained mixture by combined grinding, stirring, spraying drying and lyophilization. Also, invention relates to a pharmaceutical composition eliciting an anti-inflammatory and analgetic activity. Invention provides preparing a pharmaceutical composition for oral administration and medicinal agents based on thereof for treatment of inflammatory diseases.

EFFECT: improved preparing method, valuable medicinal properties of composition.

10 cl, 1 tbl, 10 ex

FIELD: medicine, phytotherapy, pharmaceutical industry and technology, pharmacy.

SUBSTANCE: invention relates to a method for preparing agent eliciting immunocorrecting and anti-inflammatory activity. Method for preparing the phytopreparation eliciting immunocorrecting and anti-inflammatory activity involves milling common horse radish fresh roots, extraction of raw by maceration method in the definite ratio of ratio raw : extractant for definite time at room temperature, at periodic stirring, clarification of extract under definite conditions and filtration. Method provides preparing the phytopreparation from common horse radish roots for carrying out pharmacotherapy of immune deficient states and inflammatory diseases.

EFFECT: improved preparing method, valuable medicinal properties of preparation.

4 tbl, 3 ex

FIELD: medicine, pharmacology, pharmacy.

SUBSTANCE: invention relates to the agent comprising the following components: lidazum (16-32 U), proserinum 0.05% solution, 0.00025-0.0005 g; methylprednisolone succinate sodium, 0.02-0.04 g; lidocaine 10% solution, 0.05-0.1 g, and glucose 40% solution, 3-4 ml. Also, invention relates to a method for administration of agent and a method for treatment of inflammatory diseases. Invention provides expanding assortment of medicinal agents and improving the regional transport of medicinal preparations.

EFFECT: improved and valuable properties of agent.

6 cl, 5 ex

FIELD: pharmaceutical industry.

SUBSTANCE: rectal- and vaginal-administration suppositories contain 1,3-diethylbenzimidazolium triiodide as active principal, polyvinylpyrrolidone as solubilizer and stabilizer, and lipophilic base with specified proportions of components.

EFFECT: extended therapeutical activity and reduced occurrence of side effects.

4 cl, 2 ex

FIELD: medicine.

SUBSTANCE: the present innovation deals with curative ointments of antiphlogistic and wound-healing action and could be applied for treating hemorrhoid, burns, bruises, fractures, wounds, chronic bronchitis, polyarthritis, periarthritis, radiculitis, trophic ulcer and psoriasis. Three variants of ointments have been suggested. They contain oily extract of plant components and, also, honey, mumiye, propolis and bear oil. This innovation enables to widen the assortment of ointments of antiphlogistic and wound-healing and the range of curative action upon a body.

EFFECT: higher efficiency of application.

3 cl, 11 ex

Therapeutic agents // 2246294

FIELD: medicine, pharmacy.

SUBSTANCE: invention describes composition of a pressed tablet comprising multiple of hardened melted granules of nonsteroid anti-inflammatory preparation (NSAID) with a melting point in the range 30-300oC and comprising a loosening agent dispersed uniformly in it. Granules comprise a continuum phase of indicated nonsteroid anti-inflammatory preparation and the table composition comprises additionally silicon dioxide in the amount 0.05-5.0% of composition mass. Preferably, the composition comprises also a nongranulated component containing silicon dioxide and excipient. The preferable NSAID represents ibuprofen that has a melting point in the range 75-77oC. Method provides preparing tablet showing useful industrial properties and ability for dissolving.

EFFECT: improved preparing method, valuable pharmaceutical properties of agent.

34 cl, 16 tbl, 64 ex

FIELD: pharmaceutics.

SUBSTANCE: the present innovation deals with cryoprotective ointment containing recombinant interferon-α2. The suggested cryoprotective ointment contains recombinant interferon-α2, glycerol, polyethylene glycol 300-6000, polyglucin, buffered 0.02%-Trilon B solution at pH of 5.5-7.0 and ointment foundation at a certain content of components per 1.0 g ointment. Additionally, cryoprotective ointment could contain glycine 3,7-bis(dimethylamino)phenothiazonium chloride, dry immunoglobulin preparation or dry immunoglobulin preparation for enteral application. Ointment foundation of cryoprotective ointment could contain water-free lanolin, Vaseline and Vaseline oil, at the following ratio of components: 2.5;3.5:1 - 6.5:0.5:1. The innovation provides maximal safety of recombinant interferon-α2 activity in cryoprotective ointment at multiple alteration of positive and negative environmental temperature and at keeping cryoprotective ointment under these conditions.

EFFECT: higher efficiency of application.

8 cl, 8 ex

FIELD: medicine and immunology, in particular treatment and prevention immunodeficiency conditions and diseases associated with bacterial or viral aggression.

SUBSTANCE: claimed method includes administration to a patient immunoglobulin drug (e.g., pharmaceutical composition containing 6-12 % of specific heterologous secreted immunoglobulin A, isolated from milk or foremilk of immunized ungulates). Administration is performed parenterally wherein single dose is at least 10 IU/kg of patient weight for treatment or at least 5 IU/kg for prophylaxis; or perorally in dose of 0.2-0.5 g and/or topically one-two times per day for 1-5 days. Method of present invention makes it possible to decrease dose of administrating immunoglobulin due to prolonged retention of its high titers in body fluids.

EFFECT: enlarged range of application and assortment of immunoglobulin drugs.

4 cl, 5 ex

FIELD: microbiology and immunology, in particular immunodiagnosis.

SUBSTANCE: atypical strain of melioidose Burkholderia pseudomallei-111-6-1 with altered phenotype defected with respect to synthesis of 8 antigen and acting as immunosuppressor is used as antigen for animal immunization. Immune serum is obtained after 2 immunization cycles of animal-producer with titer in gel immunodiffusion reaction not less than 1:128.

EFFECT: immune serum with increased specific activity.

2 tbl, 2 ex

FIELD: immunology.

SUBSTANCE: the innovation deals with new immunogenic conjugates of beta-propionamide-bound polysaccharide and N-propionamide-bound oligosaccharide with protein, and the method to obtain these conjugates has been suggested, as well. Conjugates should be applied to obtain vaccines against infectious diseases and cancer that enables to broaden the number of preparations applied in treating the above-mentioned diseases.

EFFECT: higher efficiency.

1 dwg, 2 ex, 8 tbl

FIELD: medicine, pharmacy.

SUBSTANCE: invention relates to a composition eliciting an antibacterial effect. Composition comprises hydrophilic conglomerate of immunoglobulins consortium adsorbed with polyethylene glycol 4000-6000 and a special additive taken among the following substances: glycine, glucose, maltose, sodium chloride taken in the definite ratio of components. Invention provides sufficient desorption of biologically active substances in resuspending the composition eliciting an antibacterial effect and comprising consortium of immunoglobulins.

EFFECT: valuable medicinal properties of composition.

5 ex

FIELD: medicine, pharmaceutical industry and technology, pharmacy.

SUBSTANCE: invention relates to a composition eliciting an antiviral effect. The composition comprises hydrophilic conglomerate of immunoglobulins consortium adsorbed with polyethylene glycol 4000-6000, recombinant interferon-α2 and a special additive taken among the following substances: glycine, glucose, maltose, sodium chloride taken in the definite ratio of components. Invention provides elevating solubility of composition eliciting an antiviral effect and enhanced release of biologically active substances to solution.

EFFECT: valuable medicinal properties of composition.

5 ex

FIELD: genetic engineering, immunology, medicine.

SUBSTANCE: invention relates to new antibodies directed against antigenic complex CD3 and can be used in therapeutic aims. Antibody IgG elicits the affinity binding with respect to antigenic complex CD3 wherein heavy chain comprises skeleton of the human variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 2, 4 and 6 and their corresponding conservatively modified variants. Light chain comprises skeleton of the rodent variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 8, 10 and 12 and their corresponding conservatively modified variants. Antibody is prepared by culturing procaryotic or eucaryotic cell co-transformed with vector comprising recombinant nucleic acid that encodes antibody light chain and vector comprising recombinant nucleic acid that encodes antibody heavy chain. Antibody is administrated in the patient suffering with malignant tumor or needing in immunosuppression in the effective dose. Invention provides preparing chimeric antibodies against CD3 that are produced by expression systems of procaryotic and eucaryotic cells with the enhanced yield.

EFFECT: improved preparing methods, valuable medicinal properties of antibody.

33 cl, 5 dwg, 1 ex

The invention relates to biotechnology, in particular to the immunoglobulin E (IgE), IgE antagonists, anti-IgE antibodies capable of binding to human IgE, and to a method of improving polypeptides, including anti-IgE antibodies

The invention relates to medicine, in particular to the treatment of infectious diseases, and for the treatment of acute viral hepatitis In

The invention relates to the field of medicine, namely biotechnology, diagnostic products, and can be used in the manufacture of medical immunobiological preparations intended for the detection of specific antibodies and antigens in biological substrates

FIELD: pharmaceutical chemistry.

SUBSTANCE: invention relates to (i) essentially crystalline melagatran in the form of hydrate, which is characterized by x-ray diffraction pattern on powder having crystalline peaks with following d values: 21.1, 10.5, 7.6, 7,0, 6.7, 6.4, 6.2, 5.7, 5.4, 5.3, 5.22, 5,19, 5.07, 4.90, 4.75, 4,68, 4.35, 4.19, 4.00, 3.94, 3.85, 3.81, 3.73, 3.70, 3.63, 3.52, 3.39, 3.27, 3,23, 3.12, 3.09, 3.06, 2.75, 2.38, and 2.35 Å and/or water content 4.3%; and (ii) essentially crystalline melagatran in the form of anhydrate, which is characterized by x-ray diffraction pattern on powder having crystalline peaks with following d values: 17.8, 8.9, 8.1, 7.5, 6.9, 6.3, 5.9, 5.6, 5.5, 5.4, 5.3, 5.2, 5.0, 4.71, 4.43, 4.38, 4.33, 4.14, 4.12, 4.05, 3.91, 3.73, 3.61, 3.58, 3.56, 3.47, 3.40, 3.36, 3,28, 3.24, 3.17, 3.09, 3.01, 2.96, 2.83, 2.54, 2.49, 2.41, 2.38, and 2.35 Å. Invention also relates to a method for preparation of indicated form, a method for interconversion of anhydrite form, to use of indicated compounds as pharmaceutical agent, and to preparation of drugs. Pharmaceutical preparation is suitable for treatment of condition, in case of which inhibition of thrombin is needed or desirable. Invention provides a method for treatment of such condition.

EFFECT: increased chemical stability and solid state stability as compared to amorphous forms of melagatran.

14 cl, 4 dwg, 3 tbl, 9 ex

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