Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4716—Muscle proteins, e.g. myosin, actin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/22—Hormones
  • A61K38/28—Insulins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4702—Regulators; Modulating activity
  • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
  • Y10S530/827—Proteins from mammals or birds
  • Y10S530/839—Nerves; brain
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
  • Y10S530/827—Proteins from mammals or birds
  • Y10S530/843—Digestive system
  • Y10S530/844—Stomach; intestine

Definitions

• the present invention relates to a substantially pure mammalian protein, hereinafter "zonulin", that is a physiological modulator of mammalian tight junctions, as well as methods for the use of the same.
• ZO zonula occludens
• ZO-1 and ZO-2 exist as a heterodimer (Gumbiner et al , Proc. Natl. Acad. Sci., USA, 8 . :3460-3464 (1991)) in a detergent-stable complex with an uncharacterized 130 kD protein (ZO-3) .
• ZO-3 uncharacterized 130 kD protein
• Rab 13 a small GTP binding protein has also recently been localized to 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, i.e., rho regulates actin-membrane attachment in focal contacts (Ridley et al, Cell , 20:389-399 (1992)), and rac regulates growth factor-induced membrane ruffling (Ridley et al, Cell , _70 . :401-410 (1992)).
• MARCKS myristoylated alanine-rich C kinase substrate
• PLC protein kinase C
• MARCKS Activated PKC phosphorylates MARCKS, which is released from the membrane (Rosen et al, J. Exp . Med. , 171:1211-1215 (1990); and Thelen et al , Nature, 151:320-322 (1991)).
• the actin linked to MARCKS is likely to be spatially separated from the membrane and be more plastic.
• MARCKS is dephosphorylated, it returns to the membrane where it once again crosslinks actin (Hartwig et al, supra; and Thelen et al, supra) .
• ZOT zonula occludens toxin
• ZOT increases the intestinal permeability by modulating the structure of intercellular tj (Fasano et al , supra) . It has been found that as a consequence of modification of the paracellular pathway, the intestinal mucosa becomes more permeable. It also was found that ZOT does not affect Na + -glucose coupled active transport, is not cytotoxic, and fails to completely abolish the transepithelial resistance (Fasano et al, supra) .
• ZOT is capable of reversibly opening tj in the intestinal mucosa, and thus ZOT, when co-administered with a therapeutic agent, is able to effect intestinal delivery of the therapeutic agent, when employed in an oral dosage composition for intestinal drug delivery
• a ZOT receptor has been identified and purified from an intestinal cell line.
• the ZOT receptor represents the first step of the paracellular pathway involved in the regulation of intestinal and nasal permeability.
• the blood-brain barrier is an extremely thin membranous barrier that is highly resistant to solute free diffusion, and separates blood and the brain. In molecular dimensions, the movement of drugs or solute through this membrane is essentially nil, unless the compound has access to one of several specialized enzyme-like transport mechanisms that are embebbed within the BBB membrane.
• the BBB is composed of multiple cells rather than a single layer of epithelial cells. Of the four different types of cells that compose the BBB (endothelial cells, perycites, astrocytes, and neurons) the endothelial cell component of the capillaries represents the limiting factor for the permeability of the BBB.
• endothelial tj are responsible for the limited permeability of the BBB.
• Lipid-soluble drugs with a molecular weight less than 600 Daltons, e.g., progesterone, aldosterone, orcortisol;
• Drugs that bind to plasma proteins e.g., propranolol, bupivacaine, imipramine, diazepam, or cyclosporin;
• Nutrients that bind to special carrier-mediated transport systems that are embebbed within both the lumenal and antilumenal membranes of the BBB e.g., glucose, glutamate, or phenylalanine
• Peptides that bind to specific receptors that mediate the peptide transcytosis through the BBB e.g., insulin and transferrin.
• mammalian proteins that are immunologically and functionally related to ZOT, and that function as the physiological modulator of mammalian tight junctions have been identified and purified. These mammalian proteins, referred to as "zonulin", are useful for enhancing absorption of therapeutic agents across tj of intestinal and nasal mucosa, as well as across tj of the BBB.
• An object of the present invention is to identify a mammalian protein that is immunologically and functionally related to ZOT, and that functions as the physiological modulator of mammalian tight junctions.
• Another object of the present invention is to purify and characterize said mammalian protein. Still another object of the present invention is to use said mammalian protein as an enhancer of the delivery of a therapeutic agent across mammalian tight junctions . Yet another object of the present invention is to use said mammalian protein as an enhancer of intestinal delivery of a therapeutic agent.
• An additional object of the present invention is to use said mammalian protein as an enhancer of nasal delivery of a therapeutic agent.
• Still another object of the present invention is to use said mammalian protein as an enhancer for the delivery of a therapeutic agent across the blood-brain barrier.
• a further object of the present invention is to provide pharmaceutical compositions effective for delivery of a therapeutic agent.
• substantially pure zonulin having an apparent molecular weight of about 47 kDa, as determined by SDS-polyacrylamide gel electrophoresis, which is recognized by both anti-tau polyclonal antibody and by anti-ZOT polyclonal antibody, and is capable of reversibly opening mammalian tight junctions.
• substantially pure zonulin having an apparent molecular weight of about 47 kDa, as determined by SDS-polyacrylamide gel electrophoresis, and an N-terminal amino acid sequence selected from the group consisting of:
• composition for delivery of a therapeutic agent comprising:
• zonulin has an apparent molecular weight of about 47 kDa, as determined by SDS-polyacrylamide gel electrophoresis, which is recognized by both anti-tau polyclonal antibody and by anti-ZOT polyclonal antibody, and is capable of reversibly opening mammalian tight junctions, and preferably, an N-terminal amino acid sequence selected from the group consisting of:
• Figure 1 shows the effect of zonulin purified from rabbit intestine ( ⁇ ) , as compared to various negative controls (Fraction 2 (O) ; Fraction 3 (•) ; Fraction 4 (A) ; and Fraction 5 (D) from a Q-Sepharose column) , on the tissue resistance (Rt) of CaCo2 cell monolayers .
• Figure 2 shows the effect of zonulin purified from rabbit intestine ( ⁇ ) , as compared to the negative control (D) , on the tissue resistance (Rt) of rabbit ileum mounted in Ussing chambers.
• Figure 3 shows the effect of zonulin purified from rabbit intestine ( ⁇ ) , as compared to the negative controls (zonulin + anti-ZOT antibody (D) ; zonulin + anti-tau antibody ( ⁇ ) ; and tau (A) ) , on the tissue resistance (Rt) of rabbit ileum mounted in
• Figures 4A and 4B show the effect of zonulin purified from either human heart (A) or human brain (D) , as compared to the negative control ( ⁇ ) , on the tissue resistance (Rt) of Rhesus monkey jejunum
• Figures 5A and 5B show the effect of zonulin purified from either human heart (A) or human brain (D) , as compared to the negative control ( ⁇ ) , on the tissue resistance (Rt) of rabbit jejunum (Figure
• Figure 5A shows a comparison of the N-terminal sequence of zonulin purified from rabbit and human tissues .
• the above-described object of the present invention have been met by substantially pure zonulin having an apparent molecular weight of about 47 kDa, as determined by SDS-polyacrylamide gel electrophoresis, which is recognized by both anti-tau polyclonal antibody and by anti-ZOT polyclonal antibody, and is capable of reversibly opening mammalian tight junctions, and preferably, an N-terminal amino acid sequence selected from the group consisting of: (1) Asn Gin Arg Pro Pro Pro Ala Gly Val Thr
• Zonulin is produced by, or found in, various mammalian cells and tissues, e.g., rabbit or human cells/tissue.
• mammalian cell/tissue type from which zonulin is derived is not critical to the present invention. Examples of such mammalian tissue types include heart, lung, intestine, liver, brain, kidney, spleen and pancreas.
• Zonulin can be obtained and purified, e.g., by affinity-purification chromatography using anti-ZOT antibodies, as described in Example 3 below.
• zonulin biological activity means the ability to reversibly increase permeability by modulating the structure of intercellular tj .
• a pharmaceutical composition for delivery of a therapeutic agent comprising: (A) a therapeutic agent; and
• the pharmaceutical composition is preferably an oral dosage composition for intestinal delivery, a nasal dosage composition for nasal delivery, or an intravenous dosage composition for the delivery through the blood-brain barrier.
• the oral dosage composition for intestinal delivery of a therapeutic agent comprises: (A) a therapeutic agent; and
• oral dosage compositions for small intestinal delivery e.g., small intestinal delivery
• Tablets are made gastroresistent by the addition of, e.g., either cellulose acetate phthalate or cellulose acetate terephthalate .
• Capsules are solid dosage forms in which the therapeutic agent (s) is enclosed in either a hard or soft, soluble container or shell of gelatin.
• the gelatin used in the manufacture of capsules is obtained from collagenous material by hydrolysis .
• Type A derived from pork skins by acid processing
• Type B obtained from bones and animal skins by alkaline processing.
• the use of hard gelatin capsules permit a choice in prescribing a single therapeutic agent or a combination thereof at the exact dosage level considered best for the individual subject.
• the hard gelatin capsule consists of two sections, one slipping over the other, thus completely surrounding the therapeutic agent. These capsules are filled by introducing the therapeutic agent, or gastroresistent beads containing the therapeutic agent, into the longer end of the capsule, and then slipping on the cap.
• Hard gelatin capsules are made largely from gelatin, FD&C colorants, and sometimes an opacifying agent, such as titanium dioxide.
• the USP permits the gelatin for this purpose to contain 0.15% (w/v) sulfur dioxide to prevent decomposition during manufacture.
• oral dosage compositions for small intestinal delivery also include liquid compositions which contain aqueous buffering agents that prevent the therapeutic agent and zonulin from being significantly inactivated by gastric fluids in the stomach, thereby allowing the therapeutic agent and zonulin to reach the small intestines in an active form.
• aqueous buffering agents which can be employed in the present invention include bicarbonate buffer (pH 5.5 to 8.7, preferably about pH 7.4).
• the oral dosage composition is a liquid composition, it is preferable that the composition be prepared just prior to administration so as to minimize stability problems. In this case, the liquid composition can be prepared by dissolving lyophilized therapeutic agent and zonulin in the aqueous buffering agent .
• the nasal dosage composition for nasal delivery of a therapeutic agent comprises: (A) a therapeutic agent; and
• Nasal dosage compositions for nasal delivery are well-known in the art.
• Such nasal dosage compositions generally comprise water-soluble polymers that have been used extensively to prepare pharmaceutical dosage forms (Martin et al, In : Physical Chemical Principles of Pharmaceutical Sciences, 3rd Ed., pages 592-638
• Microparticles with a diameter of about 50 ⁇ m are expected to deposit in the nasal cavity (Bjork et al , Tnt. J. Pharm . , 62 :187-192 (1990)); and Ilium et al, Int . J. Pharm. , 39:189-199 (1987) , while microparticles with a diameter under 10 ⁇ m can escape the filtering system of the nose and deposit in the lower airways. Microparticles larger than 200 ⁇ m in diameter will not be retained in the nose after nasal administration (Lewis et al, Proc . Int . Symp . Control Rel . Bioact . Ma ter . , 12:280-290 (1990) ) .
• the particular water-soluble polymer employed is not critical to the present invention, and can be selected from any of the well-known water-soluble polymers employed for nasal dosage forms .
• a typical example of a water-soluble polymer useful for nasal delivery is polyvinyl alcohol (PVA) .
• PVA polyvinyl alcohol
• This material is swellable hydrophiiic polymer whose physical properties depend on the molecular weight, degree of hydrolysis, cross-linking density, and crystallinity
• PVA can be used in the coating of dispersed materials through phase separation, spray-drying, spray-embedding, and spray-densation (Ting et al, supra) .
• the intravenous dosage composition for delivery of a therapeutic agent through the blood-brain barrier comprises:
• Intravenous dosage compositions for delivery to the brain are well-known in the art.
• Such intravenous dosage compositions generally comprise a physiological diluent, e.g., distilled water, or 0.9% (w/v) NaCl.
• a “nasal" delivery composition differs from an “intestinal” delivery composition in that the latter must have gastroresistent properties in order to prevent the acidic degradation of the active agents (e.g., zonulin and the therapeutic agent) in the stomach, whereas the former generally comprises water-soluble polymers with a diameter of about 50 ⁇ m in order to reduce the mucociliary clearance, and to achieve a reproducible bioavailability of the nasally administered agents.
• An “intravenous" delivery composition differs from both the “nasal” and intestinal” delivery compositions in that there is no need for gastroresistance or water-soluble polymers in the "intravenous” delivery composition.
• the above-described objects of the present invention have been met by a method for delivery of a therapeutic agent comprising administering, to a subject in need thereof, said pharmaceutical composition.
• the mode of administration is not critical to the present. Although, it is preferable that the mode of administration is orally, for the intestinal delivery composition; intranasally, for the nasal delivery composition; and intravenously for delivery through the blood-brain barrier.
• the particular therapeutic agent employed is not critical to the present invention, and can be, e.g., any drug compound, biologically active peptide, vaccine, or any other moiety otherwise not absorbed through the transcellular pathway, regardless of size or charge .
• drug compounds which can be employed in the present invention include drugs which act on the cardiovascular system, drugs which act on the central nervous system, antineoplastic drugs and antibiotics .
• drugs which act on the cardiovascular system which can be employed in the present invention include lidocaine, adenosine, dobutamine, dopamine, epinephrine, norepinephrine and phentolamine .
• drugs which act on the central nervous system include doxapram, alfentanil, dezocin, nalbup ine, buprenorphine , naloxone, ketorolac, midazolam, propofol , metacurine, mivacurium and succinylcholine .
• antineoplastic drugs which can be employed in the present include cytarabine, mitomycin, doxorubicin, vincristine and vinblastine.
• antibiotics which can be employed in the present include methicillin, mezlocillin, piperacillin, cetoxitin, cefonicid, cefmetazole and aztreonam.
• biologically active peptides which can be employed in the present invention include hormones, lymphokines, globulins, and albumins.
• hormones which can be employed in the present invention include testosterone, nandrolene, menotropins, progesterone, insulin and urofolltropin.
• globulins which can be employed in the present invention include ⁇ -globulins, 3-globulins and ⁇ -globulins (immunoglobulin) .
• immunoglobulins which can be employed in the present invention include polyvalent IgG or specific IgG, IgA and IgM, e.g., anti-tetanus antibodies .
• An example of albumin which can be employed in the present invention is human serum albumin and ovalbumin .
• peptide antigens which can be employed in the present invention include the B subunit of the heat-labile enterotoxin of enterotoxigenic E. coli , the B subunit of cholera toxin, capsular antigens of enteric pathogens, fimbriae or pili of enteric pathogens, HIV surface antigens, dust allergens and acari allergens.
• Attenuated microorganisms and viruses which can be employed in the present invention include those of enterotoxigenic Escherichia coli , enteropathogenic Escherichia coli , Vibrio cholerae, Shigella flexneri , Salmonella typhi , Helicobacter pylori and rotavirus (Fasano et al, In : Le Vaccinazioni in Pediatria, Eds. Vierucci et al , CSH, Milan, pages 109-121 (1991) ; Guandalini et al, In : Management of Digestive and Liver Disorders in Infants and Children, Elsevior, Eds.
• the pharmaceutical composition of the present invention is useful for the treatment of diabetes.
• the amount of therapeutic agent employed is not critical to the present invention and will vary depending upon the particular agent selected, the disease or condition being treated, as well as the age, weight and sex of the subject being treated.
• the amount of zonulin employed is also not critical to the present invention and will vary depending upon the age, weight and sex of the subject being treated. Generally, the final concentration of zonulin employed in the present invention to enhance absorption of the therapeutic agent is in the range of about 10 "5 M to 10 "14 M, preferably about 10 "6 M to 5.0 x 10 "8 M. To achieve such a final concentration in, e.g., the intestines, nose or blood, the amount of zonulin in a single pharmaceutical composition of the present invention will generally be about 40 ng to 1000 ng, preferably about 400 ng to 800 ng.
• the ratio of therapeutic agent to zonulin employed is not critical to the present invention and will vary depending upon the amount of therapeutic agent to be delivered within the selected period of time. Generally, the weight ratio of therapeutic agent to zonulin employed in the present invention is in the range of about 1:10 to 3:1, preferably about 1:5 to 2:1.
• Zonulin can be also be used as an immunogen to obtain antibodies, either polycolonal or monoclonal, having binding specificity for zonulin, using techniques well-known in the art (Abrams, Methods Enzymol . , 121:107-119 (1986)). These antibodies can in turn can be used to assay for zonulin in body tissue or fluids, or in affinity-purification of zonulin.
• the zot gene was fused in frame with the maltose binding protein (hereinafter "MBP") gene to create a MBP-ZOT fusion protein.
• MBP maltose binding protein
• the MBP vector pMAL-c2 (Biolab) was used to express and purify ZOT by fusing the zot gene to the malE gene of E. coli .
• This construct uses the strong, inducible tac promoter, and the malE translation initiation signals to give high level expression of the cloned zot gene.
• the vector pMAL-c2 has an exact deletion of the malE signal sequence, which leads to cytoplasmic expression of the fusion protein. Affinity chromatography purification for MBP was used to facilitate isolation of the fusion protein (Biolab) .
• vector pMAL-c2 was linearized with EcoRI (that cuts at the 3' end of the malE gene) , filled in with Klenow fragment, and digested with Xbal
• Plasmid pBB241 was digested with BssHII, filled in with Klenow fragment, and digested with Xbal. Then, the blunt-X al fragment was subcloned into pMAL-c2 to give plasmid pLClO-c. Since both the insert, and the vector had blunt and sticky ends, the correct orientation was obtained with the 3' end of malE fused with the 5' terminus of the insert. pLClO-c was then electroporated into E. coli strain DH5 ⁇ . In pBB241, the BssHII restriction site is within the zot orf. Thus, amino acids 1-8 of ZOT are missing in the MBP-ZOT fusion protein. In order to purify the MBP-ZOT fusion protein,
• the bacteria were then pelleted and resuspended in 20 ml of ice cold "column buffer" comprising 20 mM Tris-HCl, 0.2 M NaCl, 1.0 mM EDTA, 10 mM 2 -ME, 1.0 mM NaN 3 .
• the bacterial suspension was lysed by french press treatment and spun for 30 min at 13,000 x g at 4°C.
• the supernatant was collected, diluted 1:5 with column buffer and loaded into a 1 X 10 column of amylose resin (Biolabs, MBP-fusion purification system), pre-equilibrated with column buffer. After washing the column with 5 volumes of column buffer, the MBP-ZOT fusion protein was eluted by loading 10 ml of
• the MBP fusion partner of the purified MBP-ZOT fusion protein was then cleaved off using 1.0 ⁇ g of Factor Xa protease (Biolabs) per 20 ⁇ g of MBP-ZOT.
• Factor Xa protease cleaves just before the amino terminus of ZOT.
• the ZOT protein so obtained was run on a 8.0% (w/v) SDS-PAGE gel, and electroeluted from the gel using an electroseparation chamber (Schleicher & Schuell, Keene, NH) .
• the resulting purified ZOT When tested in Ussing chambers, the resulting purified ZOT induced a dose-dependent decrease of Rt , with an ED 50 of 7.5 x 10 "8 M.
• GST glutathione S-transferase
• oligonucleotide primers were used to amplify the zot orf by polymerase chain reaction (PCR) using plasmid pBB241 (Baudry et al , supra) as template DNA.
• the forward primer (TCATCACGGC GCGCCAGG, SEQ ID NO: 5) corresponded to nucleotides 15-32 of zot orf
• the reverse primer (GGAGGTCTAG AATCTGCCCG AT, SEQ ID NO: 6) corresponded to the 5' end of ctxA orf. Therefore, amino acids 1-5 of ZOT were missing in the resulting fusion protein.
• pGEX-2T is a fusion-protein expression vector that expresses a cloned gene as a fusion protein with GST of Schistosoma japonicum.
• the fusion gene is under the control of the tac promoter. Upon induction with IPTG, derepression occurs and GST fusion protein is expressed.
• the resulting recombinant plasmid was electroporated in E. coli DH5c_.
• 10 ml of Luria Bertani broth containing 100 ⁇ g/ml ampicillin were inoculated with a single colony containing pLCll, 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 shaking, to about 1.0 x 10 8 cells/ml.
• 0.2 mM IPTG was then added to induce the GST-ZOT expression, and the culture was incubated at 37°C for additional 3 hr.
• the bacteria were then pelleted, resuspended in 20 ml of ice cold PBS (pH 7.4), and lysed by the french press method.
• the GST-ZOT fusion protein was not soluble under these conditions as it sedimented with the bacterial pellet fraction. Therefore, the pellet was resuspended in Laemli lysis buffer 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% (v/v) glycerol, and subjected to electrophoresis on a 8.0% (w/v) PAGE-SDS gel, and stained with Coomassie brilliant blue.
• a band of about 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) .
• the rabbit antiserum was found to recognize ZOT, as well as MBP-ZOT and GST-ZOT fusion proteins, but not the MBP negative control.
• the anti-ZOT antibodies were affinity-purified using an MBP-ZOT affinity column. More specifically, a MBP-ZOT affinity column was prepared by immobilizing, overnight at room temperature, 1.0 mg of purified MBP-ZOT, obtained as described in Example 1 above, to a pre-activated gel (Aminolink, Pierce) . The column was washed with PBS, and then loaded with 2.0 ml of anti-ZOT rabbit antiserum.
• the column was washed with 14 ml of PBS, and the specific anti-ZOT antibodies were eluted from the column with 4.0 ml of a solution comprising 50 mM glycine (pH 2.5), 150 mM NaCl, and 0.1% (v/v) Triton X-100.
• the pH of the 1.0 ml eluted fractions was immediately neutralized with 1.0 N NaOH.
• ZOT interacts with a specific epithelial surface receptor, with subsequent activation of a complex intracellular cascade of events that regulate tj permeability
• ZOT may mimic the effect of a physiological modulator of mammalian tj .
• ZOT, and its physiological analog (zonulin) would be functionally and immunologically related. Therefore, affinity-purified anti-ZOT antibodies and the Ussing chamber assay were used in combination to search for zonulin in various rabbit and human tissues.
• zonulin was purified from rabbit intestine.
• the tissue was disrupted by homogenization in PBS.
• the resulting cell preparations were than centrifuged at 40,000 rpm for 30 min, the supernatant collected and lyophilized.
• the resulting lyophilized product was subsequently reconstituted in PBS
• the fractions obtained from the Q-Sepharose column were then tested for their tissue resistance effects on both CaCo2 monolayers, and rabbit small intestine in Ussing chambers.
• CaCo2 cells were grown in cell-culture flasks (Falcon) under humidified atmosphere of 95% 0 2 /5% C0 2 at 37°C in Dulbecco's modified Eagle's medium containing 10% (v/v) fetal-calf serum, 40 ⁇ g/1 penicillin and 90 ⁇ g/1 streptomycin.
• the cells were subcultured at a surface ratio of 1:5 after trypsin treatment every 5 days, when they had reached 70-80% confluence.
• the passage number of the cells used in the this study varied between 15 and 30.
• the CaCo2 monolayers were grown to confluence
• the zonulin-containing fraction induced a significant reduction of CaCo2 monolayers' resistance, as compared to zonulin-negative fractions.
• Ussing chamber assays were carried out using ileum from 2-3 kg adult male New Zealand white rabbits, which were sacrificed by cervical dislocation. A 20 cm segment of ileum was removed, rinsed free of the intestinal content, opened along the mesenteric border, and stripped of muscular and serosal layers. Eight sheets of mucosa so prepared were then mounted in lucite Ussing chambers (1.12 cm 2 opening) , connected to a voltage clamp apparatus
• the zonulin-containing fraction induced a significant reduction in rabbit small intestinal resistance, as compared to a zonulin-negative fraction. This effect was completely reversible once zonulin was withdrawn from the reservoir.
• the zonulin-positive fraction was also subjected to 8.0% (w/v) SDS-PAGE, followed by Western immunoblotting using the anti-ZOT antibodies.
• the protein bands separated by SDS-PAGE were then transferred onto PVDF filter (Millipore) using CAPS buffer comprising 100 ml of (3- [cyclohexylamino] -1 propanesulfonic acid) lOx, 100 ml of methanol, 800 ml of distilled water.
• the protein that aligned to a single band that was detected by Western immunoblotting had an apparent molecular weight of about 47 kDa.
• the rabbit zonulin N-terminal sequence was compared to other protein sequences by 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 tau protein from Homo sapiens .
• rabbit zonulin induced the typical decrease of tissue resistance that was readily reversible once the protein was withdrawn from the Ussing chambers . This activity was completely neutralized by pre-treatment with anti-ZOT antibodies, but not by pre-treatment with anti-tau antibodies. On the other hand, there was no significant effect on tissue resistance in tissues exposed to tau protein.
• Rabbit zonulin was also detected in various other rabbit tissues, i.e., rabbit heart, brain, muscle, stomach, spleen, lung, kidney, as well as various portions of rabbits intestines, i.e., distal jejunum, proximal jejunum, ileum, caecum and colon. That is, when these rabbit tissues were processed in the same manner as the rabbit intestine, discussed above, and subjected to 8.0% (w/v) SDS-PAGE, followed by Western immunoblotting using affinity-purified anti-ZOT antibodies obtained as described in Example 2 above, a single band of approximately 47 kDa in size was detected in all of the tissues tested.
• Zonulin was also purified from several human tissues, including intestine, heart, and brain. Both fetal and adult tissues were used. The tissues were disrupted by homogenization in PBS. The resulting cell preparations were than centrifuged at 40,000 rpm for 30 min, the supernatant 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 immunoblotting using the affinity-purified anti-ZOT antibodies obtained as described in Example 2 above .
• Fraction 2 (40% (w/v) NaCl) showed two additional bands of 35 kDa and 15 kDa in size in the Western immunoblot assay.
• Fraction 3 (60% (w/v) NaCl)
• Fraction 4 80% (w/v) showed only the 35 kDa and 15 kDa bands.
• Fraction 1 from both human heart and brain tissues
• Fraction 4 from heart tissue obtained from the Q-Sepharose column were then tested for their tissue resistance effects on both rabbit intestine and Rhesus monkey intestine in Ussing chambers.
• Ussing chamber assays were carried out using different tracts of intestine, including jejunum, ileum, or colon from either 2-3 kg adult male New Zealand white rabbits, or 5-6 kg adult male Rhesus monkeys. After the animals were sacrificed, different segments of intestine, including jejunum, ileum, and colon, were removed, rinsed free of the intestinal content, opened along the mesenteric border, and stripped of muscular and serosal layers. Eight sheets of mucosa so prepared (three jejunum, three ileum, and two colon) were then mounted in lucite Ussing chambers
• the bathing solution was maintained at 37°C with water-jacketed reservoirs connected to a constant-temperature circulating pump and gassed with 95% 0 2 /5% C0 2 .
• zonulin purified from human heart (Fraction 1) induced a significant reduction in monkey intestinal resistance (both jejunum ( Figure 4A) and ileum ( Figure 4B) , as compared to the PBS negative control. No significant changes were observed when zonulin purified from human heart was tested in the colon.
• Figures 4A and 4B also show that no significant effect on both monkey jejunum (Figure 4A) and monkey ileum (Figure 4B) was observed when zonulin purified from human brain (Fraction 1) was tested.
• Fraction 4 of zonulin purified from human heart also induced a significant decrease in monkey small intestinal tissue resistance.
• Figures 5A and 5B similar results were obtained when rabbit intestine was used.
• zonulin purified from human heart showed a significant effect on tissue resistance both in the rabbit jejunum (Figure 5A) and rabbit ileum (Figure 5B) , but not in the colon.
• Figures 5A and 5B also show that no significant effect on both rabbit jejunum (Figure 5A) and rabbit ileum (Figure 5B) was observed when zonulin purified from human brain (Fraction 1) was tested.
• zonulin increases the oral delivery of insulin, in vi tro model experiments using rabbit intestine were performed. Briefly, adult male New Zealand white rabbits (2-3 kg) were sacrificed by cervical dislocation.
• the bathing solution was maintained at 37°C with water- jacketed reservoirs connected to a constant-temperature circulating pump and gassed with 95% 0 2 /5% C0 2 .
• Potential difference (PD) was measured, and short-circuit current (Isc) and tissue resistance (Rt) were calculated.
• a 1.0 ml aliquot from the serosal side and a 50 ⁇ l aliquot from the mucosal side were immediately obtained to establish baseline values. Samples from the serosal side were then collected at 20 min intervals for the following 100 min.
• Fraction 1 of zonulin purified from human heart and Fraction 1 of zonulin purified from human brain were also subjected to 8.0% (w/v) SDS-PAGE, followed by Western immunoblotting using the anti-ZOT antibodies obtained as described in Example 2 above.
• the protein bands separated by SDS-PAGE were then transferred onto PVDF filter using CAPS buffer comprising 100 ml of (3 - [cyclohexylamino] -1 propanesulfonic acid) lOx, 100 ml of methanol, 800 ml of distilled water.
• the protein that aligned to a single band that was detected by Western immunoblotting had an apparent molecular weight of about 47 kDa.
• the first nine amino acids from the N-terminal sequence of zonulin purified from adult human intestine (SEQ ID NO: 7) were also sequenced, and found to be identical to the first nine amino acids of zonulin purified from human heart shown in SEQ ID NO: 2
• N-terminal sequence of zonulin purified from human fetal intestine were also sequenced: Met Leu Gin Lys
• N-terminal sequence of zonulin purified from adult human brain was completely different than the N-terminal of zonulin purified from both heart and intestine (SEQ ID NO: 2) (see Figure 6) . This difference is believed to explain the tissue-specificity of zonulin in determining the permeability of tissues, such as the intestine, demonstrated above .
• N-terminal sequences of human zonulin purified from heart, intestine, and brain all differ from the N-terminal sequence of zonulin purified from rabbit intestine ( Figure 6) .
• tissues from both rabbit and human were subjected to 8.0% (w/v) SDS-PAGE, followed by Western immunoblotting using either anti-ZOT or anti-tau antibodies.
• the 47 kDa zonulin bands purified from both rabbit and human tissues (including brain, intestine, and heart) which were found to be recognized by the anti-ZOT antibodies, were also found to cross-react with anti-tau antibodies.
• the different fractions of zonulin purified from human brain obtained by salt chromatography were also subjected to Western immunoblotting using either anti-ZOT antibodies or anti-tau antibodies. While anti-ZOT antibodies recognized the intact 47 kDa protein and both of the 35 kDa and 15 kDa breakdown fragments, the anti-tau antibodies only recognized the intact 47 kDa protein and the 35 kDa fragment, while the anti-tau antibodies did not recognize the 15 kDa fragment.
• 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 (SEQ ID NO: 8) .
• This sequence is different from the N-terminal sequence of the intact human brain zonulin (SEQ ID NO: 3) .
• the zonulin domain recognized by the anti-tau antibodies is toward the C-terminus of the protein, is common to the different isoforms of zonulin from either human or rabbit tissues (while the N-terminal portion may vary) , and is probably involved in the permeabilizing effect of the protein (based on the observation that tau binds to ?-tubulin with subsequent rearrangement of the cell cytoskeleton, and the effect of Fraction 4 on monkey small intestinal tissue resistance) .
• the N-terminal sequence of human zonulin purified from both the heart and intestine was compared to other protein sequences by 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 heavy variable chain of IgM from Homo sapiens .
• 1.0 mm of the PVDF filter containing zonulin purified from human heart was placed in a plastic tube previously washed with 0.1% (w/v) trifluoracetic acid (TFA) , and rinsed with methanol.
• 75 ⁇ l of a buffer solution comprising 100 mM Tris (pH 8.2), 10% (v/v) CH 3 CN, and 1.0% (v/v) dehydrogenated Triton X-100 was added, and incubated with the membrane at 37°C for 60 min.
• 150 ng of trypsin was then added, and an additional 24 h incubation period at 37°C was carried out.
• the resulting solution was sonicated for 10 min, and the supernatant decanted.
• the internal sequence of human zonulin purified from adult human heart was found to be : Leu Ser Glu Val Thr Ala Val Pro Ser Leu Asn Gly Gly (SEQ ID NO: 9) .
• the human zonulin internal sequence was compared to other protein sequences by BLAST search analysis. 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 IgM from Homo sapiens .
• Example 3 demonstrate that (1) zonulin represents the physiological modulator of the paracellular pathway; (2) the N-terminal sequence of rabbit zonulin is highly homologous to the
• zonulin and tau are two distinct moieties that are immunologically related, yet functionally different;
• the N-terminal sequence of human zonulin obtained from heart and intestine is highly homologous to the N-terminal sequence of the heavy chain of the variable region of IgM; (5) human zonulin and IgM are two distinct moieties 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 .
• ADDRESSEE SUGHRUE, MION, ZINN, MACPEAK & SEAS
• MOLECULE TYPE synthetic DNA
• HYPOTHETICAL NO
• ANTI -SENSE NO
• SEQUENCE DESCRIPTION SEQ ID NO : 5 :
• MOLECULE TYPE synthetic DNA
• HYPOTHETICAL NO
• ANTI -SENSE NO
• SEQUENCE DESCRIPTION SEQ ID NO : 6 :

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