MXPA01001321A

MXPA01001321A - Peptide antagonists of zonulin and methods for use of the same

Info

Publication number: MXPA01001321A
Application number: MXPA/A/2001/001321A
Authority: MX
Inventors: Alessio Fasano
Original assignee: University Of Maryland Baltimore
Priority date: 1998-08-03
Filing date: 2001-02-02
Publication date: 2002-07-25

Abstract

Peptide antagonists of zonulin are disclosed, as well as methods for the use of the same. The peptide antagonists bind to the zonula occludens receptor, yet do not physiologically modulate the opening of mammalian tight junctions.

Description

ZONULIN PEPTIDE ANTAGONISTS AND METHODS FOR USING THEMSELVES • The development of the present invention was sponsored by the University of Maryland, Baltimore, Maryland. The invention described herein was supported by the finding of National Institutes of Health (NIH DK-48373). The government has certain rights over it.

FIELD OF THE INVENTION # < The present invention relates to zonulin peptide antagonists, as well as methods for the use thereof. Said peptide antagonists bind to the zonule occlusion receptor, which does not yet physiologically modulate the opening of junctions hermetic in mammals.

BACKGROUND OF THE INVENTION »I. Function and regulation of intestinal tight junctions. The hermetic junctions ("tj") of zonule occlusions (hereinafter "ZO") are the important points of the absorption and secretion epithelium (Madara, J. Clin. Invest., 83: 1089-1094 (1989 ) and Madara, Textbook of Secretory Diarrhea Eds. Lebenthal et al, Chapter 11, pages 125-138 (1990) .As a barrier between apical and basolateral compartments selectively regulate the passive diffusion of water-soluble ions and solutes through the paracellular pathway (Gumbiner, Am. J. Physiol., 253 (Cell Physiol.22): C749-C758 (1987)). This barrier maintains any gradient • generated by the activity of trajectories associated with the transcellular route (Diamond, Physiologístic, 20: 10-18 (1977)) Variations in transepithelial conductance can usually be attributed to changes in the permeability of the paracellular path, since the resistances of enterocyte plasma membranes are relatively high (Madara, supra). The ZO • represents the main barrier in this paracellular trajectory, and the electrical resistance of epithelial tissues seems to depend on a number of strands of transmembrane protein, and its complexity in ZO, as observed through electron microscopy of freeze fracture (Madara et al. al, J. Cell Biol., 101: 2124-2133 (1985)) There is great evidence that ZO, once considering static structures, are actually dynamic and easily adapt to a variety of developments (Magnunson et al, Dev. Biol., 67: 214-224 (1978)).; Revel et al, Cold Spring Harbor Symp. Quant.

Biol., 40: 443-455 (1976) and Schneeberger et al, J. Cell. Sci., 32: 307-324 (1978)), physiological circumstances (Gilula et al, Dev. Biol., 50: 142-168 (1976); Madara et al, J. Membr. Biol., 100: 149-164 (1987), Mazariegos et al, J. Cell Biol., 98: 1865-1877 (1984), and Sardet et al, J. Cell Biol., 80: 96-117 (1979)), and pathological (Milks et al. , J.

Cell Biol., 103: 2729-2738 (1986); Nash et al, Lab. Invest., 59: 531-53 (1988); and Shasby et al, Am. J. Physiol., 255 (Cell Physiol., 24: C781-788 (1988)), The regulatory mechanisms underlining this adaptation are still not fully understood. However, it is evident, in the presence of Ca2 +, the assembly of ZO is the result in cellular interactions that activate a complex cascade of biochemical events that ultimately lead to the formation and modulation of an organized network of elements of ZO, the composition of the which has only been partially characterized (Diamond, Physiologist, 20: 10-18 (1977)). A candidate for the transmembrane protein strands occludin has recently been identified (Furuse et al, J. Membr.Biol., 87: 141-150 (1985)) Six proteins have been identified in contacts of an underlying submembrane plate membrane cytoplasmic, but its function remains established (Diamond, supra). ZO-1 and ZO-2 exist as heterodimers (Gumbiner et al, Proc. Nati. Acad.

Sci., USA, 88: 3460-3464 (1991)) in a detergent-stable complex with an uncharacterized 130 kD protein (ZO-3). The majority of microscopic and immunoelectric studies have located ZO-1 just below membrane contacts (Stevenson et al, Molec, Cell Biochem., 83: 129-145 (1988)). Two other proteins, cingulin (Citi et al, Nature (London), 333: 272-275 (1988)) and the 7H6 antigen (Zhong et al, J. Cell Biol., 120: 477-483 (1993)) are also located from the membrane and have not been cloned. Rab 13, a recently small GTP binding protein has also been localized in the junction region (Zahraoui et al, J. Cell Biol., 124: 101-115 (1994)), Other small GTP binding proteins are known to regulate the cortical cytoskeleton, that is, rho regulates the actin-membrane binding in • focal contacts (Ridley et al., Cell, 70: 389-399 (1992)), and rae regulates membrane-induced growth factor-induced membrane ripple (Ridley et al, Cell, 70: 401-410 (1992)). Based on the analogy with the known functions of plaque proteins in the best characterized cell junctions, focal contacts (Guan et al, Nature, 358: 690-692 (1992)), and adherent junctions (Tsukita et al, J. Cell Biol., 123: 1049-1053 (1993)), hypotheses have been made that • Plate proteins associated with tj are involved in the transduction of signals in both directions through the cell membrane, and in the regulation of cortical actin cytoskeleton links. 15 To satisfy the many physiological and pathological challenges to which the epithelium is subjected, ZO must be capable of rapid and coordinated responses that require the presence of a complex regulation system. The precise characterization of the mechanisms involved in the assembly and regulation of ZO is a active research area. There is now a body of evidence that there are structural and functional links between tj between the actin cytoskeleton and the tj complex of absorption cells (Gumbíner et al, supra, Madara et al, supra, and Drenchahn et al, J. Cell Biol., 107: 1037-1048 (1988)). The actin cytoskeleton is composed of a complicated network of microfilaments, whose precise geometry is regulated by a large array of actin-binding proteins. An example of how the phosphorylation state of an actin-binding protein can regulate cytoskeletal binding to the cell plasma membrane is the substrate of cystase C rich in methalylated alanine (hereinafter "MARCKS"). MARCKS is a specific protein C-kinase substrate (hereafter "PKC") that is associated with the cytoplasmic face of the plasma membrane (Aderem, Elsevier Sci. Pub. (UK), page 438-443 (1992 )). In its non-phosphorylated form, MARCKS is intertwined with the actin of the membrane. In this way, it is likely that the actin network associated with the membrane through MARCKS is relatively rigid (Hartwig et al, Nature, 356: 618-622 (1992)). Activated PKC phosphorylates MARCKS which is released from the membrane (Rosen et al, J. Exp. Med., 172: 1211-1215 (1990)); and Telen et al, Nature, 351: 320-322 (1991)). The actin bound to MARCKS is probably spatially separated from the membrane and is more plastic. When MARCKS is dephosphorylated, it returns to the membrane where it once again intertwines with actin (Hartwig et al, supra, and Thelen et al, supra). These data suggest that the F-actin network can be rearranged through a PKC-dependent phosphorylation process involving actin-binding proteins (MARCKS being one of them). I have shown that a variety of intracellular mediators alter the function and / or structure of tj. The hermetic junctions of the gallbladder of amphibians (Duffey et al, Nature, 204: 451-452 (1981)), and the intestine of both the golden fish (Bakker et al, Am. J. Physiol., 246: G213-G217 (1984)) as sole (Krasney et al, Fed. Proc., • 42: 1100 (1983)), exhibit improved resistance to passive ion flow as intracellular cAMP rises. Also, exposure of the gallbladder of amphibians to the Ca2 ionophore seems to improve the resistance of tj, and induces alterations in the structure of tj (Palant et al, Am. J. Physiol., 245: C203-C212 (1983)) . In addition, the activation of PKC through phorbol esters increases the # ° paracellular permeability in both kidneys (Ellis et al, C. Am. J. Physiol., 263 (Renal Fluid Electrolyte Physiol., 32): F293-F300 (1992)), as in intestines (Stenson et al, C. Am. J. Physiol., 265 (Gastrointest. Physiol., 28): G955-G962 (1993)), in epithelial cell lines. 15 II The Blood-Brain Barrier The blood-brain barrier (BBB) is an extremely thin membranous barrier that is highly resistant to solute-free diffusion, and separates blood and brain. In molecular dimensions the movement of drugs or solute through this The membrane is essentially nil, unless the compound has access to one of several specialized enzyme-type transport mechanisms that are embedded within the membrane of the blood-brain barrier. The blood-brain barrier is composed of multiple cells instead of a single layer of cells epithelial. Of the four different types of cells that make up the blood-brain barrier (endothelial cells, pericytes, astrocytes and neurons), the endothelial cell component of capillaries represents the limiting factor for permeability. • of the blood-brain barrier. The capillary endothelium in the brain and spinal cord of vertebrates is endowed with tj, which closes the enteroendothelial pores that normally exist in microvascular endothelial barriers in peripheral tissues. Finally, endothelial tj is responsible for the limited permeability of the blood-brain barrier. 0 lll Toxina de Ocludenos de Zónula. Most candidates for the Vibrio cholerae vaccine constructed by removing the ctxA gene decode cholera toxin (CT) are available to produce high antibody responses, but more than half of the vaccines continue to develop diarrhea moderate (Levine et al, Infect. Immun., 56 (1): 161-167 (1988)). Given the magnitude of diarrhea induced in the absence of CT, it was hypothesized that V. cholerae produces other enterotoxigenic factors, which are still present in strains deleted from the ctxA sequence (Levine et al, supra). As a result, a second toxin, zonula occluden toxin (hereinafter "ZOT") developed by V cholerae and which contributes to residual diarrhea, was discovered (Fasano et al, Proc.Nat.Acid.Sci., USA, 8: 5242-5246 (1991)). The zot gene is located immediately adjacent to the ctx genes. The high percentage of concurrency of zot gene with ctx genes between V. cholerae strains (Jonson et al, C. Clin. Microb., 31/3: 732-733 (1993)) suggests a possible synergistic role of ZOT in the cause of diarrhea from dehydration acute typical of anger. Recently, the zot gene has also been • identified in other enteric pathogens (Tschape, 2nd Asian- 5 Pacific Symposium on Typhoid fever and other Salmonellosis, 47 (Abstr.) (1994)). It has previously been found that, when tested in rabbit ileum mucosa, ZOT increases intestinal permeability by modulating the intracellular tj structure (Fasano et al, supra). It has been found that as a consequence of the modification of the paracellular trajectory, the intestinal mucosa becomes more permeable. It has also been found that ZOT does not affect active transport coupled to Na * -glucose, is not toxic, and fails to completely abolish transepithelial resistance (Fasano et al, supra). More recently, it has been found that ZOT is capable of reversibly opening tj in the intestinal mucosa, and thus, ZOT, when co-administered with a therapeutic agent, is capable of effecting the intestinal delivery of the therapeutic agent, when employed in an oral dose composition for intestinal drug delivery (WO 96/37196; U.S. Patent Application Serial Number 08 / 443,864, filed May 24, 1995; and U.S. Patent No. 5,665,389; and Fasano et al, J. Clin. Invest., 99: 1158-1164 (1997); each of which is incorporated herein by reference in its entirety). It has also been found that ZOT is capable of reversibly opening tj in the nasal mucosa, and thus ZOT, when coadministered with a therapeutic agent, is capable of improving the nasal absorption of a therapeutic agent (US Patent Application Series No. 08/781 /, 057, filed January 9, 1997, which is incorporated herein by reference in its entirety). In the patent application of E.U.A series number 08 / 803,364, filed on February 20, 1997; which is hereby incorporated by reference in its entirety, a ZOT receptor has been identified and purified from an intestinal cell line, i.e. CaCo2 cells. In addition, in the patent application serial number 09 / 024,198, filed on February 17, 1998; which is hereby incorporated by reference in its entirety, the ZOT receptors of intestinal, cardiac and human brain tissue have been identified and purified. The ZOT receptors represent the first step of the paracellular pathway involved in the regulation of intestinal and nasal permeability. IV Zonulina In the pending US patent application serial number 098 / 859,931, filed on May 21, 1997, which is incorporated herein by reference in its entirety, mammalian proteins that are immunologically and functionally related to mammalian proteins have been identified and purified. ZOT, and that function as the physiological modulator of hermetic mammalian joints. These mammalian proteins, designated as "zonulin", are useful for improving the absorption of therapeutic agents through tj from the intestinal mucosa and nasal mucosa, as well as through tj of the blood-brain barrier. In the present invention, zonulin peptide antagonists have been identified for the first time. Said peptide antagonists bind to the ZOT receptor, which does not yet function to physiologically modulate the opening of the mammalian tight junctions. Peptide antagonists competitively inhibit the binding of ZOT and zonulin to the ZOT receptor, thus inhibiting the ability of ZOT and zonulin to physiologically modulate the opening of mammalian tight junctions.

COMPENDIUM OF THE INVENTION An object of the present invention is to identify zonulin peptide antagonists. Another object of the present invention is to synthesize and purify said peptide antagonists. Yet another object of the present invention is to use said peptide antagonists as anti-inflammatory agents in the treatment of gastrointestinal inflammation. Yet another object of the present invention is to use said peptide antagonists to inhibit the breakdown of the blood-brain barrier. This and other objects of the present invention, which will be apparent from the detailed description of the invention provided below, have been satisfied, in one embodiment, through a zonulin peptide antagonist comprising a selected amino acid sequence. of the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID • NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO : 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 SEQ ID NO: 21, SEQ ID NO: 22 SEQ ID NO: 23 SEQ ID NO: 24, and SEQ ID NO: 35, wherein said peptide antagonist binds to a ZOT receptor, which does not yet physiologically modulate the opening of mammalian tight junctions.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the effect of purified zonulin of the rabbit intestine (0), as compared to several negative controls (Fraction 2 (fi), Fraction 3 (* .Fraction 4 (p), and Fraction 5 () of a Q-Sepharose column), in the resistance of tissue (Rt) of monolayers of CaCo2 cell .. Figure 2 shows the effect of purified zonulin from rabbit intestine (ES), as compared to the negative control (S), in the tissue resistance (Rt) of rabbit ileus mounted in Ussing chamber. Figure 3 shows the effect of purified zonulin from rabbit intestine (El), as compared to controls negatives (zonulin + anti-ZOT antibody (S); zonulin + anti-tau antibody (p); and tau (p), in tissue resistance (Rt) of rabbit ileus mounted in Ussing chambers. A and 4 B show the effect of purified zonulin • either human brain (p), human intestine (• *), or human heart (O), as compared to the negative control (?), In the tissue resistance (Rt) of the jejunum of the Rhesus monkey ( Figure 4 A) and monkey ileus (Figure 4 B) mounted on Ussing cameras. Figures 5 A and 5 B show the effect of purified zonulin either from human heart (p) or human brain (ffl), according to • OR purchased with the negative control ((S), in the tissue resistance (Rt) of rabbit jejunum (Figure 5 A) and rabbit ileum (Figure 5 B) mounted in Ussing chambers Figure 6 shows a comparison of a purified N-terminal sequence of zonulin various human and rabbit tissues.

Figure 7 shows a comparison of the N-terminal sequence of purified zonulin from various human tissues and the heavy chain of IgM with the N-terminal sequence of the biologically active fragment (amino acids 288-399) of ZOT. Figure 8 shows the effect of ZOT, zonulina, zonulinah, and either alone (closed bars), or in combination with the peptide antagonist FZI / 0 (open bars) or in combination with FZI / 1 (shaded bars), as compared to the negative control in the tissue resistance (Rt) of the rabbit ileus mounted on Ussing cameras. N equal 3-5; and * equal p < 0.01. DETAILED DESCRIPTION OF THE INVENTION • As discussed above, in one embodiment, the above-described object of the present invention has been met through a zonulin peptide antagonist comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO : 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 , SEQ ID NO: 19, SEQ ID NO: 20 SEQ ID NO: 21, SEQ ID NO: 22 SEQ ID NO: 23 SEQ ID NO: 24, and SEQ ID NO: 35, wherein said peptide antagonist is attached to the ZOT receiver, which has not yet modulated15 philologically the opening of hermetic joints of mammals. The size of the peptide antagonist is not critical to the present invention. In general, the size of the peptide antagonist will vary from 8 to 110 amino acids, preferably from 8 to 40 amino acids, most preferably it will be 8 amino acids. Peptide antagonists can be chemically synthesized and purified using well known techniques, such as described in High Performance Liked Chromatography of Peptides and Proteins: Separation Analysis and Conformation, Ed. Mant et al, C.R.C. Press (1991), and a peptide synthesizer, such as Symphony (Protein Technologies, Inc); or using recombinant DNA techniques, ie, wherein the nucleotide sequence encoding the peptide is inserted into an appropriate expression vector, eg, an E. coli or yeast expression vector, expressed in the respective host cell , and purified from it using well-known techniques. Peptide antagonists can be used as anti-inflammatory agents for the treatment of gastrointestinal inflammation that gives rise to increased intestinal permeability. In this manner, the peptide antagonists of the present invention are useful, for example, in the treatment of intestinal conditions that cause protein loss enteropathy. Protein loss enteropathy may arise due to: Infection, eg, C. difficile infection, enterocolitis, shigellosis, viral gastroenteritis, parasite infestation, bacterial overgrowth, Whipple's disease; diseases with mucosal erosion, for example, gastritis, gastric cancer, collagenous colitis, inflammatory bowel disease; Diseases marked by lymphatic obstruction, for example, congenital intestinal lymphagiectasia, sarcoidosis lymphoma, mesenteric tuberculosis and after surgical correction of congenital heart disease with the Fontan operation; Diseases of the mucosa without ulceration, for example, Ménétrier's disease, celiac disease, eosinophilic gastroenteritis; and Immune diseases, for example, systemic lupus erythematosus or food allergies, primarily to milk (see also Table 40-2 of Pediatric Gastrointestinal Say Pathophysiology Diagnosis Management, Eds. Wyllie et al, Saunders Co. (1993), pages 536- 543, which is incorporated herein by reference in its entirety). Therefore, in another embodiment, the present invention relates to a method for the treatment of gastrointestinal inflammation, which comprises administering to a subject with the • the need for such treatment, a pharmaceutically effective amount of a zonulin peptide antagonist, wherein said peptide antagonist comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NOM, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 , SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 SEQ ID NO: 21, SEQ ID NO: 22 SEQ ID NO: 23 SEQ ID NO: 24, wherein said peptide antagonist binds to the ZOT receptor in the The intestine of said subject, which does not yet physiologically modulate the opening of hermetic junctions in said intestine. Up to this point, peptide antagonists can be administered as oral dose compositions, for delivery to the small intestine. Said oral dose compositions for the Delivery to the small intestine is well known in the art, and generally comprises gastro-resistant tablets or capsules (Remington's Pharmaceutical Sciences, 16th Ed., Eds. Osol, Mack Publishing Co., Chapter 89 (1980); Digenis et al, J. Pharm. Sci., 83: 915-921 (1994), Vantiní et al, Clinical Therapeutics, 145: 445-451 (1993), Thoma et al, Pharmazie, 46: 331-336 (1991), Morishita et al, Drug Design and Delívery, 7: 309-319 (1991); and Lin et al, Pharmaceutical Res., 8: 919-924 (1991)); each of which is incorporated herein by reference in its entirety). Tablets are made gastro-resistant through the addition of, for example, either cellulose acetate-phthalate or cellulose acetate-terephthalate. Capsules are solid dosage forms wherein the peptide antagonist (s) is enclosed either in a container or coated with hard or soft soluble gelatin. The gelatin used in the manufacture of capsules is obtained from collagenous material through hydrolysis. There are two types of gelatin. Type A, derived from pig skin through acid processing, and type B, obtained from animal bones and skins through alkaline processing. The use of hard gelatin capsules allows selection to prescribe an individual peptide antagonist or a combination thereof at the exact dose level considered best for the individual subject. The hard gelatin capsule consists of two sections, one sliding over the other, thus completing the circumference to the peptide antagonist. These capsules are filled by introducing the peptide antagonist, or gastroresistant beads containing the peptide antagonist, into the longer end of the capsule, and then sliding over the cap. Hard gelatin capsules are made • enormously of gelatin, FD &C dye, and sometimes an opacifying agent, such as titanium dioxide. USP allows gelatin for this purpose to contain 0.15% (w / v) sulfur dioxide to prevent decomposition during manufacturing. In the context of the present invention, oral dose compositions for delivery to the small intestine also include • Or liquid compositions, which contain aqueous pH regulating agents that prevent the peptide antagonist from being significantly inactivated by gastric fluids in the stomach, thereby allowing the peptide antagonist to reach the small intestine in an active form. Examples of said agent Aqueous pH regulation, which can be employed in the present invention, include bicarbonate pH regulator (pH 5.5 to 8.7, preferably around 7.4). When the oral dose composition is a liquid composition, it is preferred that the composition be prepared just before to be administered, in order to minimize stability problems. In this case, the liquid composition can be prepared by dissolving the lyophilized peptide antagonist in the aqueous pH regulating agent. Peptide antagonists can be used to inhibit the failure of the blood-brain barrier. Thus, the peptide antagonists of the present invention are useful, for example, in the treatment of conditions associated with blood-brain barrier failure. Examples of such conditions include osmotic damage, for example, cerebral ischemia, shock or cerebral edema; hypertension; carbon dioxide; convulsive attack; chemical toxins; uremia (kidney failure); meningitis, encephalitis, encephalomyelitis, for example, infectious (viral (SRV, HIV, etc.), or bacterial (TB, H. Influenzae, meningococcal, etc.) or allergic, tumors, traumatic brain damage, brain damage by radiation immaturity and cernicterus, demyelination diseases, for example multiple sclerosis or Guillian-Barre syndrome.Therefore, in another embodiment, the present invention relates to a method for the treatment of conditions associated with failure of the blood-brain barrier, which comprises administering to a subject in need of such treatment, a pharmaceutically effective amount of a zonulin peptide antagonist, wherein said peptide antagonist comprises the amino acid sequence SEQ ID NO: 35, wherein said peptide antagonist is attached to the ZOT receptor in the brain of said subject, which does not yet physiologically modulate the opening of hermetic junctions in the brain. peptide can be administered as intravenous dose compositions to be delivered to the brain. Such compositions are well known in the art, and the compositions generally comprise a physiological diluent, eg, distilled water, or 0.9% (w / v) NaCl. The pharmaceutically effective amount of the antagonist of • The peptide used is not critical to the present invention and will vary depending on the disease or condition to be treated, as well as the age, weight and sex of the subject to be treated. Generally, the amount of peptide antagonist employed in the present invention used to inhibit gastrointestinal inflammation or to inhibit hematoencephalic barrier failure, for example, to #l (inhibit the biological activity of zonulin, is on the scale of approximately 7.5 x 10'6 M to 7.5 x 10-3 M, preferably around 7.5 x 10"6 M to 7.5 x 10" 4 M. To achieve this final concentration in, for example, the intestines or blood, the amount of the peptide antagonist in a dose composition The individual oral composition of the present invention will generally be about 1.0 μ to 1000 μg, preferably about 1.0 μg to 100 μg. Peptide antagonists can also be used as an immunogen to obtain antibodies, either polyclonal or Monoclonal antibodies, having specific binding character for zonulin, using techniques well known in the art, (Abrams, Methods Enzymol., 121: 107-119 (1986)). These antibodies, in turn, can be used to analyze zonulin in body tissue or fluids, or in affinity-purification of zonulin, or alternatively, to bind to zonulin, and thereby inhibit zonulin activity, for example, to inhibit gastrointestinal inflammation or to inhibit the breakdown of the blood-brain barrier. The following examples are provided for illustrative purposes only, and are in no way intended to limit the scope of the present invention.

EXAMPLE 1 Purification of ZOT 5000 ml of the obtained supernatant fraction was concentrated after cultivating V. cholerae strain CVD110 (Michalski et al, Infect. Immun., G1: 4462-4468 (1993), which had been transformed with the plasmid pZ14, 1000 times using a filter of sheet flux with a MW cutoff of 10 kDa The construction of pZ14, which contains the zot gene of Vibrio cholerae, is described in detail in, among others, WO 96/37196. The resulting supernatant was then subjected to SDS- 8.0% PAGE (p / v) Protein bands were detected through Coomassie blue staining of the SDS-PAGE gel No protein band corresponding to ZOT was detected when compared to the control supernatant of the strain CVD110 transformed with the plasmid pTTQl81 (Amersham, Arlington Heights, IL), and treated in the same manner. Therefore, although the zot gene was placed behind the strong and strong tac promoter in pZ14, the protein level in the 1000-fold concentrated pZ14 supernatant remained undetectable through the SDS-PAGE gel stained with Coomassie A. MBP-ZOT To increase the amount of ZOT produced, the zot gel was fused in frame with the maltose binding protein gel (hereinafter "MBP") to create a fusion protein of MBP-ZOT. The MBP vector pMAL-c2 (Biolab) was used to express and purify ZOT by fusing the zot gel to the malE gel of E. coli. This construct utilizes the strong tac promoter, capable of induction, and the malE translation initiation signals to give a high level expression of the cloned zot gene. The pMAL-c2 vector has an exact deletion of the malE signal sequence, which leads to the cytoplasmic expression of the fusion protein. Affinity chromatography was used for the purification of MBP to facilitate the isolation of the fusion protein (Biolab). More specifically, the pMAL-c2 vector was linearized with EcoRI (which cuts at the 3 'end of the malE gene), filled with Klenow fragment, and digested with Xbal (which has a single site of the polylinker pMAL-c2). The orf encoding ZOT was subcloned from plasmid pBB241 (Baudry et al, Infect. Immun., 60: 428-434 (1992)). Plasmid pBB241 was digested with BssHIl, filled with the Klenow fragment, and digested with Xbal. Then, the Xbal blunt fragment was subcloned in pMALc2 to give the plasmid pLC10-c. Since both the insert and the vector had blunt and sticky ends, the correct orientation was obtained with the 3 'end malE fused with the 5' of the insert. Next, pLC10-10 was electrophoresed in E. coli strain DH5a. In pBB241, the restriction site BssHIl is within zot orf. This • way, amino acids 1-8 of ZOT lack the MBP-ZOT fusion protein. In order to purify the MBP-ZOT fusion protein, 10 ml of Luria Bertani broth containing 0.2% (w / v) glucose and 100 μg / ml ampicillin were inoculated with a single colony containing pLC10-c, and they were incubated overnight at 37 ° C with shaking. The culture was diluted 1: 100 in 1.0 ml of the same fresh medium, and developed at 37 ° C while stirring, at approximately 1.0 x 10 8 cells / ml. Then, 0.2 mM of IPTG was added to induce the expression of MBP-ZOT, and the culture was incubated at 37 ° C for 3 more hours. The bacteria were then formed into pellets and resuspended in 20 ml of the ice-cooled "column pH regulator", comprising 20mM Tris-HCl, 0.2M NaCl, 1.0mM EDTA, 10mM 2-ME, 1.0mM NaN3. The bacterial suspension was used through a French press treatment and rotated for 30 minutes at 13,000 x g at 4 ° C. The supernatant was collected, was diluted, 1.2, with column pH regulator and loaded onto a 1 x 10 column of amylose resin (Bíolabs, MBP fusion purification system), pre-equilibrated with column pH regulator. After washing the column with 5 volumes of column pH regulator, the MBP-ZOT fusion protein was eluted by loading 10 ml of 10 mM maltose in the column pH regulator. The typical yield of 1.0 ml of culture was 2-3 mg of protein. The MBP fusion pattern of the purified MBP-ZOT fusion protein was then cut using 1.0 μg of the factor Xa protease (Biolabs) per 20 μg of MBP-ZOT. Factor Xa protease is divided just before the amino terminus of ZOT. The ZOT protein thus obtained was run on a 0.8% (w / v) SDS-PAGE gel, and electroeluted from the gel using an electroseparation chamber (Schleicher &Schuell, Keene, NH). • < When tested in Ussing chambers, the resulting purified ZOT induced a Rt-dependent reduction, with an ED50 of 7.5 x 10 -8 M.

B. 6xHis-ZOT 15 The zot gene was amplified through PCR with polymerase from Deep Vent (New England Biolabs), using DNA from plasmid pBB241 (Baudry et al, supra) as a template. The advance and reverse primers used were: 5-CGGGATCCCGTATGAGTATCTTT-3 '(SEQ ID NO: 39); Y 5'- 20 CCCAAGCTTGGGTCAAAATATACT-3 '(SEQ ID NO: 40), respectively. The 5 'ends of these oligonucleotides contain a restriction site of BamHI and one of HindIII, respectively. The resulting amplicon (1.2 kb) was analyzed by 8.0% (w / v) agarose gel electrophoresis, and purified from free salts and nucleotides using an extreme spin column (Pierce). The two restriction enzymes observed above were then used to digest the purified amplicon, and the resulting digested amplicon was then inserted into the vector pQE30 (Quiagen), which had previously been digested with BamHI and HindIII, to be able to contain the plasmid pSU113. PQE30 is an expression vector that provides a high level expression of a recombinant protein with a 6-poly-histidine (6xHis) tag. The expression product of plasmid pSU113, therefore, is a fusion protein of dxHis-ZOT. Then pSU113 was transformed into E. Coli DH5a. • In order to purify the 6xHis-ZOT fusion protein, the resulting transformed E. coli was grown overnight at 37 ° C in 150 ml of Luria Bertani broth containing 2.0% (w / v) glucose, 25 μg / ml of kanamycin and 200 μg / ml of ampicillin, up to that the value of A6oo was approximately 1.10. Then, 75 ml of the nocturnal cultures were added to 1000 ml of Luría Bertani broth containing 2.0 glucose (w / v), 25 μg / ml of • kanamycin and 200 μg / ml of ampicillin, was incubated for approximately 3 hours at 37 ° C, with vigorous shaking, until the value of A6oo was approximately 0.7-0.9. Then, IPTG was added to a final concentration of 2.0 mM, and the growth was allowed to continue for 5 hours at 37 ° C. Afterwards, the cells were harvested by centrifugation at 4000 xg for 20 minutes, the cells were resuspended at 5.0 ml / g weight humid pH regulator comprising 6.0 M GuHC1, 0.1 M sodium phosphate, and 0.01 M Tris-HCl (pH 8.0), was stirred for one hour at room temperature. Then, the mixture was centrifuged at 1000 x g for 30 minutes at 4 ° C, and the resulting supernatant was • added 4.0-5.0 ml / g, wet weight, of a 50% slurry of 5 SUPERFLOW resin, (QIAGEN), and stirring was carried out for one hour at room temperature. The resulting resin was loaded onto a 1.6 x 8.0 column, which was then washed sequentially with pH A regulator, pH regulator B comprising 8.0 M urea, 0.1 M sodium phosphate, and 0.01 M Tris-HCl (pH 8.0) and regulator of 0 pH c comprising 8.0 M of urea, 0.1 M of sodium phosphate, and 0.01 M Tris-HCl (pH 6.3). Each wash was performed until the Aß flux value was less than 0.01. The 6xHis-ZOT fusion protein was eluted from the column using 20 ml of the pH C buffer containing 250 mM of imidazole. After, the fractions containing the 6xHis-ZOT fusion protein were inspected through SDS-PAGE using the procedure described by Davis, Ann. N.Y. Acad. Sci., 121: 404 (1964), and the gel stained with Commassie blue. The fractions containing the dxHis-ZOT fusion protein were expressed against 8.0 M of urea, were combined, and then were diluted 100 times in PBS. Then 4.0 ml of a 50% slurry of the SUPERFLOW resin was added, stirring was carried out for 2 hours at room temperature, and the resulting resin was loaded onto a 1.6 x 8.0 column, which was then washed with 50 ml of PBS. The 6xHis-ZOT fusion protein was eluted from the column with 10 ml of PBS containing 250 ml mM imidazole. The resulting eluent was dialyzed against PBS, and the 6xHis-ZOT fusion protein was inspected through SDS-PAGE, as described above. • 5 EXAMPLE 2 PRODUCTION OF ANTI-ZOT ANTIBODIES OF PURIFIED AFFINITY To obtain specific antiserum, a glutathione S-transferase (GST) -ZOT chimeric protein was expressed and purified. More specifically, oligonucleotide primers were used to amplify zot orf through polymerase chain reaction (PCR) using plasmid pBB241 (Baudry et al, supra) as template DNA. The forward primer 15 (TCATCACGGC GCGCCAGG; SEQ ID NO: 25) corresponded to nucleotides 15-32 of zot orf, and the reverse primer (GGAGGTCTAG AATCTGCCCG AT, SEQ ID NO: 26) corresponded to the 5 'end of ctxA orf . Therefore, the 1-5 amino acids of ZOT were missing in the resulting fusion protein. The amplification product 20 was inserted into the polylinker (Smal site) located at the end of the GST gene in pGEX-2T (Pharmacia, Milwaukee, Wl). PGEX-2T is a fusion protein vector that expresses a gene cloned as a GST fusion protein from Schistosoma japonicum. The fusion gene is under the control of the tac promoter. After fusion with IPTG, the de-repression occurred and the GST fusion protein was expressed. The resulting recombinant plasmid, designated pLC11, was electrophoresed in E. coli DH5a. In order to purify the GST-ZOT fusion protein, 10 ml of Luria Bertani broth containing 100 μg / ml ampicillin was inoculated with a single colony containing pLC11, and incubated overnight at 37 ° C with shaking. The culture was diluted 1: 100 in 1.0 ml of the same fresh medium and grown at 37 ° C while stirring, at approximately 1.0 x 10 8 cells / ml. Then 0.2 mM c IPTG was added to induce the expression of GST-ZOT, and the culture was incubated at 37 ° C for 3 more hours. Afterwards, the bacteria were formed into pellets, resuspended in 20 ml of ice-cold PBS (pH 7.4), and used through the French press method. The GST-ZOT fusion protein was not soluble under these conditions since it was sedimented with the bacterial pellet fraction. Therefore, the pellet was resuspended in pH buffer of Laemli lysis comprising 0.00625 M Tris-HCl (pH 6.8), 0.2 M 2-ME, 2.0% (w / v) SDS, 0.025% (w / v ) bromophenol blue and 10% glycerol (v / v), and electrophoresed on a 8.0% (w / v) SDS-PAGE gel and shaded with Coomassie brilliant blue. A band of approximately 70 kDa (26 kDa of GST + 44 kDa of ZOT), corresponding to the fusion protein, was electroeluted from the gel using an electroseparation chamber (Schleicher &Schuell, Keene, NH). 10μg of the resulting eluted protein (10-20 μg) was injected into a rabbit, mixed with an equal volume of Freund's complete adjuvant. Doses were reinforcement were administered with incomplete Freund's assistant, 4 and 8 weeks later. One month later, the • rabbit was bled. To determine the production of specific 10"10 M ZOT antibodies, together with two MBP-ZOT and GST-ZOT fusion proteins, were transferred onto a nylon membrane and incubated with a 1: 5000 dilution of rabbit antiserum overnight at 4 ° C with moderate agitation. The filter was then washed 15 minutes, 4 times with PBS containing 0.05% (v / v) of Tween 20 (hereinafter "PBS-T"), and incubated with a 1: 30,000 dilution of anti IgG. -Goat rabbit, conjugated in horseradish peroxidase for 2 hours at room temperature. The filter was washed again for 15 minutes, 4 times with PBS containing 0.01% (v / v) of Tween, and detected immunoreactive bands using enhanced chemiluminescence (Amersham). In the immunostaining, the rabbit antiserum was found to recognize ZOT, as well as the MBP-ZOT and GST-ZOT fusion proteins, but not the negative control of MBP. In addition, to confirm the production of appropriate anti-ZOT antibodies, neutralization experiments were conducted in Ussing chambers. When pre-incubated with pZ14 supernatant at 37 ° C for 60 minutes, the specific antiserum in ZOT (1: 100 dilution), was able to completely neutralize the reduction in Rt induced by ZOT in the rabbit ileum mounted on Ussing cameras. Next, anti-ZOT antibodies were purified by affinity using a MBP-ZOT affinity column. Plus • specifically, an affinity column of MBP-ZOT was prepared by immobilizing, overnight at room temperature, 1.0 mg of purified MBP-ZOT, obtained as described in example 1 above, for a preactivated gel (Aminolink, Pierce). The column was washed with PBS, and then loaded with 2.0 ml of anti-ZOT rabbit antiserum. After an incubation period of 90 minutes at room temperature, the column was washed with 14 ml of PBS, and anti-ZOT antibodies were eluted from the column with 4.0 ml of a solution comprising 50 mM glycine (pH 2.5), 150 mM of NaCl, and 0.1% (v / v) of Triton X-100. The pH of the elution fractions of 1.0 ml was immediately neutralized with 1.0 N NaOH. EXAMPLE 3 PURIFICATION OF ZONULIN Based on the observation in the patent application of E.U.A series number 08 / 803,364, filed on February 20, 1997, that ZOT interacts with a specific epithelial surface receptor, with subsequent activation of a complex intracellular cascade of events regulating the permeability of tj was postulated in the present invention that ZOT can mimic the effect of a physiological modulator of tj of mammals. It was postulated in the patent application of E.U.A. series No. 08 / 859,931, filed on May 21, 1997, that ZOT and its physiological analogue (zonuhna), could be functionally and immunologically related. Therefore, as described there, affinity-purified anti-ZOT antibodies and the Ussing chamber assay were used in combination to search for zonulin in various rabbit and human tissues.

A. Rabbit tissues Initially, zonulin was purified from the rabbit intestine. The tissue was divided through homogenization in PBS. The resulting cell preparations were then centrifuged at 40,000 rpm for 30 minutes, the supernatant was collected and lyophilized. The resulting lyophilized product was subsequently reconstituted in PBS (10: 1 (v / v)), filtered through a 0.45 mm membrane filter, loaded onto a Sephadex G-50 chromatographic column, and eluted with PBS . Then, 2.0 ml fractions obtained from the column were subjected to standard Western immunostaining using the affinity purified anti-ZOT antibodies obtained as described in Example 2 above. The positive fractions, that is, those to which the anti-ZOT antibodies were bound, were combined, lyophilized, reconstituted in PBS (1: 1 (v / v)), and subjected to salt gradient chromatography through a column of Q-Sepharose. The salt gradient was 0-100% (v / v) NaCl in pH buffer of 50 mM Tris (pH 8.0). Five fractions of 20 ml were collected and subjected to standard Western immunostaining using the anti-ZOT antibodies purified by affinity obtained as • described in example 2 above. Fraction 1 (20% w / v) NaCl) was the only fraction that was found to be positive in the Western immunoassay assay. The fractions obtained from the Q-Sepharose column were then tested for their tissue resistance effects in both monolayers of CaCo2, and in the small intestine of the • rabbit in Ussing cameras. More specifically, CaCo2 cells were grown in cell culture flasks (Falcon) under a humid atmosphere of 95% O2 / 5% CO2 at 37 ° C in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum ( v / v) 40μg / 1 of penicillin and 90 μg / 1 streptomycin. The cells were subcultured at a surface ratio of 1: 5 after trypsin treatment every 5 days, when they reached 70-80% confluence. The passage number of the cells used in this study varied between 15 and 30. 20 The monolayers of CaCo2 were developed confluence (12-14 days after placing them on plates at a surface ratio of 1: 2.5) on treated polycarbonate filters with tissue culture firmly attached to a polystyrene ring (6.4 mm diameter, Transwell Costar). The filters were placed in an insert hermetically adjusted by separating the serosal and mucosal compartment from a Ussing chamber, and the experiments were performed as described by Fasano et al., Proc. Nati Acad. Sci., USA, 8: 5242-5246 (1991), for rabbit intestines in chambers • from Ussing. The results are as shown in Figure 1. As shown in Figure 1, the fraction containing zonulin induced a significant reduction in resistance of the monolayers of CaCo2, as compared to negative fractions of zonulin. Afterwards, Ussing chamber tests were performed using ileum of New Zealand white rabbits, adult males, weighing 2-3 kg, which were sacrificed through cervical dislocation. A 20 cm segment of the ileum was removed, rinsed free of intestinal contents, opened along the mesenteric border, and divided into muscle and serosal layers. After, 8 sheets of mucosa thus prepared were mounted in Ussíng del lucita chambers (opening of 1.12 cm2), connected to a voltage clamping apparatus (EVC 4000 WPI, Saratosa, FL), and bathed with a recently prepared Ringer solution. , comprising 53 mM NaCl, 5.0 mM KCl, 30.5 mM mannitol, 1.69 mM Na2HPO4, 0.3 mM NaH2PO4, 1.25 mM CaCl2) 1.1 mM MgCl2 > and 25 mM NaHCO3. The washing solution was maintained at 37 ° C with water-jacketed tanks connected to a constant temperature circulation pump and with gas application of 95% O2 / 5% CO2. 100μg of purified zonulin was added from the intestine rabbit beside the mucosa. The potential difference (PD) was measured every 10 minutes, and short circuit current (Isc) and tissue resistance (Rt) were calculated as described by Fasano et al., Supra. Due to tissue variability, data such as? Rt (Rt at time x) - (Rt at time 0) were calculated. The results are as shown in Figure 2. As shown in Figure 2, the fraction containing zonulin induced a significant reduction in the small intestine resistance of the rabbit, as compared to a negative fraction of zonulin. This effect was completely reversible once the zonulin was removed from the deposit. • The positive fraction of zonulin was also subjected to 8.0% (w / v) SDS-PAGE, followed by a Western immunostaining using anti-ZOT antibodies. The protein bands separated through SDS-PAGE were then transferred onto a PVDF filter (Millipore) using CAPS pH dispenser comprising 100 ml of (3- [cyclohexylamino] -1-propanesulfonic acid), 10x, 100 ml of methanol, 800 ml of distilled water. The protein that was aligned to an individual band that was detected through Western immunostaining had an apparent molecular weight of approximately 47 kDa. This band was cut from PVDF filter and subjected to N-terminal sequencing as described by Hunkapiller, In: Methods of Protein Microcharacterization, Ed. Shibley, Chapters 11-12, Humana Press, p. 315-334 (1985), using a Perkin-Elmer Applied Biosystems model 494 apparatus. The purified zonulin N-terminal sequence of rabbit intestine is shown in SEQ ID NO: 27. The N-terminal sequence of rabbit zonulin was compared with other protein sequences through BLAST search analysis. The result of this analysis revealed that the N-terminal sequence of rabbit zonulin is 85% identical, and 100% similar to the N-terminal sequence of the tau protein of Homo sapiens. As a result, to determine if the rabbit zonuüna and tau are the same portion, cross neutralization experiments conducted in Ussing chambers were conducted. More specifically 10 μg / ml of rabbit zonulin was added to mucosal side of rabbit ileum either untreated or preincubated for 60 minutes at 37 ° C with anti-tau antibodies (1:10 dilution) (Sigma). Both the 10 μg / ml of rabbit zonulin preincubated with anti-ZOT antibodies (1:10 dilution) (example 2) and 0.4 μg / ml of purified tau (Sigma), were used as controls. The results are as shown in Figure 3. As shown in Figure 3, rabbit zonulin induced the typical reduction in tissue resistance that was easily reversible once the protein was removed from the Ussing chambers. This activity was completely neutralized through pre-treatment with anti-ZOT antibodies, but not through pretreatment with anti-tau antibodies. On the other hand, there was no significant effect on tissue resistance in tissues exposed to tau protein. The rabbit zonulin was also detected in several other rabbit tissues, ie, heart, brain, muscle, stomach, vessel, lung, rabbit kidney, as well as several portions of intestines of rabbits, ie the distant jejunum, jejunum near, • ileus, caecum and colon. That is, when these rabbit tissues were processed in the same manner as the rabbit intestine discussed above, and subjected to 0.8% (w / v) SDS-PAGE, followed by Western immunostaining using affinity purified anti-ZOT antibodies obtained As described in Example 2 above, an individual band with a size of Or approximately 47 kDa in all tested tissues. • B. Tissues of humans Zonulin was also purified from various tissues of humans, including the intestine, heart and brain. They were used tissues, both fetal and adult. The tissues were divided through homogenization in PBS. The resulting cell preparations were then centrifuged at 40,000 rpm for 30 minutes, the supernatant was collected and lyophilized. The resulting lyophilized product was subsequently reconstituted in PBS (10: 1 v / v), filtered through a 0.45 mm membrane filter was loaded onto a Sephadex G-50 chromatographic column and eluted with PBS. Then, 2.0 ml fractions obtained from the column were subjected to standard Western immunostaining using the anti-ZOT antibodies purified by affinity obtained as described in Example 2 above.

The positive fractions, that is, those to which the anti-ZOT antibodies were bound, were combined, lyophilized, reconstituted in PBS (1: 1 v / v), and subjected to salt gradient chromatography through a column. of Q-Sepharose. The salt gradient was 0-100% (w / v) of NaCl in pH buffer of 50 mM Tris (pH 7.4). Five fractions of 20 ml were collected, and subjected to standard Western immunostaining using the affinity purified anti-ZOT antibodies obtained as described in example 2 above. Fraction 1 (20% (w / v) NaCl) showed a single band with a size of 45 kDa in the assay of • Western immunostaining. Fraction 2 (40% (w / v) NaCl) showed two additional bands with a size of 35 kDa and 15 kDa in the Western immunostaining assay. Fraction 3 (60% (w / v) NaCl) and fraction 4 (80% (w / v) showed only bands with a size of 35 kDa and 15 kDa. These results suggest that zonulin can be subjected to degradation through proteases, probably present in the human tissues used, and that the fault products were eluted from the column at higher salt concentrations as compared to holoprotein. 20 Fraction 1 (from human heart, intestine and brain tissues) and fraction 4 (from heart tissue) obtained from the Q-Sepharose column were then tested for their tissue resistance effects in both the rabbit intestine and the intestine of the Rhesus monkey in Ussing chambers. 25 Ussing chamber tests were performed using different tracts of intestine, including, jejunum, ileus or colon of New Zealand white rabbits adult males weighing 2-3 kg, or adult male Rhesus monkeys weighing 5-6 kg. After the animals were sacrificed, different intestine segments were removed, including the jejunum, ileum and colon, rinsed free of intestinal contents, opened along the mesenteric boundary, and divided from muscle and serosal layers. Then, 8 sheets of mucosa prepared in this way (3 of the jejunum, 3 of ileus and 2 of the colon) were mounted in Ussing chambers of lucita (opening of 1.12 cm2), connected to an apparatus for restraining • voltage (EVC 4000 WPI, Saratosa, FL), and washed with a freshly prepared Ringer's solution, comprising 53 mM NaCl, 5.0 mM KCl, 30.5 mM mannitol, a.69 mM Na2HPO4, 0.3 mM NaH2PO4, 1.25 mM CaCl2l 1.1 mM MgCl2, and 23 mM NaHCO3. The washing solution was maintained at 37 ° C with water-jacketed tanks connected to a constant temperature circulation pump and with gas application of 95% O2 / 5% CO2. 100 μl of fraction 1 of purified zonulin from human heart or fraction 1 of zonulin was added to the mucosal side purified from human brain, or purified zonulin fraction from human intestine, or purified fraction 4 from human heart. The potential difference (PD) was measured every 10 minutes. And short circuit current (Isc) and tissue resistance (Rt) were calculated as described by Fasano et al., Supra. The data was calculated as Rt for Figures 4 A and 4 B; but due to the tissue variability, the data were calculated as? Rt (Rt at time x) - (Rt at time 0) for figures 5 A and 5 B. The results are as shown in figures 4 A and 4 B (monkey intestine) and figures 5 A and 5 B (rabbit intestine). As shown in Figures 4A and 4B, the purified zonulin of human heart and intestine (fraction 1) induced a significant reduction in the intestinal resistance of the monkey (both jejunum (Figure 4 A) and ileus (Figure 4 B)), according to purchased with the negative control of PBS. No major changes were observed when the purified zonuin either from human heart or from human intestine was tested in the colon. Figures 4A and 4B also show that no significant effect occurred in both the jejunum monkey (figure 4A) and the monkey ileum (figure 4B) when purified zonulin was tested in the human brain (fraction 1). Fraction 4 of purified zonulin from human heart also induced a significant reduction in the resistance of the small intestinal tissue of the monkey. As shown in Figures 5A and 5B, similar results were obtained when rabbit intestine was used. That is, zonulin purified from human heart (Fraction 1) showed an important effect on tissue resistance in both the rabbit jejunum (Figure 5A) and rabbit ileum (Figure 5B), but not in the colon. Figures 5A and 5B also had no significant effect on either the rabbit jejunum (Figure 5A) or the rabbit ileum (Figure 5B) when purified human brain zonulin was tested (Fraction 1).

To know if zonulin increases the oral insulin supply, in vitro model experiments were performed using rabbit intestine. In summary, male New Zealand white rabbits (2-3 kg) were sacrificed through cervical dislocation. Segments of the rabbit's small intestine (either jejunum or ileus) were removed, rinsed free of intestinal contents, opened along the mesenteric border, and separated from muscle and serosal layers. Then 8 sheets of mucosa prepared in this way were mounted in Ussing chambers of lucite (opening of O 1.12 cm2), connected to a voltage clamping device (EVC 4000) • WPI, Sarasota, Fl), and washed with freshly prepared pH buffer containing 53 mM NaCl, 5.0 mM KCl, 30.5 mM mannitol, 1.69 mM Na2HPO4, 0.3 mM NaH2PO4. 1.25 mM CaCl2, 1.1 mM MgCl2, and 25 mM NaHCO3. The washing solution was maintained at 37 ° C with tanks jacketed with water connected to a constant temperature circulation pump and with gas application of 95% O2 / 5% CO2. The potential difference (PD) was measured, and the short circuit current (Isc) and the tissue resistance (Rt) were calculated. Once the tissues reached a condition of stable state, the tissues in pairs, based on their resistance were exposed luminally to 10"11 M 125 I-insulin (Amersham, Arlington Heights, IL; 2-0 μCi = 10" 12 M), alone or in the presence of 100 μl of heart zonulin from fraction 1. An aliquot of 1.0 ml of serosal side and a 50 μl aliquot of the side of mucosa were immediately obtained to establish baseline values. Samples of the serosal broth were then collected at 20 minute intervals for the next 100 minutes. It was found that heart zonulin increased ia • intestinal absorption of insulin in both the jejunum (0.058 ± 0.003 fmol / cm2, min vs. 0.12 ± 0.005 fmol / cm2, min, untreated tissues versus zonulin-treated respectively, p = 0.001), as in ileus (0.006 ± 0.0002 fmol / cm2, min vs. 0.018 ± 0.005 fmol / cm2, min, untreated tissues vs. zonulin, respectively, p = 0.05) in a time-dependent manner . Fraction 1 of purified zonulin from human heart, fraction 1 of purified zonulin from human intestine, fraction 1 of zonulin purified from human brain were also subjected to 8.0% (w / v) SDS-PAGE, followed by Western immunostaining using the anti-ZOT antibodies obtained as described in example 2 above. The separated protein bands mediating SDS-PAGE were then transferred onto the PVDF filter using CAPS pH regulator, comprising 100 ml of 10x (3- [cyclohexylamino] -1-propanesulfonic acid), 100 ml of methanol, 800 ml of distilled water. The protein that was aligned to a band The individual that was detected by Western immunostaining had an apparent molecular weight of approximately 47 kDa. This band was cut from the PDVF filter, and subjected to N-terminal sequencing as described by Hunkapiller, In: Methods of Protein Microcharacterization, Ed. Shibley, Chapters 11-12, Humana Press, p. 315-334 (1985), using an apparatus of Perkin-Elmer Applied Biosystems model 494. The N-terminal sequence of purified zonulin from adult human heart is shown in SEQ ID NO: 28, the N-terminal sequence of purified zonulin from adult human brain is shown in SEQ ID NO: 29, and the N-terminal sequence of purified zonulin from adult fetal brain is shown in SEQ ID NO: 36. The first 9 amino acids of the purified zonulin N-terminal sequence of adult human intestine (SEQ ID NO: 31) were also sequenced, and found to be identical to the first 9 amino acids of purified zonulin from human heart • 'shown in SEQ ID NO: 28 (see Figure 6). The first 20 amino acids of the purified N-terminal sequence of zonulin from human fetal intestine were also sequenced: Met Leu Gln Lys Ala Glu Ser Gly Val Leu Val Gln Pro Gly Xaa Ser Asn Arg Leu (SEQ ID NO: 30), and found to be nearly identical to the purified human heart zonulin amino acid sequence shown in SEQ ID NO: 28 (see Figure 6). The N-terminal sequence of purified zonulin from adult human brain (SEQ ID NO: 29) and fetal human brain (SEQ ID NO: 29) NO: 36) was completely different than the N-terminus of purified zonulin from each of the heart (SEQ ID NO: 28), fetal gut (SEQ ID NO: 30) and adult intestine (SEQ ID NO: 31) (see Figure 6-7). This difference is believed to explain the specific character of zonulin in tissues to determine the permeability of tissues, such as the intestine, as demonstrated above.

The N-terminal sequences of purified human zonulin from heart, intestine and brain, all differ from the N-terminal sequence of purified zonulin from rabbit intestine (Figure 6). To establish whether these proteins represent different isoforms of a family of tau proteins, rabbit and human tissues were subjected to 8.0% (w / v) SDS-PAGE, followed by Western immunostaining using either anti-ZOT or anti-ZOT antibodies. - tau The purified 47 kDa zonulin bands from rabbit and human tissues (including brain, intestine and heart) Or, which were found to be recognized by anti-ZOT antibodies, they were also found to cross-react with anti-tau antibodies. The different fractions of human brain purified zonulin obtained through salt chromatography were also subjected to Western immunostaining using either anti-ZOT antibodies or anti-tau antibodies. Although the anti-ZOT antibodies recognized the intact 47 kDa protein and the 35 kDa and the 15 kDa cleavage fragments, the anti-tau antibodies only recognized the intact 47 kDa protein and the 35 kDa fragment, whereas the antibodies anti-tau no 0 recognized the 15 kDa fragment. To establish whether the 35 kDa fragment includes the N term or the zonulin C terminus, the N-terminal sequence of the 35 kDa band was obtained and found to be: Xaa Xaa Asp Gly Thr Gly Lys Val Gly Asp Leu (SEC ID NO: 32). This sequence is different from the intact human brain zonulin N-terminal sequence (SEQ ID NO: 29). These results suggest that the 15 kDa fragment is the N-terminal portion of zonulin while the 35 kDa fragment represents the C terminal portion of zonulin. Combined together, these results suggest that the zonulin domain recognized by anti-tau antibodies is towards the C-terminus of the protein, it is common for different isoforms of zonulin from either human or rabbit tissues (although the N-terminal portion may vary ), and is probably involved in the permeabilization effect of the protein (based on the observation of tau binds to β-tubulin with subsequent rearrangement of the cell cytoskeleton, and the effect of fraction 4 on tissue resistance of the small intestine of monkey). The N-terminal sequence of purified human zonulin from both heart and intestine was compared to other protein sequences through BLAST search analysis. The result of this analysis revealed that the N-terminal sequence of human zonulin is 95% identical to the N-terminal sequence of the variable chain presumed of Homo Sapiens IgM (SEQ ID NO: 37). As a result, to determine if purified human zonulin from heart and IgM are the same portion, a partial digestion of human zonulin was performed to obtain an internal fragment which was then sequenced. More specifically, 1.0 mm of the PVDF filter containing purified human heart zonulin was placed in a plastic tube previously washed with 0.1% (w / v) trifluoroacetic acid (TFA), and rinsed with methanol. 75 μl of a pH buffer solution comprising 100 mM Tris (pH 8.2), 10% (v / v) CH3CN, and 1.0% (v / v) of dehydrogenated triton X-100 were added and incubated • with the membrane at 37 ° C for 60 minutes. Then 150 mg of trypsin was added and an additional incubation period of 24 hours was carried out at 37 ° C. The resulting solution was given sound for 10 minutes, and the supernatant was decanted. After 75μl of 0.1% (w / v) TFA was added, sound was applied to the solution for an additional 10 minutes, and the supernatant was decanted.

• Both aliquots were loaded on a 0.5 mm x 250 mm C18 column, with a particle size of 5.0 μm, and a pore size of 300 A. A gradient of 0.1% was developed for 2 hours and 15 minutes (p / v) from TFA to 45% CH3CN - water + 0.1% (w / v) TFA. The peaks were finally collected and sequenced. The internal sequence of human purified zonulin from adult human heart was found to be these: Leu Ser Glu Val Thr Ala Pro Ser Leu Asn Gly Gly (SEQ ID NO: 33). The internal sequence of human zonulin was compared with other protein sequences through search analysis BLAST. The result of this analysis revealed that the internal sequence of human zonulin has 0% identity to any internal sequence of the heavy variable chain of Homo sapiens IgM. The results in Example 3 above demonstrate that (1) zonulin represents the physiological modulator of the path paracellular; (2) the N-terminal sequence of rabbit zonulin is highly homologous to the N-terminal sequence of the tau protein; (3) zonulin and tau are two distinct portions that are immunologically related, yet functionally different; (4) the N-terminal sequence of human zonuin obtained from heart and intestine is highly homologous to the N-terminal sequence of the heavy chain of the IgM variable region; (5) human zonulin IgM are two different portions that are structurally related, yet functionally different; and (6) zonulin represents a family of tau-related proteins with common, active C-terminal sequences and variable N-terminal sequences.

EXAMPLE 4 ZONULINE PEPTIDE ANTAGONISTS Since ZOT, human intestinal zonulin (zonulin) and human heart zonuin (zonulinah) all act in intestinal tj (Fasano et al, Gastroenterology, 112: 839 (1997); Fasano et al, J. Clin. Invest., 96 : 710 (1995) and figures 1-5) and endothelial and that all 3 have a similar regional effect (Fasano et al (1997), supra, and figures 1-5) that coincides with the distribution of the ZOT receptor within the intestine (Fasano et al (1997), supra; and Fasano et al (1995), supra), postulated, in the present invention, that these 3 molecules interact with the same receptor binding site. In this way, a comparison of the primary amino acid structure of ZOT and human zonulins was made to provide clarifications as to the absolute structural requirements of the receptor-ligand interaction involved in the regulation of intestinal tj. He • Analysis of the N terms of these molecules revealed the following common motif (residues 8-15 of amino acid found in a box in figure 7): non-polar (Gly for intestine, Val for brain), variable, non-polar, variable , not polar, polar, variable, polar (Gly). The Gly in position 8, Val in position 12 and Gln in position 13, all are highly conserved in ZOT, zonulina ,, and zonulinah (see figure 7), which is believed to be critical for the function • receptor binding for the intestine. To verify the same, the synthetic octapeptide, Gly Gly Val Leu Val Gln Pro Gly (SEQ ID NO: 15) (designated FZI / 0, and corresponding to amino acid residues 8-15 of human fetal zonulin) was chemically synthesized.

Afterwards, the rabbit ileus mounted in Ussing chambers was exposed as described above, at 100 μg of 6xHis-ZOT (obtained as described in example 1), 1.0 μg of zonulin (obtained as described in example 3), or 1.0 μg of zonulinah (obtained as described in example 3) alone; or they were pre-exposed for 20 minutes to 100 μg of FZI / 0 or FZI / 1, at that time, 1.0 μg of 6xHis-ZOT, 1.0 μg of zonulin, was added, or 1. 0 μg of zonulinah. • Rt after calculation as described above. The results are shown in Figure 8. As shown in Figure 8, FZI / 0 did not induce any change significant in Rt (0.5% as compared to the negative control) (see closed bar). Otherwise, pre-treatment for 20 minutes with FZI / 0 reduced the effect of ZOT, zonulin, and zonulina on Rt by 75%, 97% and 100% (see open bars). Also as shown in Figure 8, this inhibitory effect was completely cut off when a second synthetic peptide (FZI / 0) was chemically synthesized by changing the Gly at the 8 position, the Val at the 12 position, and the Gln at the 13 position. (as designated for zonulin,) with the corresponding amino acid residues of zonulinab (Val, Gly, and Arg, respectively) was used (see shaded bars). The above results demonstrate that there is a region that expands between residues 28 and 15 of the N-terminal end of ZOT and the zonulin family that is crucial for binding to the target receptor, and that the amino acid residues in position 8, 12 and 13 determine the specific character of this union. Although the invention has been described in detail, and with reference to its specific embodiments, it will be apparent to those aspects in the art that various changes and modifications may be made therein without departing from the spirit and scope of the invention.

LIST OF SEQUENCES (1) GENERAL INFORMATION: • (i) APPLICANT: FASANO, Alessio 5 (ii) TITLE OF THE INVENTION: ANTAGONISTS OF ZONULIN PEPTIDE AND METHODS TO USE THEM (iii) NUMBER OF SEQUENCES: 40 (iv) ADDRESS OF CORRESPONDENCE: (A) RECIPIENT: SUGHRU, MION, ZINN, MACPEAK & SEAS, PLLC • (B) STREET: 2100 Pennsylvania Avenue, N.W., Suite 800 (C) CITY: Washington, D.C. (D) STATE: D.C. (E) COUNTRY: E.U.A. 15 (F) POSTAL CODE: 20037 (v) MEDIA LEGIBLE BY COMPUTER: (A) TYPE OF MEDIUM: flexible disk (B) COMPUTER: IBM PC compatible (C) OPERATUVO SYSTEM: PC-DOS / MS-DOS 20 (D) SOFTWARE: Patentln Reléase # 1.0 Version # 1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER (B) SUBMISSION DATE: AUGUST 3, 1998 (C) CLASSIFICATION: 25 (viii) EMPOWERED / AGENT INFORMATION: (A) NAME: KIT, Gordon (B) REGISTRATION NUMBER: 30,764 (C) REFERENCE / No. OF APPORERADO: A-7242 • (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (202) 293-7060 (B) TELEFAX: (202) 293-7860 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (i) type of molecule: peptide (iii) HYPOTHETICAL : No 15 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 Gly Arg Val Cys Gln Pro Gly 1 5 (2) INFORMATION FOR SEQ ID NO: 2: 20 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ií) type of molecule: peptide 25 (iii) HYPOTHETIC: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Gly Arg Val Cys Val Gln Asp Gly 1 5 • (2) INFORMATION FOR SEC ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear 10 (ii) molecule type: peptide (iii) HYPOTHETIC: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Gly Arg Val Leu Val Gln Pro Gly 1 5 15 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid 20 (D) TOPOLOGY: linear (ii) type of molecule: peptide ( I) HYPOTHETIC: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Gly Arg Val Leu Val Gln Asp Gly (2) INFORMATION FOR SEC ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids • (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) molecule type: peptide (iii) HYPOTHETIC: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: Gly Arg Leu Cys Val Gln Pro Gly 1 5 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids 15 (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) type of molecule: peptide (iii) HYPOTHETICAL : No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Gly Arg Leu Cys Val Gln Asp Gly 1 5 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: 25 (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) type of molecule: peptide (iii) HYPOTHETICAL : No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: Gly Arg Leu Leu Val Gln Pro Gly 1 5 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ií) type of molecule: peptide (iii) HYPOTHETICAL: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Gly Arg Leu Leu Val Gln Asp Gly 1 5 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) type of molecule: peptide (ii) HYPOTHETICAL : No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: Gly Arg Gly Cys Val Gln Pro Gly • 1 5 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) type of molecule: peptide (ni) HYPOTHETIC: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: Gly Arg Gly Cys Val Gln Asp Gly 15 1 5 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids 20 (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) type of molecule: peptide (iii) HYPOTHETICAL : No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11 Gly Arg Gly Leu Val Gln Pro Gly 1 5 • (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) type of molecule: peptide (iii) HYPOTHETICAL : No • (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: Gly Arg Gly Leu Val Gln Asp Gly 1 5 (2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear 20 (ii) type of molecule: peptide (iii) HYPOTHETIC: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: Gly Gly Val Cys Val Gln Pro Gly 1 5 25 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) molecule type: peptide (iii) HYPOTHETIC: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: Gly Gly Val Cys Val Gln Asp Gly 1 5 (2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) type of molecule: peptide (iii) HYPOTHETICAL: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: Gly Gly Val Leu Val Gln Pro Gly 1 5 (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) type of molecule: peptide • (iii) HYPOTHETICAL : No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: Gly Gly Val Leu Val Gln Asp Gly 1 5 (2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS: • (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) molecule type: peptide 15 (iii) HYPOTHETICAL: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: Gly Gly Leu Cys Val Gln Pro Gly • fifteen (2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear 25 (ii) type of molecule: peptide (iii) HYPOTHETIC: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: Gly Gly Leu Cys Val Gln Asp Gly • 1 5 (2) INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear • (i) type of molecule: peptide (iii) HYPOTHETICAL: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: Gly Gly Leu Leu Val Gln Pro Gly 15 1 5 (2) INFORMATION FOR SEQ ID NO: 20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids 20 (B) TYPE: amino acid (D) TOPOLOGY: linear (ií) type of molecule: peptide (iii) HYPOTHETICAL : No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: Gly Gly Leu Leu Val Gln Asp Gly 1 5 • (2) INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ií) type of molecule: peptide (iii) HYPOTHETICAL : No • < (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21 Gly Gly Gly Cys Val Gln Pro Gly 1 5 (2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids • (B) TYPE: amino acid (D) TOPOLOGY: linear 20 (ii) molecule type: peptide (iii) HYPOTHETIC: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: Gly Gly Gly Cys Val Gln Asp Gly 1 5 (2) INFORMATION FOR SEQ ID NO: 23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids • (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) type of molecule: peptide (iii) ) HYPOTHETICAL: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: Gly Gly Gly Leu Val Gln Pro Gly 1 5 • (2) INFORMATION FOR SEQ ID NO: 24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids 15 (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) type of molecule: peptide (iii) HYPOTHETICAL : No • (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: Gly Gly Gly Leu Val Gln Gly Asp 1 5 (2) INFORMATION FOR SEQ ID NO: 25: (i) SEQUENCE CHARACTERISTICS: 25 (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear # (ii) type of molecule: synthetic DNA (iii) HYPOTHETIC: No (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: TCATCACGGC GCGCCAGG 18 JO (2) INFORMATION FOR SEC ID NO: 26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRING STRUCTURE: individual 15 (D) TOPOLOGY: linear (ii) molecule type: synthetic DNA (iii) HYPOTHETIC: No (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: 20 GGGGTCTAG AATCTGCCCG AT 22 (2) INFORMATION FOR SEQ ID NO: 27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids 25 (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) type of molecule: peptide (iii) HYPOTHETICAL : No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: Asn Gln Arg Pro Pro Pro Aia Gly Val Thr Wing Tyr Asp Tyr Leu Val lie 1 5 10 15 Gln (2) INFORMATION FOR SEQ ID NO: 28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (i) type of molecule: peptide (iii) HYPOTHETICAL : No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 1 5 10 15 Leu Arg Leu 20 (2) INFORMATION FOR SEQ ID NO: 29: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) type of molecule: peptide (iii) HYPOTHETIC: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: Val Thr Phe Tyr Thr Asp Ala Val Ser 1 5 (2) INFORMATION FOR SEQ ID NO: 30: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) type of molecule: peptide (iii) HYPOTHETICAL: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30: Met Leu Gln Lys Ala Glu Ser Gly Gly Val Leu Val Gln Pro Gly Xaa Ser 1 5 10 15 Asn Arg Leu 20 (2) INFORMATION FOR SEQ ID NO: 31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) type of molecule: peptide (iii) HYPOTHETIC: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31 • Glu Val Gln Leu Val Glu Ser Gly Gly Xaa Leu 1 5 10 (2) INFORMATION FOR SEQ ID NO: 32: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 amino acids 0 (B) TYPE: amino acid • (D) TOPOLOGY: linear (ii) type of molecule: peptide (iii) HYPOTHETICAL: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: Xaa Xaa Asp Gly Thr Gly Lys Val Gly Asp Leu 1 5 10 # (2) INFORMATION FOR SEQ ID NO: 33: (i) SEQUENCE CHARACTERISTICS: 20 (A) LENGTH: 13 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ií) type of molecule: peptide (iii) HYPOTHETICAL: No 25 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: Leu Ser Glu Val Thr Ala Val Pro Ser Leu Asn Gly Gly 1 5 10 • (2) INFORMATION FOR SEQ ID NO: 34: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ií) type of molecule: peptide (iii) HYPOTHETICAL : No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34 Val Gly Val Leu Gly Arg Pro Gly 1 5 (2) INFORMATION FOR SEQ ID NO: 35: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear 20 (ii) type of molecule: peptide (iii) HYPOTHETIC: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35: Val Asn Gly Phe Gly Arg lie Gly 1 5 25 (2) INFORMATION FOR SEQ ID NO: 36: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids • (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) molecule type: peptide (iii) HYPOTHETIC: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: Xaa Gly Lys Val Lys Val Gly Val Asn Gly Phe Gly Arg lie Gly Arg lie O 1 5 10 15 • Gly Arg lie Gly Arg Leu Val lie 20 (2) INFORMATION FOR SEQ ID NO: 37: 15 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids (B) TYPE: amino acid • (D) TOPOLOGY: linear (ii) molecule type: peptide 20 (iii) HYPOTHETIC: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg Ser 1 5 10 15 Leu Arg Leu 25 20 (2) INFORMATION FOR SEQ ID NO: 38: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) ) type of molecule: peptide (iii) HYPOTHETIC: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: Phe Cys He Gly Arg Leu Cys Val Gln Asp Gly Phe Val Thr 1 5 15 (2) INFORMATION FOR SEQ ID NO: 39: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) ) type of molecule: synthetic DNA (iii) HYPOTHETIC: No (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39: CGGGATCCCG TATGAGTATC TTT 23 (2) INFORMATION FOR SEQ ID NO: 40: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) CHAIN STRUCTURE: individual • (D) TOPOLOGY: linear ( ii) type of molecule: synthetic DNA (iii) HYPOTHETIC: No (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40: CCCAAGCTTG GGTCAAAATA TACT 24 • •

Claims

1. - A zonulin peptide antagonist comprising a • amino acid sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO : 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 , SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 SEQ ID NO: 21, SEQ ID NO: 22 SEQ ID 0 NO: 23 SEQ ID NO: 24 and SEQ ID NO: 35, wherein said antagonist • The peptide binds to a zonula ocludenin toxin receptor, which still does not physically modulate the opening of mammalian tight junctions. 2.- A method for the treatment of inflammation Gastrointestinal comprising administering to a subject in need of such treatment, a pharmaceutically effective amount of a zonulin peptide antagonist, wherein said peptide antagonist comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEC ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO. : 19, SEQ ID NO: 20 SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, in Wherein said peptide antagonist binds to a zonula ocludenin toxin receptor in the intestine of said subject, which still does not physically modulate the opening of hermetic junctions in said intestine. • 3.- A method for the treatment of a condition associated with the breakdown of the blood-brain barrier, which comprises administering to a subject in need of such treatment, a pharmaceutically effective amount of a zonulin peptide antagonist, wherein the antagonist of peptide comprises the amino acid sequence SEQ ID NO: 35, wherein said peptide antagonist binds to the zonula ocludenin toxin receptor in the brain of said subject, which still does not physically modulate the opening of tight junctions in said brain.