Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/08—Peptides having 5 to 11 amino acids
  • A61K38/09—Luteinising hormone-releasing hormone [LHRH], i.e. Gonadotropin-releasing hormone [GnRH]; Related peptides
• • 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
• • 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
  • A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
  • A61K47/6921—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
  • A61K47/6927—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
  • A61K47/6929—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
  • A61K47/6931—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
  • A61K47/6935—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
  • A61K47/6937—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol the polymer being PLGA, PLA or polyglycolic acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
  • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
  • A61K9/51—Nanocapsules; Nanoparticles
  • A61K9/5107—Excipients; Inactive ingredients
  • A61K9/513—Organic macromolecular compounds; Dendrimers
  • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
  • A61K9/5153—Polyesters, e.g. poly(lactide-co-glycolide)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
  • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/06—Antimigraine agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
  • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/06—Antianaemics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/12—Antihypertensives
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
  • C07K1/047—Simultaneous synthesis of different peptide species; Peptide libraries
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
• • 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
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/10—Tetrapeptides
  • C07K5/1002—Tetrapeptides with the first amino acid being neutral
  • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
  • C07K5/1013—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/10—Tetrapeptides
  • C07K5/1002—Tetrapeptides with the first amino acid being neutral
  • C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• the present invention relates generally to random peptides capable of specific binding to gastro-intestinal tract (GIT) transport receptors.
• this invention relates to peptide sequences and motifs, as well as derivatives thereof, which enhance drug delivery and transport through tissue, such as epithelial cells lining the lumenal side of the gastro-intestinal tract (GIT) .
• Production of peptides, derivatives and antibodies is also provided.
• the invention further relates to pharmaceutical compositions, formulations and related methods.
• M13 phage and, in particular, protein pIII of M13.
• the viral capsid protein of M13, protein III (pill) is responsible for infection of bacteria.
• pill protein III
• the C-terminus anchors the protein to the viral coat, while portions of the N-terminus of pill are essential for interaction with the E. coli pillin protein (Crissman, J.W. and Smith, G.P., 1984, Virology 132 : 445- 455) .
• viral capsid proteins for expression of non-viral DNA on the surface of phage particles.
• the major capsid protein pVIII was so used by Cesareni, G., 1992, FEBS Lett. 307 : 66-70.
• Other bacteriophage than M13 have been used to construct peptide libraries.
• Four and six amino acid sequences corresponding to different segments of the Plasmodium falciparum major surface antigen have been cloned and expressed in the filamentous bacteriophage fd (Greenwood, J., et al., 1991, J. Mol. Biol. 120: 821-827).
• Kay et al . , 1993, Gene 128 : 59-65 discloses a method of constructing peptide libraries that encode peptides of totally random sequence that are longer than those of any prior conventional libraries.
• the libraries disclosed in Kay encode totally synthetic random peptides of greater than about 20 amino acids in length.
• Such libraries can be advantageously screened to identify peptides, polypeptides and/or other proteins having binding specificity for a variety of ligands. (See also U.S. Patent No. 5,498,538 dated March 12, 1996; and PCT Publication No. WO 94/18318 dated August 18, 1994.)
• peptide libraries are excellent sources for identifying epitopes or epitope-like molecules of that antibody (Yayon et al . , 1993, Proc. Natl. Acad. Sci. USA 90:10643-10647) . McCafferty et al .
• the heavy and light chain V-C regions were engineered to combine in the periplasm to produce an antibody-like molecule with a functional antigen binding site.
• Infection of cells harboring this phagemid with helper phage resulted in the incorporation of the antibody-like molecule on the surface of phage that carried the phagemid DNA. This allowed for identification and enrichment of these phage by screening with the antigen. It was suggested that the enriched phage could be subject to mutation and further rounds of screening, leading to the isolation of antibody-like molecules that were capable of even stronger binding to the antigen.
• naive antibody genes might be cloned into phage display libraries. This would be followed by random mutation of the cloned antibody genes to generate high affinity variants.
• Intravenous drug administration suffers from numerous limitations, including (i) the risk of adverse effects resulting from rapid accumulation of high concentrations of drug, (ii) repeated injections which can cause patient discomfort; and (iii) the risk of infection at the site of repeated injections.
• Subcutaneous injection is not generally suitable for delivering large volumes or for irritating substances.
• oral administration is generally more convenient, it is limited where the therapeutic agent is not efficiently absorbed by the gastrointestinal tract.
• Site specific drug delivery or drug targeting can be achieved at different levels, including (i) primary targeting to a specific organ, (ii) secondary targeting to a specific cell type within that organ and (iii) tertiary targeting where the drug is delivered to specific intracellular structures ⁇ e . g. , the nucleus for genes)
• DDS Drug Delivery Systems
• anticancer drugs are toxic to the body as well as to malignant cells. If a drug, or a delivery system, can be modified so that it "homes in” on the tumor, then by maximizing the drug concentration at the disease site, the anti-cancer effect can be exploited to the full, while toxicity is greatly reduced.
• Tumors contain antigens which provoke the body to respond by producing antibodies designed to attach to the antigens and destroy them.
• Monoclonal antibodies are being used as both delivery vehicles targeted to tumor cells (reviewed by Pietersz, G.A., 1990, Bioconjugate Chem. 1:89-95) and as imaging agents to carry molecules of drug or imaging agent to the tumor surface.
• the epithelial cells lining the lumenal side of the GIT are a major barrier to drug delivery following oral administration.
• transport pathways which can be exploited to facilitate drug delivery and transport: the transcellular, paracellular , carrier- mediated, and transcytotic pathways.
• the ability of a conventional drug, peptide, protein, macromolecule or nano- or microparticulate system to "interact" with one of these transport pathways may result in increased delivery of that drug or particle from the GIT to the underlying circulation.
• the receptor-mediated, carrier- mediated or transcytotic transport pathways some of the uptake signals have been identified.
• These signals include, inter alia , folic acid, which interacts with the folate receptor, and cobalamin, which interacts with Intrinsic Factor.
• leucine- and tyrosine-based peptide sorting motifs or internalization sequences exist, such as YSKV, FPHL, YRGV, YQTI , TEQF, TEVM, TSAF, and YTRF (SEQ ID NOS:203, 204, 205, 206, 207, 208, 209, and 210, respectively) , which facilitate uptake or targeting of proteins using specific membrane receptors or binding sites to identify peptides that bind specifically to the receptor or binding site.
• Non-receptor based assays to discover particular ligands have also been used. For instance, a strategy for identifying peptides that alter cellular function by scanning whole cells with phage display libraries is disclosed in Fong et al . , Drug Development Research 33:64-70 (1994) . However, because whole cells, rather than intact tissue or polarized cell cultures, are used for screening phage display libraries, this procedure does not provide information regarding sequences whose primary function includes affecting transport across polarized cell layers.
• Phage from a random phage library is plated onto or brought into contact with a first side, preferably the apical side, of a tissue sample, either in vitro, in vivo or in si tu, or polarized tissue cell culture.
• the phage which is transported to a second side of the tissue opposite the first side, preferably the basolateral side, is harvested to select transported phages .
• the transported phages are amplified in a host and this cycle is repeated (using the transported phage from the most recent cycle) to obtain a selected phage library containing phage which can be transported from the first side to the second side.
• the present invention relates generally to random peptides and peptide motifs capable of specific binding to GIT transport receptors.
• Such proteins can be identified using any random peptide library, e . g. , a chemically synthesized peptide library or a biologically expressed peptide library.
• a biological peptide expression library is used, the nucleic acid which encodes the peptide which binds to the ligand of choice can be recovered, and then sequenced to determine its nucleotide sequence and hence deduce the amino acid sequence that mediates binding.
• the amino acid sequence of an appropriate binding domain can be determined by direct determination of the amino acid sequence of a peptide selected from a peptide library containing chemically synthesized peptides.
• direct amino acid sequencing of a binding peptide selected from a biological peptide expression library can also be performed.
• this invention relates to proteins (e . g. , peptides) that are capable of facilitating transport of an active agent through a human or animal gastrointestinal tissue, and derivatives (e.g., fragments) and analogs thereof, and nucleotide sequences coding for said proteins and derivatives.
• proteins e . g. , peptides
• derivatives e.g., fragments
• the tissue through which transport is facilitated is of the duodenum, jejunum, ileum, ascending colon, transverse colon, descending colon, or pelvic colon.
• the tissue is most preferably epithelial cells lining the lumenal side of the GIT.
• the proteins of the invention have use in facilitating transport of active agents from the lumenal side of the GIT into the systemic blood system, and/or in targeting active agents to the GIT.
• a protein of the invention to an orally administered drug, the drug can be targeted to specific receptor sites or transport pathways which are known to operate in the human gastrointestinal tract, thus facilitating its absorption into the systemic system.
• the invention also relates to derivatives and analogs of the invention which are functionally active, i.e., they are capable of displaying one or more known functional activities associated with a full-length peptide.
• Such functional activities include but are not limited to antigenicity (ability to bind or to compete with GIT transport receptor-binding peptides for binding to an anti- GIT transport receptor antibody) and ability to bind or compete with full-length peptide for binding to a GIT transport receptor.
• the invention further relates to fragments of (and derivatives and analogs thereof) GIT transport receptor- binding peptides which comprise one or more motifs of a GIT transport receptor-binding peptide.
• Antibodies to GIT transport receptor-binding peptides and GIT transport receptor-binding peptide derivatives and analogs are additionally provided.
• the present invention also relates to therapeutic methods, pharmaceutical compositions and formulations based on GIT transport receptor-binding peptides.
• Formulations of the invention include but are not limited to GIT transport receptor-binding peptides or motifs and derivatives (including fragments) thereof; antibodies thereto; and nucleic acids encoding the GIT transport receptor-binding peptides or derivatives associated with an active agent.
• the active agent is a drug or drug-containing nano- or microparticle .
• the GIT transport-receptor binding proteins of the invention can also be used to determine levels of the GIT transport receptors in a sample by binding thereto.
• the GIT transport-receptor binding proteins can also be used to identify molecules that bind thereto, by contacting candidate test molecules under conditions conducive to binding, and detecting any binding that occurs.
• Figure 1 shows the human PEPT1 predicted amino acid sequence determined from the sequence of the cDNA clone coding for human PEPT1 (SEQ ID NO: 176) (Liang R. et al. J. Biol. Chem. 270 (12) : 6456-6463 (1995)), including the extracellular domain from amino acid 391 to 573 (Fei et al . , Nature 368:563 (1994)).
• Figures 2A-2C shows the human PEPT1 predicted amino acid sequence determined from the sequence of the cDNA clone coding for human PEPT1 (SEQ ID NO: 176) (Liang R. et al. J. Biol. Chem. 270 (12) : 6456-6463 (1995)), including the extracellular domain from amino acid 391 to 573 (Fei et al . , Nature 368:563 (1994)).
• Figures 2A-2C shows the human PEPT1 predicted amino acid sequence determined from the sequence of the cDNA clon
• Figures 2A-2C show the DNA sequence of the cDNA coding for the human intestinal peptide-associated transporter HPT1 and the corresponding putative amino acid sequence (bases 1 to 3345; Medline : 94204643 ) (SEQ ID NOS: 177 and 178, respectively) .
• Figures 3A-3B show the putative Human Sucrase-isomaltase complex (hSI) amino acid sequence determined from the sequence of the cDNA clone coding for human sucrase-isomaltase complex (SEQ ID NO: 179) (Chantret I., et al . , Biochem. J. 2JL5(Pt 3) :915-923 (1992).
• hSI Human Sucrase-isomaltase complex
• Figures 4A-4B Figures 4A-4B show the D2H nucleotide and deduced amino acid sequence for the human D2H transporter (SEQ ID NOS:180 and 181, respectively) (Wells, R.G. et al.,J. Clin. Invest. 9J3: 1959-1963 (1993) .
• Figures 5A-5C Figure 5A is a schematic summary of the cloning of the DNA insert present in gene III of the phages selected from the phage display libraries into the expression vector pGex-4T-2. The gene insert in gene III of the phages was amplified by PCR using DNA primers which flank the gene insert and which contained recognition sequences for specific restriction endonucleases at their extreme 5' sides.
• the digested PCR fragments were ligated into the digested plasmid pGex-4T-2 using T4 DNA Ligase and the ligated products were transformed into competent Escherichia coli , with selection of transformants on agar plates containing selection antibiotic.
• the selected clones were cultured, the plasmids were recovered and the in-frame sequence of the DNA insert in the plasmids was confirmed by DNA sequencing.
• FIG. 5B shows the series of full-length P31 (designated P31) (SEQ ID NO: 43) and truncated peptides derived from P31 (clones # 101, 102, 103 and 119), (SEQ ID NOS:183, 184, 185, and 186, respectively) full-length PAX2 (designated PAX2) (SEQ ID NO: 55) and truncated peptides derived from PAX2 (clones # 104, 105, 106) (SEQ ID NOS:170, 187, and 188, respectively) and full-length DCX8 (DCX8) (SEQ ID NO: 23) and series of truncated peptides derived from DCX8 (clones # 107, 108, 109) (SEQ ID NOS: 189, 190, and 191, respectively) that were expressed as fusion proteins to GST.
• Figure 5A shows the construction of these GST- fusion proteins.
• Figure 5C shows the series of full-length P31 (designated P31) (SEQ ID NO:43) and truncated peptides derived from P31 (clones # 103, 110, 119, 111, and 112) (SEQ ID NOS:185, 192, 193, 194, and 195, respectively) , full-length PAX2 (designated PAX2) (SEQ ID NO: 55) and truncated peptides derived from PAX2 (clones # 106, 113, 114, 115) (SEQ ID NOS:188, 196, 197, and 198, respectively) and full-length SNilO (designated SNilO) (SEQ ID NO: 4) and series of truncated peptides derived from SNilO (clones # 116, 117, 118) (SEQ ID NOS:199, 200, and 201, respectively) that were expressed as fusion proteins to GST
• Figures 6A-6B show the binding of GST and GST- fusion proteins to recombinant hSI and to fixed C2BBel fixed cells as detected by ELISA assays.
• Figure 6A shows the binding of the control protein GST, which does not contain a fusion peptide, and the GST-fusion proteins from SNilO (designated GST-SNilO) and SNi34 (designated GST-SNi34) to recombinant hSI .
• Figure 6B shows the binding of the control protein GST, which does not contain a fusion peptide, and the GST-fusion proteins from SNilO (designated GST-SNilO) and SNi34 (designated GST-SNi34) to fixed C2BBel cells.
• Figures 7A-7M show the binding of GST peptide and truncated fusion proteins to fixed Caco-2 cells, fixed C2BBel cells, and fixed A431 cells or to recombinant GIT transport receptors D2H, HPT1 , hPEPTl or to BSA using increasing concentrations (expressed as ⁇ g/ml on the X-axis) of the control GST protein and the GST- fusion proteins, as detected by ELISA assays.
• Figure 7A shows the binding of the control protein GST, which does not contain a fusion peptide, and the series of GST-fusion proteins from P31 including the fusion to full-length P31 peptide (designated P31) (SEQ ID NO:43) and clone # 101 (designated P31,101), clone # 102 (designated P31, 102) and clone # 103 (designated P31,103).
• Figure 7B shows the binding of the control protein GST, which does not contain a fusion peptide, and the series of GST- fusion proteins from PAX2 including the fusion to full-length PAX2 peptide (designated PAX2) and clone # 104 (designated PAX2,104), clone # 105 (designated PAX2 , 105) and clone # 106 (designated PAX2,106) (SEQ ID NOS:55, 170, 187, and 188, respectively) .
• Figure 7C shows the binding of the control protein GST, which does not contain a fusion peptide, and the series of GST- fusion proteins from DCX8 including the fusion to full-length DCX8 peptide (designated DCX8) and clone # 107 (designated DCX8,107), clone # 108 (designated DCX8 , 108) and clone # 109 (designated DCX8,109) (SEQ ID NOS: 23, 189, 190, and 191, respectively) .
• Figure 7D shows the binding of the control protein GST, which does not contain a fusion peptide, and the GST- fusion proteins from DCX8 (designated GST-DCX8) and DCX11 (designated GST-DCX11) to recombinant D2H.
• Figure 7E shows the binding of the control protein GST, which does not contain a fusion peptide, and the GST-fusion proteins from DCX8 (designated GST-DCX8) and DCX11 (designated GST-
• FIG. 7F shows the binding of the control protein GST, which does not contain a fusion peptide, and the GST-fusion proteins from P31 (designated GST-P31) and 5PAX5 (designated GST-5PAX5) to recombinant hPEPTl .
• Figure 7G shows the binding of the control protein GST, which does not contain a fusion peptide, and the GST- fusion proteins from P31 (designated GST-P31) and 5PAX5 (designated GST-5PAX5) to fixed C2BBel cells.
• Figure 7H shows the binding of the control protein GST, which does not contain a fusion peptide, and the GST- fusion proteins from HAX42 (designated GST-HAX42) and PAX2 (designated GST-PAX2) to recombinant HPT1.
• Figure 71 shows the binding of the control protein GST, which does not contain a fusion peptide, and the GST- fusion proteins from HAX42 (designated GST-HAX42) and PAX2 (designated GST-PAX2) to fixed C2BBel cells.
• Figure 7J shows the binding of the control protein GST, which does not contain a fusion peptide, and the GST- fusion proteins from P31 (designated GST-P31) and truncated derivatives clone # 101 (designated GST-P31-101) , clone # 102 (designated GST- P31-102) , clone # 103 (designated GST-P31-103) to either recombinant hPEPTl or to BSA.
• Figure 7K shows the binding of the control protein GST, which does not contain a fusion peptide, and the GST-fusion proteins from P31 (designated GST-P31) and truncated derivatives clone # 101 (designated GST-P31-101) , clone # 102 (designated GST-P31-102) , clone # 103 (designated GST-P31-103) to either fixed C2BBel cells or to fixed A431 cells.
• Figure 7L shows the binding of the control protein GST, which does not contain a fusion peptide, and the GST-fusion proteins from PAX2 (designated GST-PAX2) and truncated derivatives clone # 104 (designated GST-PAX2- 104) , clone # 105 (designated GST-PAX2-105) , clone # 106 (designated GST-PAX2-106) to either recombinant hPEPTl or to BSA.
• PAX2 designated GST-PAX2
• truncated derivatives clone # 104 designated GST-PAX2- 104
• clone # 105 designated GST-PAX2-105
• clone # 106 designated GST-PAX2-106
• Figure 7M shows the binding of the control protein GST, which does not contain a fusion peptide, and the GST- fusion proteins from PAX2 (designated GST-PAX2) and truncated derivatives clone # 106 (designated GST-PAX2-106) to either fixed Caco-2 cells or to fixed A431 cells.
• Figures 8A-8D show the transport of GST or GST- peptide fusion derivatives across polarized Caco-2 cells in an apical to basolateral direction as a function of time (1-4 hours) as detected by ELISA assays.
• Figure 8A shows the transport of either GST, the GST fusion to full-length P31 peptide (designated P31) (SEQ ID NO: 43) and the GST clone derivative clone # 103 (designated P31.103) across polarized Caco-2 cells in an apical to basolateral as a function of time (in hours) following initial administration of the proteins to the apical medium of polarized Caco-2 cells.
• the line designated No Protein corresponds to control assays in which buffer control was applied to the apical medium of polarized Caco-2 cells followed by sampling of the basolateral medium as a function of time (hours) and assay for GST by the ELISA assay.
• Figure 8B shows the transport of either GST, the GST fusion to full-length PAX2 peptide (designated PAX2) and the GST clone derivative clone # 106 (designated PAX2.106) across polarized Caco-2 cells in an apical to basolateral as a function of time (in hours) following initial administration of the proteins to the apical medium of polarized Caco-2 cells.
• the line designated No Protein corresponds to control assays in which buffer control was applied to the apical medium of polarized Caco-2 cells followed by sampling of the basolateral medium as a function of time (hours) and assay for GST by the ELISA assay.
• Figure 8C shows the transport of either GST, the GST fusion to full-length DCX8 peptide (designated DCX8), and the GST clone derivatives clone # 107 (designated DCX8.107) and clone # 109 (designated DCX8.109) across polarized Caco-2 cells in an apical to basolateral as a function of time (in hours) following initial administration of the proteins to the apical medium of polarized Caco-2 cells.
• the line designated No Protein corresponds to control assays in which buffer control was applied to the apical medium of polarized Caco-2 cells followed by sampling of the basolateral medium as a function of time (hours) and assay for GST by the ELISA assay.
• Figure 8D shows the amount of the GST and GST- fusion proteins (GST fusions to P31, P31-103, PAX2 , PAX2.106, DCX8 , DCX8-107, DCX8-109) , used in the experiments shown in panels A-C above, in the apical medium of the polarized Caco-2 cells as detected by ELISA assay.
• Figures 9A-9B show the inhibition of GST-P31 binding to C2BBel fixed cells with varying concentration of competitors while holding the concentration of GST-P31 constant at 0.015 ⁇ M; the peptide competitors are ZElan024 which is the dansylated peptide version of P31 (SEQ ID NO: 43) and ZElan044, ZElan049 and ZElan050 which are truncated, dansylated pieces of P31 (SEQ ID NO:43) . Data is presented as O.D. versus peptide concentration ( Figure 9A) and as percent inhibition of GST-P31 binding versus peptide concentration ( Figure 9B) .
• Figures 10A-10C present a compilation of the results of competition ELISA studies of GST-P31, GST- PAX2, GST-SNilO and GST-HAX42 versus listed dansylated peptides on fixed C2BBel cells ("Z” denotes e -amino dansyl lysine) .
• Z denotes e -amino dansyl lysine
• the pi of the dansylated peptides is also included.
• Estimated IC 50 values are in ⁇ M and where present, IC 50 ranges refer to results from multiple assays. If the IC 50 value could not be determined, a ">" or " ⁇ ” symbol is used.
• the GST/C2BBel column shows GST protein binding to fixed C2BBel cells .
• Figures 11A-11B show the transport of GST or GST-peptide fusion derivatives across polarized Caco-2 cells in an apical to basolateral direction at 0, 0.5, 2 and 4 hours as detected by ELISA assays and described elsewhere in the text in full detail.
• the proteins used in the assay included GST, GST-P31 fusion, GST-5PAX5 fusion, GST-DCX8 fusion, GST-DCX11 fusion, GST-PAX2 fusion, GST-HAX42 fusion, GST-SNi34 fusion and GST-SNilO fusion.
• the column designated No protein refers to control experiments in which buffer was applied to the apical medium of the cells and ELISA assay was performed on the corresponding basolateral medium of these cells at 0, 0.5, 2 and 4 hours post buffer addition.
• Figure 11B shows the internalization of GST or GST-peptide fusion derivatives within polarized Caco-2 cells following administration of the GST or GST-fusion protein derivatives to the apical medium of polarized Caco-2 cells and subsequent recovery of the cells from the transwells and detection of the GST or GST fusions within the recovered cell lysates as detected by ELISA assays and as described elsewhere in the text in full detail .
• the proteins used in the assay included GST, GST-P31 fusion, GST-5PAX5 fusion, GST-DCX8 fusion, GST- DCX11 fusion, GST-PAX2 fusion, GST-HAX42 fusion, GST-SNi34 fusion and GST-SNilO fusion.
• the column designated No protein refers to control experiments in which buffer was applied to the apical medium of the cells and ELISA assay was performed on the corresponding cell lysates of these cells at the end of the experiment .
• Figure 12 shows the binding of GST and GST- fusion proteins to fixed Caco-2 cells, and the corresponding proteins following digestion with the protease Thrombin which cleaves at a recognition site between the GST portion and the fused peptide portion of the GST- fusion protein.
• the symbol "-" refers to proteins which were not digested with thrombin and the symbol “+” refers to proteins which were digested with thrombin prior to use in the binding assay.
• the binding of the proteins to the fixed Caco-2 cells was detected by ELISA assays.
• Figures 13A-13B Figures 13A-13B show binding of peptide- coated nanoparticles to fixed Caco-2 cells.
• Figures 14A-14B show the binding of (A) dansylated peptide SNilO to the purified hSI receptor and BSA and (B) dansylated peptides and peptide-loaded insulin- containing PLGA particles to fixed C2BBel cells.
• Figure 14B depicts binding of dansylated peptides corresponding to P31 (SEQ ID NO:43), PAX2 , HAX42, and SNilO to fixed C2BBel cells, as well as the insulin-containing PLGA particles adsorbed with each of these peptides.
• Figures 15A-15B Figure 15 shows the binding of peptide- coated particles to A) S100 and B) P100 fractions harvested from Caco-2 cells.
• the dilution series 1:2 - 1:64 represents particle concentrations in the range 0.0325-0.5 ⁇ g/well .
• Data is presented with background subtracted. The particles are identified as follows: 939, no peptide; 1635, scrambled PAX2; 1726, P31 D-Arg 16-mer (ZElan053); 1756, HAX42; 1757, PAX2; 1758, HAX42/PAX2.
• Figures 16A-16B Figure 16 shows the binding of dansylated peptides to P100 fractions harvested from Caco-2 cells.
• Peptides were assayed in the range 0.0032-2.5 ⁇ g/well . Data is presented with background subtracted. A) HAX42, P31 D-form (ZElan 053) and scrambled PAX2 ; B) PAX2 , HAX42 and scrambled PAX2.
• Figures 17A-17B show (A) the systemic blood glucose and (B) insulin levels following intestinal administration of control (PBS) ; insulin solution; insulin particles; all 8 peptides mix particles and study group peptide-particles according to this invention (lOOiu insulin loading) .
• Figures 18A-18B show the (A) systemic blood glucose and (B) insulin levels following intestinal administration of control (PBS) ; insulin solution; insulin particles and study group peptide-particles according to this invention (300iu insulin loading) .
• Figure 19 shows the enhanced plasma levels of leuprolide upon administration of P31 (SEQ ID NO: 43) and PAX2 coated nanoparticles loaded with leuprolide relative to subcutaneous injection.
• Group 1 was administered leuprolide acetate (12.5 ⁇ g) subcutaneously.
• Group 2 was administered intraduodenally uncoated leuprolide acetate particles (600 ⁇ g, 1.5 ml) .
• Group 3 was intraduodenally administered leuprolide acetate particles coated with PAX2 (600 ⁇ g; 1.5 ml) .
• Group 4 was administered intraduodenally leuprolide acetate particles coated with P31 (SEQ ID NO:43) (600 ⁇ g, 1.5 ml) .
• Figure 20 lists P31 (SEQ ID NO: 43) known protein homologies .
• Figures 21A-21C list DCX8 known protein homologies .
• Figure 22 lists DAB10 known protein homologies.
• Figure 23 shows the DNA sequence (SEQ ID NO-211) and the corresponding amino acid sequence (SEQ ID NO: 212) for glutathione S-transferase (Smith and Johnson, 1988, Gene 7:31-40) .
• the present invention relates to proteins ( e . g. , peptides) that bind to GIT transport receptors and nucleic acids that encode such proteins.
• the invention further relates to fragments and other derivatives of such proteins. Nucleic acids encoding such fragments or derivatives are also within the scope of the invention.
• the invention further relates to fragments (and derivatives and analogs thereof) of GIT transport receptor-binding peptides which comprise one or more domains of the GIT transport receptor-binding peptides.
• the invention also relates to derivatives of GIT transport receptor-binding proteins and analogs of the invention which are functionally active, i . e .
• Such functional activities include but are not limited to ability to bind to a GIT transport receptor, antigenicity [ability to bind (or compete with peptides for binding) to an anti-GIT transport receptor-binding peptide antibody] , immunogenicity (ability to generate antibody which binds to GIT transport receptor-binding peptide), etc.
• the invention relates to peptides that bind GIT transport receptors and derivatives (including but not limited to fragments) and analogs thereof.
• such peptides that 0 bind to GIT transport receptor include but are not limited to those containing as primary amino acid sequences, all or part of the amino acid sequences substantially as depicted in
• the GIT transport receptor-binding peptides are encoded by the nucleic acids having the nucleotide sequences set forth in Table 8 infra (SEQ ID NO: 1]
• Proteins whose amino acid sequence comprise, or alternatively, consist of SEQ ID NOS: 1-55 or a portion thereof that mediates binding to a GIT transport receptor are provided.
• the derivative or analog is functionally active, i.e., capable of exhibiting one or more functional activities associated with a full-length GIT transport receptor-binding peptide.
• the derivatives or analogs which have the desired immunogenicity or antigenicity can be used, in immunoassays, for immunization, etc.
• a specific embodiment relates to a GIT transport receptor-binding peptide fragment that can be bound by an anti-GIT transport receptor-binding peptide antibody.
• the derivatives or ,_ analogs have the ability to bind to a GIT transport receptor.
• GIT transport receptor-binding peptides can be tested for the desired activity by procedures known in the art, including binding to a GIT transport receptor domain or to Caco-2 cells, in vi tro, or to intestinal tissue, in vivo . (See the Examples infra . )
• derivatives can be made by altering GIT transport receptor-binding peptide sequences by substitutions, additions or deletions that provide for functionally equivalent molecules.
• nucleotide coding sequences Due to the degeneracy of nucleotide coding sequences, other nucleotide sequences which encode substantially the same amino acid sequence may be used in the practice of the present invention. These include but are not limited to nucleotide sequences which are altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change.
• the GIT transport receptor-binding peptide derivatives of the invention include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of a GIT transport receptor-binding peptide including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change.
• one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration.
• Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs.
• the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine .
• the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
• the positively charged (basic) amino acids include arginine, lysine and histidine.
• the negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
• proteins consisting of or, alternatively, comprising all or a fragment of a GIT transport receptor-binding peptide consisting of at least 5, 10, 15, 20, 25, 30 or 35 (contiguous) amino acids of the full-length GIT transport receptor-binding peptide are provided. In a specific embodiment, such proteins are not more than 20, 30, 40, 50, or 75 amino acids in length.
• Derivatives or analogs of GIT transport receptor-binding peptides include but are not limited to those molecules comprising regions that are substantially homologous to GIT transport receptor-binding peptides or fragments thereof ( e . g.
• At least 50%, 60%, 70%, 80% or 90% identity (e.g., over an identical size sequence or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art) or whose encoding nucleic acid is capable of hybridizing to a coding GIT transport receptor-binding peptide sequence, under stringent, moderately stringent, or nonstringent conditions.
• the GIT transport receptor-binding derivatives of the invention are not known proteins with homology to the GIT transport receptor-binding peptides of the invention or portions thereof.
• the GIT transport receptor-binding peptide derivatives and analogs of the invention can be produced by various methods known in the art. The manipulations which result in their production can occur at the gene or protein level.
• the cloned GIT transport receptor- binding peptide gene sequence can be modified by any of numerous strategies known in the art (Maniatis, T., 1990, Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York) .
• the sequence can be cleaved at appropriate sites with restriction endonuclease (s) , followed by further enzymatic modification if desired, isolated, and ligated in vi tro .
• nucleic acid sequences encoding the GIT transport receptor-binding peptides can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vi tro modification.
• Any technique for mutagenesis known in the art can be used, including but not limited to, chemical mutagenesis, in vi tro site-directed mutagenesis (Hutchinson, C, et al . , 1978, J. Biol.
• GIT transport receptor-binding peptide sequences may also be made at the protein level . Included within the scope of the invention are GIT transport receptor-binding peptide fragments or other derivatives or analogs which are differentially modified during or after translation or chemical synthesis, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc.
• any of numerous chemical modifications may be carried out by known techniques, including but not limited to specific chemical cleavage by cyanogen bromide, trypsin, chy ⁇ notrypsin, papain, V8 protease, NaBH 4 ; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.
• the amino- and/or carboxy-termini are modified.
• GIT transport receptor-binding peptides and analogs and derivatives thereof can be chemically synthesized.
• a peptide corresponding to all or a portion of a GIT transport receptor-binding peptide which comprises the desired domain or which mediates the desired activity in vi tro can be synthesized by use of a peptide synthesizer.
• nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the GIT transport receptor-binding peptide sequence.
• Non-classical amino acids include but are not limited to the D-isomers of the common amino acids, ⁇ -amino isobutyric acid, 4-aminobutyric acid, Abu, 2 -amino butyric acid, ⁇ -Abu, e-Ahx, 6 -amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, -alanine, fluoro-amino acids, designer amino acids such as -methyl amino acids, Cc.- methyl amino acids, N ⁇ -methyl amino acids, and amino acid analogs in general.
• the amino acid can be D (dextrorotary) or L (levorotary) .
• the GIT transport receptor-binding peptide derivative is a chimeric, or fusion, peptide comprising a GIT transport receptor-binding peptide or fragment thereof (preferably consisting of at least a domain or motif of the GIT transport receptor-binding peptide, or at least 6, 10, 15, 20, 25, 30 or all amino acids of the GIT transport receptor-binding peptides or a binding portion thereof) joined at its amino- or carboxy-terminus via a peptide bond to an amino acid sequence of a different peptide.
• such a chimeric peptide is produced by recombinant expression of a nucleic acid encoding the protein (comprising a transport receptor-coding sequence joined in-frame to a coding sequence for a different protein) .
• a nucleic acid encoding the protein comprising a transport receptor-coding sequence joined in-frame to a coding sequence for a different protein.
• Such a chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the chimeric product by methods commonly known in the art.
• such a chimeric product may be made by protein synthetic techniques, e.g., by use of a peptide synthesizer. Chimeric genes comprising portions of GIT transport receptor fused to any heterologous protein-encoding sequences may be constructed.
• a specific embodiment relates to a chimeric protein comprising a fragment of GIT transport receptor- binding
• the GIT transport receptor-binding peptide derivative is a molecule comprising 5 a region of homology with a GIT transport receptor-binding peptide.
• a first protein region can be considered "homologous" to a second protein region when the amino acid sequence of the first region is at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or
• 15 molecule can comprise one or more regions homologous to a GIT transport receptor-binding peptide domain (see infra) or a portion thereof.
• the GIT transport receptor-binding proteins and derivatives thereof of the invention can be assayed for
• the invention relates to
• GIT transport receptor-binding peptide derivatives and analogs in particular GIT transport receptor-binding peptide fragments and derivatives of such fragments, that comprise, or alternatively consist of, one or more domains of a GIT transport receptor-binding peptide.
• examples in particular, examples
• the peptides and derivatives of the present invention may be chemically synthesized or synthesized using recombinant DNA techniques .
• Solid Phase Synthesis Peptides may be prepared chemically by methods that are known in the art. For example, in brief, solid phase peptide synthesis consists of coupling the carboxyl group of the C-terminal amino acid to a resin and successively adding N-alpha protected amino acids.
• the protecting groups may be any known in the art . Before each new amino acid is added to the growing chain, the protecting group of the previous amino acid added to the chain is removed.
• the coupling of amino acids to appropriate resins is described by Rivier et al . , U.S. Patent No. 4,244,946. Such solid phase syntheses have been described, for example, by Merrifield, 1964, J. Am. Chem. Soc. 85:2149; Vale et al .
• peptides can be synthesized on an Applied Biosystems Inc. ("ABI") model 431A automated peptide synthesizer using the "Fastmoc” synthesis protocol supplied by ABI, which uses
• Fmoc amino acids (1 mmol) are coupled according to the Fastmoc protocol.
• the following side chain protected Fmoc amino acid derivatives are used: FmocArg(Pmc)OH; FmocAsn (Mbh) OH; FmocAsp ⁇ Bu) OH; FmocCys (Acm) OH; FmocGlu ⁇ Bu) OH; FmocGln (Mbh) OH; FmocHis (Tr) OH,• FmocLys (Boc)OH; FmocSer ( fc Bu) OH; FmocThr ( fc Bu) OH; FmocTyr ( fc Bu) OH.
• Acm Acm, acetamidomethyl ; Boc, tert-butoxycarbonyl ; "Bu, tert-butyl; Fmoc,
• N, N-dimethylformamide DMF
• Deprotection of the Fmoc group is effected using approximately 20% piperidine in NMP. At 0 the end of each synthesis the amount of peptide present is assayed by ultraviolet spectroscopy . A sample of dry peptide resin (about 3-10 mg) is weighed, then 20% piperidine in DMA (10 ml) is added. After 30 min sonication, the UV (ultraviolet) absorbance of the dibenzofulvene-piperidine 5 adduct (formed by cleavage of the N-terminal Fmoc group) is recorded at 301 nm. Peptide substitution (in mmol g "1 ) can be calculated according to the equation:
• cleavage and deprotection can be carried out as follows:
• the air-dried peptide resin is treated with ethylmethyl-sulfide (EtSMe) , ethanedithiol (EDT) , and thioanisole (PhSMe) for __ approximately 20 min. prior to addition of 95% aqueous trifluoracetic acid (TFA) .
• EtSMe ethylmethyl-sulfide
• EDT ethanedithiol
• PhSMe thioanisole
• a total volume of approximately 50 ml of these reagents per gram of peptide-resin is used.
• the following ratio is used: TFA:EtSMe :EDT : PhSMe (10:0.5:0.5:0.5) .
• the mixture is stirred for 3 h at room temperature under an atmosphere of N 2 .
• Purification of the synthesized peptides can be carried out by standard methods including chromatography (e.g., ion exchange, affinity, and sizing column 15 chromatography, high performance liquid chromatography
• Biological peptide libraries can be used to express and identify peptides that bind to GIT transport receptors. According to this second approach, involving recombinant DNA techniques, peptides can, by way of example, be expressed in biological systems as either soluble fusion proteins or viral
• _ n inventions that specifically bind to GIT transport receptors are identified by screening a random peptide library by contacting the library with a ligand selected from among
• HPT1, hPEPTl, D2H, or hSI or a molecule consisting essentially of an extracellular domain thereof or fragment of
• a process to identify the peptides of the present method utilizes a library of recombinant vectors constructed by methods well known in the art and comprises screening a library of recombinant vectors 5 expressing inserted synthetic oligonucleotide sequences encoding extracellular GIT transport receptor domains, for example, attached to an accessible surface structural protein of a vector to isolate those members producing peptides that bind to HPT1, hPEPTl, D2H, or hSI .
• the nucleic acid sequence 0 of the inserted synthetic oligonucleotides of the isolated vector is determined and the amino acid sequence encoded can be deduced to identify a binding domain that binds the ligand of choice (e.g., HPT1 , hPEPTl, D2H, or hSI) .
• the ligand of choice e.g., HPT1 , hPEPTl, D2H, or hSI
• the present invention encompasses a method for
• identifying a peptide which binds to a ligand selected from among HPT1, hPEPTl, D2H, or hSI comprising: screening a library of random peptides with the ligand (or an extracellular domain or fragment thereof) under conditions conducive to ligand binding and isolating the peptide which
• the methods of the invention further comprise determining the nucleotide sequence encoding the binding domain of the peptide identified to deduce the amino acid sequence of the binding domain.
• molecules consisting essentially of an extracellular domain of the desired GIT
• _ n transport receptor or a fragment of an extracellular domain are used to screen a random peptide library for binding thereto.
• a nucleic acid encoding the extracellular domain is cloned and recombinantly expressed, and the domain is then purified for use.
• the library is screened to identify peptides having binding affinity for the GIT transport receptor, e.g., HPTl, hPEPTl, D2H, or hSI .
• the library is a TSAR library (see U.S. Patent No. 5,498,538 dated March 12, 1996 and PCT Publication WO 94/18318 dated August 18, 1994, both of which are incorporated by reference herein in their entireties) .
• Screening the libraries can be accomplished by any of a variety of methods known to those of skill in the art.
• the library is screened to identify binding molecules having specific binding affinity for a ligand for a GIT transport receptor preferably selected from among HPTl, hPEPTl, D2H, or hSI . - j .
• Screening the libraries can be accomplished by any of a variety of methods known to those of skill in the art. Exemplary screening methods are described in Fowlkes et al . , 1992, BioTechniques, 11:422-427 and include contacting the vectors with an immobilized target ligand and harvesting those vectors that bind to said ligand. Such useful screening methods, are designated "panning" methods.
• the target ligand can be immobilized on plates, beads (such as magnetic beads), sepharose, beads used in columns, etc.
• the immobilized target ligand can be "tagged", e.g., using labels such as biotin, fluoroscein isothiocyanate, rhodamine, etc. e.g. for FACS sorting. Panning is also disclosed in Parmley, S.F. and Smith, G.P., 1988, Gene 73 : 305-318.
• the library can be screened with a recombinant receptor domain.
• the library can be screened successively with receptor domains and then on CaCO-2 cells.
• the solvent requirements involved in screening are not limited to aqueous solvents; thus, nonphysiological binding interactions and conditions different from those found in vivo can be exploited.
• Screening a library can be achieved using a method comprising a first "enrichment” step and a second filter lift as follows.
• a method comprising a first "enrichment” step and a second filter lift as follows.
• the following description is given by way of example, not limitation.
• Binders from an expressed library (e.g., in phage) capable of binding to a given ligand (“positives") are initially enriched by one or two cycles of panning or affinity chromatography.
• a microtiter well is passively coated with the ligand (e.g., about 10 ⁇ g in 100 ⁇ l) .
• the well is then blocked with a solution of BSA to prevent nonspecific adherence of the phage of the library to the plastic surface.
• BSA a solution of BSA to prevent nonspecific adherence of the phage of the library to the plastic surface.
• about 10 11 phage particles expressing peptides are then added to the well and incubated for several hours. Unbound phage are removed by repeated washing of the plate, and specifically bound phage are eluted using an acidic glycine-HCl solution or other elution buffer.
• the eluted phage solution is neutralized with alkali, and amplified, e . g. , by infection of E. coli and plating on large petri dishes containing Luria broth (LB) in agar. Amplified cultures expressing the binding peptides are then titered and the process repeated.
• the ligand can be covalently coupled to agarose or acrylamide beads using commercially available activated bead reagents.
• the phage solution is then simply passed over a small column containing the coupled bead matrix which is then washed extensively and eluted with acid or other eluant . In either case, the goal is to enrich the positives to a frequency of about > 1/10 5 .
• a filter lift assay is conducted. For example, when specific binders are expressed in phage, approximately 1-2 x 10 5 phage are added to 500 ⁇ l of log phase E. coli and plated on a large Luria Broth-agarose plate with 0.7% agarose in broth. The agarose is allowed to solidify, and a nitrocellulose filter (e.g., 0.45 ⁇ ) is placed on the agarose surface. A series of registration marks is made with a sterile needle to allow re-alignment of the filter and plate following development as described below. Phage plaques are allowed to develop by overnight incubation at 37 °C (the presence of the filter does not inhibit this process) .
• a nitrocellulose filter e.g. 0.45 ⁇
• the filter is then removed from the plate with phage from each individual plaque adhered in si tu .
• the filter is then exposed to a solution of BSA or other blocking agent for 1-2 hours to prevent non-specific binding of the ligand (or "probe") .
• the probe itself is labeled, for example, either by biotinylation (using commercial NHS-biotin) or direct enzyme labeling, e.g., with horse radish peroxidase or alkaline phosphatase. Probes labeled in this manner are indefinitely stable and can be re-used several times.
• the blocked filter is exposed to a solution of probe for several hours to allow the probe to bind in si tu to any phage on the filter displaying a peptide with significant affinity to the probe.
• the filter is then washed to remove unbound probe, and then developed by exposure to enzyme substrate solution (in the case of directly labeled probe) or further exposed to a solution of enzyme-labeled avidin (in the case of biotinylated probe) .
• Positive phage plaques are identified by localized deposition of colored enzymatic cleavage product on the filter which corresponds to plaques on the original plate.
• the developed filter is simply realigned with the plate using the registration marks, and the "positive" plaques are cored from the agarose to recover the phage . Because of the high density of plaques on the original plate, it may be difficult to isolate a single plaque from the plate on the first pass.
• phage recovered from the initial core can be re-plated at low density and the process can be repeated to allow isolation of individual plaques and hence single clones of phage.
• Successful screening experiments are optimally conducted using 3 rounds of serial screening.
• the recovered cells are then plated at a low density to yield isolated colonies for individual analysis.
• the individual colonies are selected and used to inoculate LB culture medium containing ampicillin. After overnight culture at 37°C, the cultures are then spun down by centrifugation. Individual cell aliquots are then retested for binding to the target ligand attached to the beads. Binding to other beads having attached thereto a non-relevant ligand, can be used as a negative control.
• One aspect of screening the libraries is that of elution.
• the following discussion is applicable to any system where the random peptide is expressed on a surface fusion molecule. It is conceivable that the conditions that disrupt the peptide-target interactions during recovery of the phage are specific for every given peptide sequence from a plurality of proteins expressed on phage. For example, certain interactions may be disrupted by acid pH but not by basic pH, and vice versa .
• elution conditions including but not limited to pH 2-3, pH 12-13, excess target in competition, detergents, mild protein denaturants, urea, varying temperature, light, presence or absence of metal ions, chelators, etc.
• Some of these elution conditions may be incompatible with phage infection because they are bactericidal and will need to be removed by dialysis (i.e., dialysis bag, Centricon/Amicon microconcentrators) .
• a phage display library of random peptides is screened to select phage expressing peptides that bind to a GIT transport receptor.
• a first step is to isolate a preselected phage library.
• the "preselected phage library” is a library consisting of a subpopulation of a phage display library. This subpopulation can be formed by initially screening against either a target GIT transport receptor (or domain thereof) so as to permit the selection of a subpopulation of phages which specifically bind to the receptor.
• the subpopulation can be formed by screening against a target cell or cell type or tissue type or tissue barrier of the gastro-intestinal tract, so as to permit the selection of a subpopulation of phages which either bind specifically to the target cell or target cell type or target tissue or target tissue barrier, or which binds to and/or is transported across (or between) the target cell or target cell type or target tissue or target tissue barrier either in si tu or in vivo .
• This preselected phage library or subpopulation of selected phages can also be rescreened against the target GIT transport receptor, permitting the further selection of a subpopulation of phages which bind to the GIT transport receptor or target cell or target cell type or target tissue or target tissue barrier or which bind to and/or is transported across the target cell, target tissue or target tissue barrier either in si tu or in vivo .
• Such rescreening can be repeated from zero to 30 times with each successive "pre-selected phage library" generating additional pre-selected phage libraries.
• a preselected phage library binding a ligand that is a GIT transport receptor preferably selected from among HPTl, hPEPTl, D2H, or hSI is obtained by an in vi tro screening step as described above, 5 and then the phage are optionally further characterized using in vi tro assays consisting of binding phage directly to the receptor domain of interest or, alternatively, to Caco-2 cells or using in vivo assays.
• in vivo assays are used that measure uptake of 0 phage by intestinal tissue or, alternatively, through the
• a GIT transport receptor-binding peptide, fragments or other derivatives, or analogs thereof may be used as an immunogen to generate antibodies which immunospecifically bind such an immunogen.
• Such antibodies include but are not limited to polyclonal , monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library.
• GIT transport receptor-binding peptide or derivative or analog various procedures known in the art may be used for the production of polyclonal antibodies to a GIT transport receptor-binding peptide or derivative or analog.
• various host animals can be immunized by injection with the native GIT transport receptor-binding peptides, or a synthetic version, or derivative (e.g., fragment) thereof, including but not limited to rabbits, mice, rats, fowl, etc.
• Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and
• mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol , and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum.
• surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol , and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum.
• any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used.
• the hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256:495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al . , 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies Colde et al . , 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) .
• monoclonal antibodies can be produced in germ- free animals utilizing recent technology (PCT/US90/02545) .
• human antibodies may be used and can be obtained by using human hybridomas (Cote et al . , 1983, Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030) or by transforming human B cells with EBV virus in vi tro (Cole et al . , 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96) .
• techniques developed for the production of "chimeric antibodies” (Morrison et al . , 1984, Proc. Natl. Acad. Sci.
• Antibody fragments which contain the idiotype of the molecule can be generated by known techniques.
• such fragments include but are not limited to: the F(ab') 2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragment, the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent, and Fv fragments.
• screening for the desired antibody can be accomplished by techniques known in the art, e.g. ELISA (enzyme-linked immunosorbent assay).
• ELISA enzyme-linked immunosorbent assay
• Antibodies specific to a domain of a GIT transport receptor-binding peptide are also provided.
• the foregoing antibodies can be used in methods known in the art relating to the localization and activity of the GIT transport receptor-binding peptide sequences of the invention, e.g., for imaging these peptides after in vivo administration ( e . g. , to monitor treatment efficacy), measuring levels thereof in appropriate physiological samples, in diagnostic methods, etc.
• antibodies or antibody fragments specific to a domain of a GIT transport receptor-binding peptide or to a derivative of a peptide, such as a dansyl group or some other epitope introduced into the peptide can be used to 1) identify the presence of the peptide on a nanoparticle or other substrate; 2) quantify the amount of peptide on the nanoparticle; 3) measure the level of the peptide in appropriate physiological samples; 4) perform im unohistology on tissue samples; 5) image the peptide after in vivo administration; 6) purify the peptide from a mixture using an immunoaffinity column or 7) bind or fix the peptide to the surface of nanoparticle.
• the antibody in such a way that the peptide is fully active.
• Abtides (or Antigen binding peptides) specific to a domain of a GIT transport receptor-binding peptide or to a derivative of a peptide, such as a dansyl group or some other epitope introduced into the peptide, can be used for the same
• _ n binding peptides, derivatives and analogs can be assayed by various methods .
• the binding can be assayed by in vivo or in vi tro assays such as described in the examples infra, or by other means that are known in the art .
• immunoassays known in the art can be used, including but not limited to competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays,
• immunoradiometric assays gel diffusion precipitin reactions, immunodiffusion assays, in si tu immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc.
• antibody binding is detected by detecting a label on the primary antibody.
• the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
• the secondary antibody is labelled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.
• compositions comprising the GIT transport receptor-binding proteins of the invention bound to a material comprising an active agent.
• Such compositions have use in targeting the active agent to the GIT and/or in facilitating transfer through the lumen of the GIT into the systemic circulation.
• the active agent is an imaging agent
• compositions can be administered in vivo to image the GIT (or particular transport receptors thereof) .
• Other active agents include but are not limited to: any drug or antigen or any drug- or antigen-loaded or drug- or antigen-encapsulated nanoparticle, microparticle, liposome, or micellar formulation capable of eliciting a biological response in a human or animal .
• Examples of drug- or antigen-loaded or drug- or antigen-encapsulated formulations include those in which the active agent is encapsulated or loaded into nano- or microparticles, such as biodegradable nano- or microparticles, and which have the GIT transport receptor-binding protein or derivative or analog adsorbed, coated or covalently bound, such as directly linked or linked via a linking moiety, onto the surface of the nano- or microparticle.
• the protein, derivative or analog can form the nano- or microparticle itself or the protein, derivative or analog can be covalently attached to the polymer or polymers used in the production of the biodegradable nano- or microparticles or drug-loaded or drug- encapsulated nano- or microparticles or the peptide can be directly conjugated to the active agent.
• conjugations to active agents include fusion proteins in which a DNA sequence coding for the peptide is fused in- frame to the gene or cDNA coding for a therapeutic peptide or protein such that the modified gene codes for a recombinant fusion protein.
• the invention provides for treatment of various diseases and disorders by administration of a therapeutic compound (termed herein "Therapeutic”).
• a therapeutic compound include but are not limited to: GIT transport receptor-binding proteins, and analogs and derivatives (including fragments) thereof (e.g., as described hereinabove) that bind to GIT transport receptors, bound to an active agent of value in the treatment or prevention of a disease or disorder (preferably a mammalian, most preferably human, disease or disorder) .
• Therapeutics also include but are not limited to nucleic acids encoding the GIT transport receptor-binding proteins, analogs, or derivatives bound to such a therapeutic or prophylactic active agent.
• the active agent is preferably a drug.
• drug includes, without limitation, any pharmaceutically active agent.
• Representative drugs include, but are not limited to, peptides or proteins, hormones, analgesics, anti-migraine agents, anti-coagulant agents, anti-emetic agents, cardiovascular agents, anti- hypertensive agents, narcotic antagonists, chelating agents, anti-anginal agents, chemotherapy agents, sedatives, anti- neoplasties, prostaglandins, and antidiuretic agents.
• Typical drugs include peptides, proteins or hormones such as insulin, calcitonin, calcitonin gene regulating protein, atrial natriuretic protein, colony stimulating factor, betaseron, erythropoietin (EPO) , interferons such as a.
• peptides, proteins or hormones such as insulin, calcitonin, calcitonin gene regulating protein, atrial natriuretic protein, colony stimulating factor, betaseron, erythropoietin (EPO) , interferons such as a.
• somatropin somatotropin, somatostatin, insulin-like growth factor (somatomedins) , luteinizing hormone releasing hormone (LHRH) , tissue plasminogen activator (TPA) , growth hormone releasing hormone (GHRH) , oxytocin, estradiol, growth hormones, leuprolide acetate, factor VIII, interleukins such as interleukin-2 , and analogs thereof; analgesics such as fentanyl, sufentanil, butorphanol, buprenorphine, levorphanol, morphine, hydromorphone , hydocodone, oxymorphone, methadone, lidocaine, bupivacaine, diclofenac, naproxen, paverin, and analogs thereof; anti-migraine agents such as heparin, hirudin, and analogs thereof; anti -coagulant agents such as scopol
• Representative drugs also include but are not limited to antisense oligonucleotides, genes, gene correcting hybrid oligonucleotides, ribozymes, aptameric oligonucleotides, triple-helix forming oligonucleotides, inhibitors of signal transduction pathways, tyrosine kinase inhibitors and DNA modifying agents.
• Drugs that can be used also include, without limitation, systems containing gene therapeutics, including viral systems for therapeutic gene delivery such as adenovirus, adeno-associated virus, retroviruses, herpes simplex virus, Sindbus virus, liposomes, cationic lipids, dendrimers, and enzymes.
• gene delivery viruses can be modified such that they express the targeting peptide 5 on the surface so as to permit targeted gene delivery.
• a Therapeutic is therapeutically or prophylactically administered to a human patient .
• the invention provides methods of treatment (and 15 prophylaxis) by administration to a subject of an effective amount of a Therapeutic of the invention.
• the Therapeutic is substantially purified.
• the subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably a human .
• any disease or disorder of interest amenable to therapy or prophylaxis by providing a drug in vivo systemically or by targeting a drug in vivo to 25 the GIT (by linkage to a GIT transport -receptor binding protein, derivative or analog of the invention) can be treated or prevented by administration of a Therapeutic of the invention.
• diseases may include but are not limited
• _ n hypertension, diabetes, osteoporosis, hemophilia, anemia, cancer, migraine, and angina pectoris, to name but a few.
• any route of administration known in the art may be used, including but not limited to oral, nasal, topical, intravenous, intraperitoneal , intradermal, mucosal, ⁇ - 5 c intrathecal, intramuscular, etc.
• administration is oral; in such an embodiment the GIT-transport binding protein, derivative or analog of the invention acts advantageously to facilitate transport of the therapeutic active agent through the lumen of the GIT into the systemic circulation.
• a GIT transport receptor-binding peptide or motif of interest is associated with a therapeutically or prophylactically active agent, preferably a drug or drug- containing nano- or microparticle.
• the active agent is a drug encapsulating or drug loaded nano- or microparticle, such as a biodegradable nano- or microparticle, in which the peptide is physically adsorbed or coated or covalently bonded, such as directly linked or linked via a linking moiety, onto the surface of the nano- or microparticle.
• the peptide can form the nano- or microparticle itself or can be directly conjugated to the active agent.
• Such conjugations include fusion proteins in which a DNA sequence coding for the peptide is fused in-frame to the gene or cDNA coding for a therapeutic peptide or protein, such that the modified gene codes for a recombinant fusion protein in which the "targeting" peptide is fused to the therapeutic peptide or protein and where the "targeting" peptide increases the absorption of the fusion protein from the GIT.
• the particles range in size from 200-600 nm.
• a GIT transport- binding protein is bound to a slow-release (controlled release) device containing a drug.
• polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres . , Boca Raton, Florida (1974) ; Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol . Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al . , Science 228:190 (1985); During et al . , Ann. Neurol .
• compositions comprise a therapeutically effective amount of a Therapeutic, and a pharmaceutically acceptable carrier.
• pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
• carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
• Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
• compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
• These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
• the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides .
• Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.
• Such compositions will contain a therapeutically effective amount of the Therapeutic, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
• the Therapeutics of the invention can be formulated as neutral or salt forms.
• Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc .
• the amount of the Therapeutic of the invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vi tro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances.
• HPTl, hPEPTl, D2H, and hSI receptors were selected for cloning as GIT receptor targets based on several criteria, including: (1) expression on surface of epithelial cells in gastro-intestinal tract (GIT) ; (2) expression along the length of small intestine (HPTl, hPEPTl, D2H) ;
• the four recombinant receptor sites screened with the peptide libraries additionally have the following characteristics : Receptor Characteristics
• Figures 1-4 show the predicted amino acid sequences for hPEPTl, HPTl, hSI and D2H, respectively.
• receptor domains were cloned and expressed as His-tag fusion proteins by standard techniques:
• the receptor proteins were expressed as His-tag fusion proteins and affinity purified under denaturing conditions, using urea or guanidine HCI, utilizing the pET His-tag metal chelate affinity for Ni-NTA Agarose (Hochuli, E., Purification of recombinant proteins with metal chelate adsorbent, Genetic Engineering, Principals and Methods (J.K. Setlow, ed.), Plenum Press, NY, Vol. 12 (1990), pp. 87-98). 6.3. Phage Libraries
• phage DC8, D38, and DC43 libraries expressing N-terminal pill fusions in M13 were used to identify peptides that bind to the GIT receptors.
• the D38 and DC43 libraries which are composed of 37 and 43 random amino acid domains, respectively, have been described previously (McConnell et al., 1995, Molecular Diversity, 1:165-176).
• the DC8 library is similar to the other two except that the random insert is 8 amino acids long flanked on each side by a cysteine residue (i.e. , CX 8 C) .
• phage clones were identified which had high absorbance in the ELISA assay and/or a good ratio of binding to target compared to binding to BSA.
• the Insulin Degrading Enzyme (IDE) and recombinant human tissue factor (hTF) were used as irrelevant controls.
• IDE Insulin Degrading Enzyme
• hTF recombinant human tissue factor
• Selection or panning methods followed one of two strategies. The first strategy involved panning the mixed libraries on the specific GIT receptor adsorbed to a solid surface. The second strategy panned the libraries twice against the GIT receptor and then against Caco-2 cells (Peterson and Mooseker, 1992, J. Cell Science 102:581-600), Selection methods are reflected in the clone nomenclature as described below: S designates the clone was identified by binding to the hSI receptor domain.
• D designates the clone was identified by binding to the D2H receptor domain.
• P designates the clone was identified by binding to the PEPT1 receptor domain.
• H designates the clone was identified by binding to the HPT-1 receptor domain.
• Phage designated Ni are from a solid phase band GIT receptor pan that used the standard procedure with the addition of Ni-NTA Agarose (Qiagen, Chatsworth, CA) .
• Receptor coated plates were blocked with 0.5% BSA/PBS containing 160 ⁇ l Ni-NTA agarose and libraries were panned in the presence of 50 ⁇ l Ni-NTA agarose.
• the receptor proteins were expressed as His-tag fusions.
• the His-tag has a high affinity for Ni-NTA Agarose. Blocking the plate and panning in the presence of Ni-NTA agarose minimized phage binding to the His-tag portion of the recombinant receptor.
• Phage with the designation AX were eluted with acid and Factor Xa. Phage were first eluted by standard acid elution then Factor Xa (New England Biolabs, Beverly, MA: l ⁇ g protease in 300 ⁇ l of 20mM Tris-HCL, lOOmM NaCI, 2mM CaCl 2 ) was added to the panning plate and incubated 2 hours . Phage from both elution methods were pooled together then plated. Phage with the designation AB were eluted with acid and base. Phage were eluted first by standard acid elution then lOOmM triethylamine pH 12.1 was added to the panning plate for 10 minutes.
• Phage from both elution methods were pooled together then plated.
• C designates panning on receptor followed by Caco-2 cells.
• First and second round pans were performed on the receptor and the third round pan was on snapwells of Caco-2 cells.
• DCX11, DCX8 and DCX33 were identified by two pans on D2H receptor, third pan on Caco-2 cells.
• the third round Factor Xa eluate from the Caco-2 cells was screened by ELISA on D2H, BSA and fixed Caco-2 cells.
• the first two rounds of panning were performed on the HPT-1 receptor and the third pan was on monolayers cultured on snapwells of Caco-2 cells.
• Phage designated 5PAX were carried through five rounds of panning after which a number of phage were sequenced prior to screening by ELISA.
• the amino acid sequence of phage inserts demonstrating a good ratio of binding to receptor domains and/or Caco-2 cells over background BSA binding were deduced from the nucleotide sequence obtained by sequencing (Sequenase ® , U.S. Biochemical Corp., Cleveland, OH) both DNA strands of the appropriate region in the viral genome .
• the third round acid eluate was screened by ELISA on HPT-1, BSA and Caco-2 fixed cells. Phage designated 5PAX were carried through five rounds of panning after which a number of phages were sequenced prior to screening by ELISA.
• One well of a 24 well plate was coated with 10 ⁇ g/ml of GIT receptor and the plate was incubated overnight at 4°C. The plate was blocked with 0.5 BSA-PBS for one hour. A mixture of the DC8, D38 and DC43 phage libraries was added to the plate and the plate was incubated for 2 to 3 hours at room temperature on a rotator. After washing the well 10 times with 1% BSA plus 0.05% Tween 20 in PBS, the well was eluted with 0.05m glycine, pH2. The phage was then eluted with 0.2M NaP0 4 . The eluted phage was titered on agar plates; the remaining phage was amplified overnight.
• the amplified phage was added to a second coated plate and the panning procedure was repeated as described above .
• the eluted phage from the second pan as well as the amplified phage from the first pan was titered on agar plates.
• the panning procedure was repeated as described above.
• the phage eluted from the third pan and the amplified phage from the second pan were then titered overnight on agar plates. Isolated phage colonies were amplified overnight prior to use in an ELISA assay. 6.6. Receptor ELISA Procedure
• 96 well plates were coated overnight with GIT receptor, BSA and, optionally, IDE (insulin degrading enzyme, an irrelevant His-fusion protein) or hTF .
• the plates were 5 blocked for one hour with 0.5% BSA-PBS.
• the amplified phage were diluted 1:100 in 1% BSA plus 0.05% Tween 20 in PBS and added to the plates.
• the plates were washed 5 times with 1% BSA plus 0.05% Tween 20 in
• Table 5 shows the results of an ELISA which assessed the binding of phage panned on the HPT-1 receptor to HPT-1 and BSA.
• the table shows the OD results as well as the ratio of HPT-1 to BSA binding.
• Table 6 shows the results of an ELISA which assessed the binding of phage panned for two rounds on the HPT-1 receptor followed by a third round pan on Caco-2 snapwells. Binding to fixed Caco-2 cells, HPT-1 and BSA was examined. The table shows the OD results as well as the ratio of HPT-1 to BSA binding.
• Phage ELISA was used as described above with the following changes.
• Diluent and wash buffer was PBS containing 1%BSA and 0.05% Tween 20 and plates were washed five times at each wash step.
• Supernatant of infected bacterial cultures was diluted 1:100 and incubated with protein coated plates for 2-3 hours with mild agitation.
• Anti-M13 Horseradish peroxidase (HRP) conjugate was diluted 1:8000.
• TARGET BINDING PHAGE INSERT SEQUENCES: t ⁇ -V . hSI ID. NO.
• DCX33 26 RWNWTVLPATGGHYWTRSTDYHAINNHRPSIPHQHPTPI DCX36 27 SWSSWNWSSKTTRLGDRATREGCGPSQSDGCPYNGRLTTVKPRT
• PAX9 31 RWPSVGYKGNGSDTIDVHSNDASTKRSLIYNHRRPLFP
• PAX14 32 RTFENDGLGVGRSIQKKSDRWYASHNIRSHFASMSPAGK
• PAX15 33 SYCRVKGGGEGGHTDSNLARSGCGKVARTSRLQHINPRATPPSR
• PAX16 34 SWTRWGKHTHGGFVNKSPPGKNATSPYTDAQLPSDQGPP PAX17 35 SQVDSFRNSFRWYEPSRALCHGCGKRDTSTTRIHNSPSDSYPTR
• PAX35 37 RSITDGGINEVDLSSVSNVLENANSHRAYRKHRPTLKRP
• PAX38 38 SSKVSSPRDPTVPRKGGNVDYGCGHRSSARMPTSALSSITKCYT
• PAX40 39 RASTQGGRGVAPEFGASVLGRGCGSATYYTNSTSCKDAMGHNYS PAX43 40 RWCEKHKFTAARCSAGAGFERDASRPPQPAHRDNTNRNA
• PAX45 41 SFQVYPDHGLERHALDGTGPLYAMPGRWIRARPQNRDRQ
• PAX2 55 STPPSREAYSRPYSVDSDSDTNAKHSSHNRRLRTRSRPN Table 8
• SNi 34 (SEQ ID NO: 61) TCTCACTCCTCGAGTCCGTGCGGGGGGTCGTGGGGGCGTTTTATGCAGGGTGGCCTTTTCG GCGGTAGGACTGATGGTTGTGGTGCCCATAGAAACCGCACTTCTGCGTCGTTAGAGCCCCC GAGCAGCGACTACTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA
• SNi AX2 (SEQ ID NO: 64) TCTCACTCCTCGAGTGATAGTGACGGGGATCATTATGGGCTTCGGGGGGGGGTGCGTTGTT CGCTTCGTGATAGGGGTTGTGGTCTGGCCCTGTCCACCGTCCATGCTGGTCCCCCCTCTTT TTACCCCAAGCTCTCCAGCCCCTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA
• SNi AX4 (SEQ ID NO: 65) TCTCACTCCTCGAGGAGCTTGGGTAATTATGGCGTCACCGGGACTGTGGACGTGACGGTTT TGCCCATGCCTGGCCACGCCAACCACCTTGGTGTCTCCTCCGCCTCTAGCTCTGATCCTCC GCGGCGCTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA
• DAB10 (SEQ ID NO: 70) TCTCACTCCTCGAGTAAGTCCGGGGAGGGGGGTGACAGTAGCAGGGGCGAGACGGGCTGGG CGAGGGTTCGGTCTCACGCCATGACTGCTGGCCGCTTTCGGTGGTACAACCAGTTGCCCTC TGATCGGTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA
• DAX24 (SEQ ID NO: 76) TCTCACTCCTCGAGGATGGAGGACATCAAGAACTCGGGGTGGAGGGACTCTTGTAGGTGGG GTGACCTGAGGCCTGGTTGTGGTAGCCGCCAGTGGTACCCCTCGAATATGCGTTCTAGCAG AGATTACCCCGCGGGGGGCCACTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA
• DCX8 (SEQ ID NO: 78)
• DCX11 (SEQ ID NO: 79)
• DCX33 (SEQ ID NO: 81) TCTCACTCCTCGAGGTGGAATTGGACTGTCTTGCCCGCCACTGGCGGCCATTACTGGACGC GTTCGACGGACTATCACGCCATTAACAATCACAGGCCGAGCATCCCCCACCAGCATCCGAC CCCTATCTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA DCX36 ( SEQ ID NO : 82 )
• DCX39 (SEQ ID NO: 83)
• DCX42 (SEQ ID NO: 84)
• DCX45 (SEQ ID NO: 85) TCTCACTCCTCGAGCGTGGGGAATGATAAGACTAGCAGGCCGGTTTCCTTCTACGGGCGCG TTAGTGATCTGTGGAACGCCAGCTTGATGCCGAAGCGTACTCCCAGCTCGAAGCGCCACGA TGATGGCTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA
• PAX2 (SEQ ID NO: 86)
• PAX9 (SEQ ID NO: 87)
• PAX14 (SEQ ID NO: 88)
• PAX15 (SEQ ID NO: 89)
• PAX16 (SEQ ID NO: 90) TCTCACTCCTCGAGTTGGACTCGGTGGGGCAAGCACANTCATGGGGGGTTTGTGAACAAGT CTCCCCCTGGGAAGAACGCCACGAGCCCCTACACCGACGCCCAGCTGCCCAGTGATCAGGG TCCTCCCTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA PAX17 ( SEQ ID NO : 91 )
• PAX18 (SEQ ID NO: 92)
• PAX35 (SEQ ID NO: 93)
• PAX38 (SEQ ID NO: 94) TCTCACTCCTCGAGTTCGAAGGTGAGCAGCCCGAGGGATCCGACGGTCCCGCGGAAGGGCG GCAATGTTGATTATGGTTGTGGTCACAGGTCTTCCGCCCGGATGCCTACCTCCGCTCTGTC GTCGATCACGAAGTGCTACACTTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA
• PAX40 SEQ ID NO: 95
• PAX43 (SEQ ID NO: 96)
• PAX45 (SEQ ID NO: 97)
• PAX46 (SEQ ID NO: 98)
• 5PAX5 (SEQ ID NO: 102) TCTCACTCCTCGAGAGGCAGTACGGGGACGGCCGGCGGCGAGCGT
• HAX42 (SEQ ID NO: 107) TCTCACTCNTNGAGTGATCACGCGTTGGGGACGAATCTGAGGTCTGACAATGCCAAGGAGC CGGGTGATTACAACTGTTGTGGTAACGGGAACTCTACCGGGCGAAAGGTTTTTAACCGTAG GCGCCCCTCCGCCATCCCCANTTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA
• HCA3 (SEQ ID NO: 108)
• Phage expressing presumed GIT binding peptide inserts were also assayed by ELISA on fixed Caco-2 or C2BBel cells as follows. Cells were plated at 1 x 10 5 cells/well on 100 ⁇ l culture media and incubated at 30°C in 5% C0 2 overnight. 100 ⁇ l 25% formaldehyde was added to each well for 15 minutes. Contents of the wells were removed by inverting the plate. The plate was then washed 3 times with DPBS . 0.1% phenylhydrazine DPBS solution was added to each well and incubated for 1 hr at 37°C. The plate was inverted and washed 3 times. The plate was blocked with 0.5% BSA-DPBS for 1 hr at room temperature.
• the plate was inverted and washed 3 times with 1% BPT (PBS containing 1% BSA and 0.05% Tween20) . Phage diluted with 1% BPT was added to wells containing fixed cells. Wells without phage added were used to determine background binding of the HRP conjugate. The plates were incubated 2-3 hours on a rotor at room temperature. Plates were washed as before. Plates were incubated with dilute anti-M13-HRP antibody in 1% BPT for 1 hour at room temperature. Following washing, TMB substrate was added and absorbance of the plates were read at 650 nm. Table 10 shows the relative binding of phage encoding peptides to fixed Caco-2 cells.
• In vivo phage selection Further selection of phage expressing peptides capable of binding to the GIT or transporting the GIT was done as follows.
• the purified library was resuspended in a buffer, such as TBS or PBS, and introduced onto one side of a tissue barrier, e.g., injected into the duodenum, jejunum, ileum, colon or other in vivo animal site using, for instance, a closed loop model or open loop model.
• samples of bodily fluids located across the tissue barrier e.g., samples of the portal circulation and/or systemic circulation, were withdrawn at predetermined time points, such as 0 to 90 minutes and/or 2 to 6 hours or more.
• a host e.g., E. coli
• the amplified phage present in the culture can be sequenced individually to determine the identity of peptides coded by the phage or, if further enrichment is desired, can be precipitated using PEG, and resuspended in PBS. The phage can then be further precipitated using PEG or used directly for administration to another animal using a closed or open GIT loop model system.
• the corresponding region of the tissue barrier can be recovered at the end of the procedures given above.
• This recovered tissue can be washed repeatedly in suitable buffers, e.g., PBS containing protease inhibitors and homogenized in, for example, PBS containing protease inhibitors.
• suitable buffers e.g., PBS containing protease inhibitors and homogenized in, for example, PBS containing protease inhibitors.
• the homogenate can be used to infect a host, such as E. coli , thus permitting amplification of phages which bind tightly to the tissue barrier (e.g., intestinal tissue) .
• the recovered tissue can be homogenized in suitable PBS buffers, washed repeatedly and the phage present in the final tissue homogenate can be amplified in E. coli . This approach permits amplification
• phages which either bind tightly to the tissue barrier (e.g., intestinal tissue) or which are internalized by the cells of the tissue barrier (e.g., epithelial cells of the intestinal tissue) .
• tissue barrier e.g., intestinal tissue
• cells of the tissue barrier e.g., epithelial cells of the intestinal tissue
• the purified phage display library (random or preselected) was diluted to 500 ⁇ l in PBS buffer and injected into the closed (or open) intestinal loop model (e.g., rat, rabbit or other species) .
• the closed (or open) intestinal loop model e.g., rat, rabbit or other species
• a sample of either the portal circulation or systemic circulation was withdrawn.
• An aliquot of the withdrawn blood was incubated with E. coli , followed by plating for phage plaques or for transduction units or for colonies where the phage codes for resistance to antibiotics such as tetracycline.
• the remainder of the withdrawn blood sample (up to 150 ⁇ l) was incubated with 250 ⁇ l of E. coli and 5 ml of LB medium or other suitable growth medium.
• coli cultures were incubated overnight by incubation at 37°C on a shaking platform. Blood samples taken at other time points (such as 15 min, 30 min, 45 min, 60 min, up to 6 hours) were processed in a similar manner, permitting amplification of phages present in the portal or systemic circulation in E. coli at these times.
• the amplified phage was recovered by PEG precipitation and resuspended in PBS buffer or TBS buffer. The titer of the amplified phage, before and after PEG precipitation, was determined. The amplified, PEG precipitated phage was diluted to a known phage titer
• This procedure of phage injection followed by collection of portal and/or systemic blood samples and amplification of phage transported into these blood samples can be repeated, for example, up to 10 times, to permit the selection of phages which are preferentially transported from the GIT into the portal and/or systemic circulation.
• Phage from random phage display libraries as well as control phage were injected into the lumen of the rat gastro-intestinal tract ( in si tu rat closed loop model) . Blood was collected over time from either the systemic circulation or portal circulation and the number of phage which were transported to the circulation was determined by titering blood samples in E. coli .
• the phage display libraries used in this study were D38 and DC43 in which gene III codes for random 38-mer and 43-mer peptides, respectively.
• the identical phage M13mpl8, in which gene III does not code for a "random" peptide sequence was used.
• Both the library phages D38 and DC43 were prepared from E. coli , mixed together, dialyzed against PBS, precipitated using PEG/NaCl and were resuspended in PBS buffer.
• the M13mpl8 control was processed in a similar manner. The titer of each phage sample was determined and the phage samples were diluted in PBS to approximately the same titers prior to injection into the rat closed loop model .
• the estimated number of transported phage has been adjusted to account for differences in volume injected into each animal (using 0.5 ml as the standard volume) .
• animals Rl , R2 and R3 received the control phage M13mpl8 and animals R4 , R5 , R6 and R7 received the test phage D38/DC43 mix.
• animals R8 , R9 and R10 received the control phage M13mpl8 and animals Rll, R12, R13 and R14 received the test phage D38/DC43 mix.
• Animal R15* received the combined phage samples from animals R4-R7 (see Table 11) which were sampled from the systemic circulation on day one, followed by amplification in E. coli , PEG precipitation and resuspension in PBS.
• the titer of this phage was found to be 100 times greater than the other phage samples used for animals R8-R14. Thus, the data presented for animal R15* is adjusted down.
• Approximately 0.4 ml of the blood was collected at each time point in each model system.
• 30 ⁇ l of the collected blood (systemic) was mixed with 100 ⁇ l of the prepared E. coli strain K91Kan, incubated at 37°C for 30 min, and plated out for plaque formation using Top Agarose on LB plates.
• Various negative controls were included in the titering experiments. The following day, the number of plaque forming units was determined.
• phage supernatant samples were collected, serially diluted (10 ⁇ 2 , 10 -4 , 10 "6 , 10 "8 ) in TBS buffer, and plated for plaques in order to determine the number of plaque forming units present in the amplified phage samples. Furthermore, an aliquot of phage was removed from the "amplified" supernatants obtained from test animals R4-R7 (samples from each time point were used) , combined, and precipitated using PEG for two hours. The precipitated phage was resuspended in PBS buffer and was injected into closed loop model of animal R15*, followed by portal sampling.
• the number of phage transported from the closed loop model into the systemic circulation is presented in Table 11 hereafter.
• the number of phage transported from the closed loop model into the portal circulation is presented in Table 12 hereafter. These numbers are corrected for phage input difference and for volume input differences.
• more phage are present in the portal samples than in the systemic samples, indicative of either hepatic or RES clearance and/or phage instability in the systemic circulation.
• the uptake of phage from the GIT into the portal circulation is quite rapid, with substantial 5 number of phages detected within 15 minutes.
• the results from the portal sampling experiments would also indicate that the kinetics of uptake of phage from the D38/DC43 libraries is quicker than that of the control phage.
• Animal R15* received the combined phage samples from animals R4-R7 (see Table 11) which were sampled from the systemic circulation on day one, followed by PEG precipitation and resuspension in PBS. On subsequent analysis, the titer of this phage was found to be 100 times greater than the other phage samples used for animals R8-R14. Thus, the data measuring phage transport into the portal circulation for animal R15* is adjusted down.
• si tu loop model may represent an attractive screening model in which to identify peptide sequences which facilitate transport of phage and particles from the GIT into the circulation.
• phage libraries now exist, including a one pass systemic phage library from animals R4-R7, a one-pass portal library from animals R11-R14, and a two pass, rapid transport, systemic-portal phage library SP-2 from animal R15*.
• HPT1 and GI -hPEPTl were constructed by pooling phage previously selected by screening random phage display libraries D38 and DC43 using the HPTl, HPEPTl, D2H and hSI receptor or binding sites located in the GIT.
• the phage pools, preselected phage libraries are shown in Table 13.
• H10 and HAX44 are the same. Also, the sequence for HAX40 is the same as that for H44. The corresponding SEQ ID NOS. are shown in Table 7.
• these preselected phage libraries together with the negative control phage M13mpl8 were injected into the rat closed loop model (6 animals per preselected phage library) , blood was collected over time from the portal circulation via the portal vein and, at the termination of the experiment, a systemic blood sample was collected from the tail vein and the intestinal tissue region from the closed loop was collected.
• phages selected in vi tro to each receptor or binding site located in the GIT were amplified in E. coli , PEG-precipitated, resuspended in TBS and the titer of each phage sample was determined by plaquing in E. coli as described above.
• each phage (8 x 10 8 phage) for each receptor site was pooled into a preselected phage library together with the negative control phage M13mpl8 and each preselected phage library was administered to 6 Wistar rats per library (rats 1-6; GI-D2H, rats 7-12; Gl-hSI, rats 13-18; GI -hPEPTl, and rats 19-24; GI- HPT1) .
• GI-D2H rats 7-12
• Gl-hSI rats 13-18
• GI -hPEPTl GI- HPT1
• 0.5 ml of preselected phage library solution was injected into the tied-off portion of the duodenum/jejunum. Blood was collected into heparinized tubes from the portal vein at 0, 15, 30, 45 and 60 minutes. A blood sample was taken from the systemic circulation at the end of the experiment. Similarly, the portion of the duodenum/jejunum used for phage injection was taken at the end of the
• coli was removed by centrifugation and the amplified phage supernatant samples were either titered directly or were PEG-precipitated, resuspended in TBS and titered. Following titration of the amplified phage, samples containing phage from each set of animals were combined, adjusting the titer of each sample to the same titer, and were plated for plaques on LB agar plates (22cm 2 square plates). Either 12,000 or 24,000 phage were plated for plaques .
• coli was removed by centrifugation and the amplified phage supernatant samples were either titered directly or were PEG-precipitated, resuspended in TBS and titered.
• samples containing phage from each set of animals were combined, adjusting the titer of each sample to the same titer, and were plated for plaques on LB agar plates (22cm 2 square plates) . Either 12,000 or 24,000 phage were plated for plaques .
• the intestinal tissue portion used in each closed loop was excised.
• tissue was cut into small segments, followed by 3 washings in sterile PBS containing protease inhibitors, and homogenized in an Ultra thorex homogeniser (Int-D samples) .
• tissue in PBS supplemented with protease inhibitors was homogenized in an Ultra thorex homogeniser.
• the phage amplified from the portal blood, systemic blood and intestinal tissue was plated for plaques.
• the plaques were transferred to Hybond-N Nylon filters, followed by denaturation (1.5M NaCI, 0.5M NaOH), neutralization (0.5M TRIS-HC1, pH7.4, 1.5M NaCI), and washing in 2X SSC buffer.
• the filters were air-dried, and the DNA was cross-linked to the filter (UV crosslinking: 2min, high setting) .
• the filters were incubated in pre-hybridization buffer (6X SSC, 5X Denhardt's solution, 0.1% SDS, 20 ⁇ g/ml yeast tRNA) at 40°C- 45°C for at least 60 min.
• PAX2 5 CTATCGACACTATAGGGCCTAC 3 ' 140
• PAX14 5 TTCTTCTG AATAGACCGGCCGA 3 ' 142
• PAX35 5 GACACACTACTCAGGTCCACCT 3' 147
• oligonucleotides (5pmol) were 5 ' end labelled with 32 P-ATP and T4 polynucleotide kinase and approximately 2.5pmol of labelled oligonucleotide was used in hybridization studies. Hybridizations were performed at 40-45°C overnight in buffer containing 6X SSC, 5X Denhardt's solution, 0.1% SDS, 20 ⁇ g/ml yeast tRNA and the radiolabeled synthetic oligonucleotide, followed by washings (20-30 min at 40-45°C) in the following buffers: (i) 2X SSC / 0.1% SDS, (ii) IX SSC / 0.1% SDS, (iii) 0. IX SSC / 0.1% SDS. The filters were air-dried and exposed for autoradiography for 15 hours, 24 hours or 72 hours.
• Hybridization data indicated that all the oligonucleotide probes bound specifically to their phage target except for the HAX9 probe which apparently was not labeled.
• a negative control probe that hybridized only to M13mpl8 DNA showed a weak to negative signal in all samples tested (data not shown) .
• Hybridization data for pools from each receptor group of rats was compiled.
• Tables 15, 16, 17 and 18 show a representative compilation of autoradiograph signals of the HSI, D2H, HPTl and hPEPTl receptor groups. These Tables show the phage absorption and uptake from the closed loop GIT model to portal and systemic circulation and phage absorption/internalization to intestinal tissue.
• Int-G refers to intestinal tissue homogenized prior to washing and recovery while Int-D refers to intestinal tissue washed prior to homogenization and phage recovery. In all cases, leading phage candidates were present in more than one animal .
• oligonucleotide to HAX9 Apart from the synthetic oligonucleotide to HAX9 , all oligonucleotides were initially confirmed to be radiolabeled, as determined by hybridization to the corresponding phage target (eg., phage S15 hybridized to the oligonucleotide S15) . In addition, under the experimental conditions used, the oligonucleotides essentially did not hybridize to the negative control phage template M13mpl8.
• Two oligonucleotides were synthesized to the phage M13mpl8 : (1) a positive oligonucleotide which hybridizes to a conserved sequence in both M13mpl8 and each of the GIT receptor or GIT binding site selected phages [designated M13 (positive) ] ; and (2) a negative oligonucleotide which only hybridizes to a sequence unique to the multiple cloning site of phage M13mpl8 and which does not hybridize to any of the GIT receptor or GIT binding site selected phages.
• phages S15, SNi-10, SNi-34 and SNi-38 were transported from the closed loop model into the portal circulation: phages S15, SNi-10, SNi-34 and SNi-38.
• phages SNi-10 and to a lesser extent phages S15 and S22 were found in the intestine samples or fractions, whereas the other phages were not.
• phages from this pool were not transported into the portal circulation, including phages DAB18, DAB24, DAX15, DAX24, DAX27, DCX26, DCX36, DCX39, DCX42, DCX45.
• phages DAB18, DAB24, DAX15, DAX24, DAX27, DCX26, DCX36, DCX39, DCX42, DCX45 There is a very low level of transport of phage DAX23 from the GIT into the portal circulation.
• phages DAB30, DCX33, DAB7, DCXll, DCX45 and to a much lesser extent phages DAB3 , DAB10, DCX8 , DCX39, DCX42.
• phages were not found in the intestinal samples, including phages DAB18, DAB24, DAX15, DAX24, DCX26, and DCX36. There was a very low presence ( ⁇ 0.1%) of the phage M13mpl8 in the Int-G samples. These results showed that phages can be further selected from pre-selected libraries, permitting the identification of phages which are transported from the GIT closed loop into the portal circulation or phages which bind to or are internalized by intestinal tissue.
• phages can be further selected from pre-selected libraries, permitting the identification of phages which are transported from the GIT closed loop into the portal and/or systemic circulation or phages which bind to or are internalized by intestinal tissue .
• the phages PAX2 and H40 were also included in this pool.
• a number of phages from this pool were found in the portal circulation, including phages P31 (SEQ ID NO:43), PAX46, PAX9, H40, PAX17, PAX40, PAX2, PAX14, 5PAX3 and 5PAX12.
• phages were not found in the portal blood including the negative control phage M13mpl8, PAX15, PAX16, PAX18, PAX35, PAX38, PAX43, PAX45, P90, 5PAX5 and 5PAX7.
• the only phage found in the systemic circulation were phages 5PAX5 and P31 (SEQ ID NO: 43) .
• phages can be further selected from pre-selected libraries, permitting the identification of phages which are transported from the GIT closed loop into the portal and/or systemic circulation or phages which bind to or are internalized by intestinal tissue. Further Characterization of Select Sequences
• phage display libraries Following initial screening of the four recombinant receptor sites (hPEPTl, HPTl, D2H, hSI) of the gastrointestinal tissue, with the phage display libraries, a series of phage were isolated which showed preferential binding to the respective target receptor sites in comparison to negative control protein BSA protein and the recombinant protein recombinant human tissue factor (hTF) (which, like the recombinant receptors of the gastrointestinal tissue, contained a poly-histidine tag at its NH 2 -terminal end) . In subsequent experiments same titers of the selected phage which bound to each target receptor site were combined into a single pool ( i . e .
• hTF human tissue factor
• phage pools were injected into a closed duodenal loop region of rat intestinal tissue and subsequently phage was harvested and recovered which was bound to and retained by the intestinal tissue and/or was absorbed from the intestinal loop into the portal and/or systemic circulation.
• a selection of the initial phages which bound to the target recombinant receptor site were analyzed for binding to either fixed Caco-2 cells and/or to fixed C2BBel cells.
• the selection of the final lead peptide sequences was based on the ability of the phage, coding for that peptide sequence (1) to bind to the target recombinant receptor site in vi tro in preference to its binding to the negative control proteins BSA and/or hTFs, (2) to bind to rat intestinal tissue following injection into a closed duodenal loop of rat intestinal tissue in preference to the negative control phage M13mpl8, (3) to be absorbed from rat intestinal tissue into either the portal and/or systemic circulation following injection into a closed duodenal loop of rat intestinal tissue in preference to the negative control phage M13mpl8, and (4) to bind to either fixed Caco-2 cells or fixed C2BBel cells in phage binding studies in preference to the negative control phage M13mpl8.
• Peptides were also selected with consideration to the ease of chemical synthesis.
• Glutathione S-transferase (GST) vectors encoding fusion proteins of GI targeting peptides were constructed in the vector pGEX4T-2 (source, Pharmacia Biotech, Piscataway, NJ) . Briefly, single-strand DNA from the clones of interest were amplified by the polymerase chain reaction. The amplified DNA was then cleaved with the restriction enzymes Xhol and Notl and then ligated into Sall/Notl cleaved pGEX4T-2. Following transformation, the DNA sequence for each construct was verified by sequencing.
• FIG. 5A-5C A diagrammatic representation of the various GST fusion protein constructs that have been synthesized is indicated in Figures 5A-5C.
• Escherichia coli BL21 cells containing GST fusion protein constructs were grown overnight in 2X YT media containing 100 ⁇ g/ml ampicillin (2X YT/amp) . Overnight cultures were diluted 1:100 in 2X YT broth (100 ml), and cells were grown to an A 600 of 0.5 at 30°C, induced with ImM isopropyl-1-thio-B-D-galactopyranoside, and grown for an additional 3 h. Cells were harvested by centrifugation and resuspended in 5 ml of PBS containing a mixture of the proteinase inhibitors (Boehringer/Mannheim) .
• the standard ELISA procedure was modified as follows. GST proteins were diluted to an appropriate concentration in PBS containing 1%BSA and 0.05% Tween20
• Figure 6 shows the binding of GST-SNilO, GST-SNi34 and GST alone to the hSI receptor and to fixed C2BBel cells.
• the standard ELISA procedure was modified as follows. GST fusion proteins and peptides were diluted to an appropriate concentration in PBS containing 1% BSA and 0.05% Tween 20. Peptides were titered, a constant concentration of diluted GST protein was added to titered peptides and the mixture was incubated one hour at room temperature . Following five washes, an anti-GST monoclonal antibody was added (Sigma, St. Louis Clone GST-2 diluted 1:10,000 in 1% BPT) and incubated one hour. After five more washes goat anti-mouse IgG2b-HRP was added (Southern Biotechnology
• Figures 9A and 9B show the inhibition of GST-P31 binding to C2BBel fixed cells.
• the peptide competitors are ZElan024 which is the dansylated peptide version of P31 (SEQ ID NO: 43) and ZElan044, ZElan049 and ZElan050 which are truncated, dansylated pieces of P31 (SEQ ID NO: 43) .
• Data is presented as O.D. vs. peptide concentration and as percent inhibition of GST-P31 binding vs. peptide concentration. Uncompeted GST-P31 binding was considered as 100% binding.
• IC 50 values are estimates using the 50% line on the percent inhibition graph.
• GST-P31 and GST-PAX2 exhibited no crossreactive binding to ZElan024 (P31) (SEQ ID NO:43) and ZElanOl ⁇ (PAX2) at the 0.5 ⁇ g/ml concentration used in competition assays.
• GST-HAX42 exhibited crossreactivity to ZElan018 (PAX2) and ZElan021 (HAX42) at the 5 ⁇ g/ml concentration used in competition assays.
• Figures 10A-10C present a compilation of data generated by competition ELISA of GST-P31, GST-PAX2, GST- SNilO and GST-HAX42 versus various dansylated peptides on fixed C2BBel cells. IC 50 values are in ⁇ M and include ranges determined from multiple assays.
• the GST/C2BBel column is a summary of GST protein binding to fixed C2BBel cells.
• Caco-2 cells were fixed, treated with phenylhydrazine and blocked as described above.
• Synthetic peptides (lOO ⁇ g/ml) were applied in duplicate to Caco-2 cells and serially diluted down the 96 -well plate.
• the corresponding GST-peptide fusion protein (lO ⁇ g) was added to each well and the plates were incubated for 2h at room temperature with agitation. Binding of the GST-peptide fusion proteins to the cells was assayed using the ELISA technique described above. GST-P31 binding was inhibited by ZElan024, ZElan028 and ZElan031 as well as the two D forms ZElan053 and ZElan054.
• GST-PAX2 binding was inhibited by ZElan032, ZElan033, and ZElan035.
• GST-HAX42 binding was not inhibited by ZElan021 (full length HAX42) but it was inhibited by ZElanOl ⁇ (PAX2) and ZElan026 and ZElan038 (scrambled PAX2 peptides) .
• Transport and uptake of GST-peptide fusions and deletion derivatives across cultured polarized Caco-2 monolayers over 4 hours in HBSS buffer was examined using an anti-GST ELISA assay.
• transport and uptake of GST-peptide fusions and deletion derivatives across cultured polarized Caco-2 monolayers over 24 hours in serum- free medium (SFM) was examined using an anti-GST ELISA assay.
• HBSS Gibco CN.14065-031 supplemented with 0.011M glucose (lg/1) , 25 mM Hepes (15 mM acid (3.575g/l; Sigma CN.H3375); lOmM base (2.603g/l; Sigma CN.H1016)].
• Chloroquine Made up as lOmM solution in water [Sigma CN C6628]
• Lysate buffer 30 mM Tris-HCl pH8.0 ; ImM EDTA Serum- free medium (SFM) is normal medium without serum.
• TER Transepithelial electrical flux (TER) across the Caco-2 monolayers grown on snapwells (passage 33; 23 days old) was measured to confirm monolayer integrity before beginning the experiment . The medium was removed and the cells were washed once with SFM.
• Figure 11A describes complete transport of GST- peptide across a polarized Caco-2 monolayer and does not necessarily refer to internalization, i.e., the GST-peptide was recovered from the basolateral reservoir of a snapwell but the proteins could have crossed the barrier by the paracellular route.
• Binding of intact and thrombin-cleaved GST-peptide fusions to fixed Caco-2 cells was compared. Reduced binding of the thrombin-cleaved GST-peptide fusions relative to intact fusions indicates that the peptide component of the fusion, and not the GST domain, mediates binding.
• Confluent Caco-2 monolayers grown in 96-well plates (p38) were fixed and treated with 0.1% phenylhydrazine before blocking with 0.1% BSA in PBS.
• Thirty micrograms of each GST-peptide was treated with bovine thrombin (l ⁇ /ml; 0.4 NIH units; Sigma CN.T9681) for 18h at room temperature in 20mM Tris-HCl pH8.0, 150mM NaCI, 2.5mM CaCl 2 . Controls were similarly treated without addition of thrombin.
• Peptides may be prepared by methods that are known in the art. For example, in brief, solid phase peptide synthesis consists of coupling the carboxyl group of the C- terminal amino acid to a resin and successively adding N- alpha protected amino acids.
• the protecting groups may be any known in the art . Before each new amino acid is added to the growing chain, the protecting group of the previous amino acid added to the chain is removed.
• the coupling of amino acids to appropriate resins is described by Rivier et al . , U.S. Patent No. 4,244,946. Such solid phase syntheses have been described, for example, by Merrifield, 1964, J. Am.
• peptides can be synthesized on an Applied Biosystems Inc. ("ABI") model
• HBTU 2- (lH-Benzotriazol-1-yl) -1,1,3,3, -tetramethyluronium hexafluorophosphate
• Fmoc amino acids (1 mmol) are coupled according to the Fastmoc protocol.
• the following side chain protected Fmoc amino acid derivatives are used:
• FmocTyr ( t Bu) OH [Abbreviations: Acm, acetamidomethyl ; Boc, tert-butoxycarbonyl ; fc Bu, tert-butyl; Fmoc,
• Synthesis is carried out using N-methylpyrrolidone (NMP) as solvent, with HBTU dissolved in N,N-dimethylformamide (DMF) . Deprotection of the Fmoc group is effected using approximately 20% piperidine in NMP. At the end of each synthesis the amount of peptide present is assayed by ultraviolet spectroscopy. A sample of dry peptide resin (about 3-10 mg) is weighed, then 20% piperidine in DMA (10 ml) is added. After 30 min sonication, the UV
• 7800 x w where A is the absorbance at 301 nm, v is the volume of 20% piperidine in DMA (in ml) , 7800 is the extinction coefficient (in molMm 3 cm “1 ) of the dibenzofulvene-piperidine adduct, and w is the weight of the peptide-resin sample (in mg) .
• N-terminal Fmoc group is cleaved using 20% piperidine in DMA, then acetylated using acetic anhydride and pyridine in DMA.
• the peptide resin is thoroughly washed with DMA, CH 2 C1 2 and finally diethyl ether.
• cleavage and deprotection can be carried out as follows:
• the air-dried peptide resin is treated with ethylmethyl-sulfide (EtSMe) , ethanedithiol (EDT) , and thioanisole (PhSMe) for approximately 20 min. prior to addition of 95% aqueous trifluoracetic acid (TFA) .
• EtSMe ethylmethyl-sulfide
• EDT ethanedithiol
• PhSMe thioanisole
• a total volume of approximately 50 ml of these reagents are used per gram of peptide-resin.
• TFA EtSMe
• EDT PhSme (10:0.5:0.5:0.5).
• the mixture is stirred for 3 h at room temperature under an atmosphere of N 2 .
• Purification of the synthesized peptides can be carried out by standard methods including chromatography (e.g., ion exchange, affinity, and sizing column chromatography, high performance liquid chromatography (HPLC) ) , centrifugation, differential solubility, or by any other standard technique.
• chromatography e.g., ion exchange, affinity, and sizing column chromatography, high performance liquid chromatography (HPLC)
• HPLC high performance liquid chromatography
• the peptides of the present invention may be linked to other molecules (e.g., a detectable label, a molecule facilitating adsorption to a solid substratum, or a toxin, according to various embodiments of the invention) by methods that are well known in the art . Such methods include the use
• the homobifunctional molecules have at least two reactive functional groups, which are the same.
• the reactive functional groups on a homobifunctional molecule include, for
• aldehyde groups and active ester groups are examples of Homobifunctional molecules having aldehyde groups.
• Homobifunctional molecules having aldehyde groups include, for example, glutaraldehyde and subaraldehyde .
• glutaraldehyde as a cross-linking agent was disclosed by Poznansky et al . , 1984, Science 223:1304-1306.
• Homobifunctional molecules having at least two active ester units include esters of dicarboxylic acids and
• N-hydroxysuccinimide N-hydroxysuccinimide .
• N-succinimidyl esters include disuccinimidyl suberate and dithio-bis-
• the heterobifunctional molecules have at least two amino acids
• heterobifunctional reagents containing reactive disulfide bonds include N-succinimidyl 3- (2-pyridyl-dithio) propionate (Carlsson et al . , 1978, Biochem J. 173:723-737), sodium S-4- succinimidyloxycarbonyl-alpha-methylbenzylthiosulfate, and
• N-succinimidyl 3- (2- pyridyldithio) propionate is preferred.
• heterobifunctional reagents comprising reactive groups having a double bond that reacts with a thiol group include succinimidyl 4- (N-maleimidomethyl) cyclohexahe-1-carboxylate and succinimidyl m-maleimidobenzoate .
• heterobifunctional molecules include succinimidyl 3 - (maleimido) propionate, sulfosuccinimidyl 4-(p- maleimido-phenyl) butyrate, sulfosuccinimidyl 4- (N- maleimidomethyl-cyclohexane) -1-carboxylate, maleimidobenzoyl-
• N-hydroxy-succinimide ester N-hydroxy-succinimide ester.
• the sodium sulfonate salt of succinimidyl m-maleimidobenzoate is preferred.
• Many of the above-mentioned heterobifunctional reagents and their sulfonate salts are available from Pierce.
• biotinylating peptides are well known in the art . Any convenient method may be employed in the practice of the invention. For example, the following procedure was used. Ten micrograms of peptide was dissolved in 100 ⁇ l of 0.1 % acetic acid. PBS (900 ⁇ l) and 3.3 mg of biotin-LC-NHS (Pierce, Rockford, IL) was added. Following incubation for 30 minutes at room temperature the biotinylated peptides were purified over a Superose 12 column (Pharmacia, Piscataway, NJ) .
• Synthetic Peptides Tables 19, 20 and 21 provide the primary structure for various synthetic peptides manufactured in the practice of the present invention.
• SIF simulated intestinal fluid
• Peptide (3.25mg) was dissolved in 3.25 ml of 10,000 fold diluted SIF solution at 37°C. Aliquots (0.7ml) of the digestion solution were then withdrawn at ⁇ lmin, lh, 3h, and 21h or 24h. The samples were quickly passed through a syringe filter (Millipore Millex-GV 0.22 ⁇ m, part# SLGV025LS, lot# H2BM95250) and 300 ⁇ L of the filtered solution was immediately injected onto a Hewlett-Packard HPLC system equipped with a C- ⁇ column (Applied Biosystems column and guard column: column- p/n 0711-0023 Spheri-5 ODS 5 ⁇ m, 220x4.6mm). The products were eluted at 1.5ml/min using an acetonitrile-water gradient. The major fluorescent peaks were collected, lyopholized and identified by MS analysis. The HPLC gradient used was:
• Samples were resuspended in sterile water at lOmg/ml and stirred with a magnet for lh at room temperature.
• Samples consisted of: (1) blank nanoparticle control, (2) scrambled PAX2-coated nanoparticles, (3) PAX2-coated nanoparticles, (4) HAX42 -coated nanoparticles, 0 (5) PAX2/HAX42 -coated nanoparticles, and (6) 8 peptide-coated nanoparticles .
• Nanoparticles were added to the cells at lOmg/ml in lOO ⁇ l 1%BSA-PBS (no Tween ⁇ O is used in this assay) and 2-fold serially-diluted. The 96-well plates were incubated for lh
• Table 25 below shows the insulin potency and level of peptides coated onto the particles (measured by fluorescense) for formulation 1 particles (formulation by the coacervation method given below) .
• Insulin Peptide mg/g ⁇ l/mg
• the standard ELISA procedure was modified as follows. Peptides and particles were diluted to an appropriate concentration in PBS containing 1%BSA (particles were sonicated to achieve a homogeneous solution) , titered and incubated one hour at room temperature. Following five washes with PBS containing 1%BSA, an in-house IgGl ⁇ anti- dansyl monoclonal antibody was added (diluted to l ⁇ g/ml in 1%BSA-PBS) and the plates were incubated for one hour. After five more washes goat anti-mouse ⁇ -HRP was added (Southern Biotechnology Associates Inc., Birmingham, AL, diluted 1:10,000 in 1%BSA-PBS) and the plates were incubated one hour.
• TMB peroxidase substrate (Kirkegard and Perry, Gaithersburg, MD) . All data is presented with background binding subtracted. Tween 20 was not added to the diluent or the washes when insulin coated PLGA particles were included in the assay.
• Figures 14A-14B show the binding of the dansylated peptide SNilO to hSI and BSA.
• Caco-2 cell membrane (P100) and cytosolic (S100) fractions were prepared using a modification of the method described in Kinsella, B. T., O'Mahony, D. J. and G. A. 5
• Dulbecco's PBS Dulbecco's PBS (DPBS) and the cells were harvested by 0 centrifugation at 1000 rpm after treatment with 10 mM EDTA-
• DPBS DPBS .
• the cells were washed 3 times in DPBS and the final cell pellet was resuspended in 3 volumes of ice cold HED buffer (20 mM HEPES (pH 7.67), 1 mM EGTA, 0.5 mM dithiothreitol, 1 mM phenylmethylsulphonyl fluoride (PMSF) ) .
• the cells were allowed to swell for 5 min on ice prior to homogenization for 30 sec. The homogenates were centrifuged at 40,000 rpm for 45 min at 4°C.
• the supernatant (S100) was removed and the pellet (P100) was resuspended in HEDG buffer (20 mM HEPES (pH 7.67), 1 mM EGTA, 0.5 mM dithiothreitol , 100 mM NaCI, 10% glycerol, 1 mM PMSF) . Protein concentrations were determined using the Bradford assay (Bradford, M. M., 5 1976, Anal. Biochem. 72 : 248-254).
• Binding of peptide and/or peptide-coated PLGA particles to membrane (P100) and cytosolic (S100) fractions was assessed by detection of the dansyl moiety incorporated in the peptide.
• Costar ninety six well ELISA plates were 0 coated with S100 and P100 fractions (100 ⁇ g/ml in 0.05 M NaHC0 3 ) overnight at 4°C. The plates were blocked with 0.5% bovine serum albumin in DPBS for 1 h at room temperature and washed 3 times in 1% BSA-DPBS. Peptide-coated particles or peptides were dispersed in the same buffer and added to the 5 plates at concentrations in the range 0.0325 - 0.5 mg/well.
• a novel assay system is provided by the instant invention for detection of binding of peptide-coated PLGA particles to membrane (P100) and cytosolic (S100) fractions derived from live Caco-2 cells.
• the absorbance readings obtained using this assay system were substantially higher 5 than those obtained using similar peptide-coated PLGA particle concentrations on fixed Caco-2 cells. This greater sensitivity together with the derivation of the S100 and P100 fractions from live Caco-2 cells suggests that this assay may be the assay system of choice for detection of peptide-coated PLGA particle binding.
• the assay was concentration dependent and peptide/particle correlation permitted differentiation between specific and non-specific binding interactions.
• Binding of peptide-coated PLGA particles was assessed using S100 and P100 fractions derived from live Caco-2 cells as described above. The fractions were coated onto 96-well plates at lO ⁇ g/well in 0.05 M NaHC0 3 and peptide-coated PLGA particles were assayed by ELISA at concentrations in the range 0.0325 - 0.5 mg/well.
• Figures 15A and 15B illustrate the data obtained on S100 and P100 fractions respectively for particles coated with no peptide, scrambled PAX2 (control) , P31 D-Arg 16 -mer (ZElan053), HAX42, PAX2 and HAX42/PAX2.
• P31 D-Arg 16 -mer ZElan053
• HAX42 HAX42
• PAX2 PAX2
• HAX42/PAX2/PAX2 HAX42/PAX2.
• P31 D-Arg 16- mer (ZElan053) coated particles to the S100 fraction may be indicative of non-specific binding due to the D-Arg modification of the P31 peptide (SEQ ID NO: 43) .
• Binding of PLGA particles coated with varying concentrations of PAX2 peptide ranging from 0.05 - 5.0 mg/g was assessed using a) fixed Caco-2 cells (P35) and b) S100 and P100 fractions (Caco-2 P33) . The particles were assayed at concentrations in the range 0.03125 - 0.0625 mg/well.
• the effect of blocking solution on binding of peptide- coated PLGA particles to P100 fractions was assessed using 1% bovine serum albumin (BSA) and 1% milk powder blocking solutions to assess background binding.
• BSA bovine serum albumin
• the following particles were assayed at concentrations in the range 0.03125 - 0.0625 mg/well: no peptide; scrambled PAX2 ; and a range of PAX2 coated particles having peptide concentrations from 5-0.05 mg/g.
• all test peptide coated particles except PAX2 coated at 0.05 mg/g exhibited comparable or greater binding to the P100 fractions than the scrambled PAX2 coated control particles.
• Binding to P100 fractions was directly proportional to the concentration of the PAX2 peptide on the particle (although in this instance PAX2 (5 mg/g) exhibited slightly lower binding than PAX2 (1 mg/g)) .
• PAX2 5 mg/g
• PAX2 exhibited slightly lower binding than PAX2 (1 mg/g)
• a similar trend was observed using 1% milk powder and a particle concentration of 0.0625 mg/well. However all absorbance readings were low when 1% milk powder was used and the binding pattern was not detectable using particles at a concentration of 0.0625 mg/well.
• Non-specific binding of peptide-coated PLGA particles to plastic was also assessed using 1% BSA and 1% milk powder blocking solutions. The binding pattern observed above could be detected when BSA was used; however, absorbance readings
• FIG. 16 illustrates the data obtained for the dansylated peptides A) HAX42, P31
• HAX42 Two consecutive assays produced substantial variations in absorbance readings. Initially, the HAX42 peptide exhibited strong binding when compared to the scrambled PAX2 control. The P31 D-form peptide (ZElan053) exhibited binding at the highest dilution only. In the repeat assay, HAX42 also exhibited significant binding compared to the scrambled
• PAX2 control was indistinguishable from the scrambled PAX2 control.
• HAX42 and PAX2 Binding Motif Sequences Peptides and GST fusion proteins of HAX42, PAX2 and various derivatives were assayed using peptide ELISA to P100 membrane fractions derived from Caco-2 cells.
• the GST-PAX2 protein and PAX2 peptide data indicate that a core binding motif lies in the amino acid sequence TNAKHSSHNRRLRTR (SEQ ID NO: ) otherwise named GST-106 and ZElan033.
• the HAX42 peptide data suggest that a core binding motif for HAX42 lies in the amino acid sequence PGDYNCCGNCNSTG (SEQ ID NO: ), otherwise named ZElan091.
• the peptides and proteins were analyzed by a dansylated peptide ELISA method in which 96 well plates were coated overnight at 4°C with lOO ⁇ l/well coating protein (normally lOO ⁇ g/ml P100 membrane fraction) in 0.05M carbonate buffer pH9.6. Nonspecific binding was blocked using 200 ⁇ l/well, 2% Marvel/PBS for 2 hours at 37°C prior to incubation with dansylated peptides. The plates were washed three times with PBS/0.05% Tween 20 and after each subsequent incubation step.
• the peptides were diluted in blocking solution at a starting concentration of lOO ⁇ g/ml and diluted 1:2 downwards, lOO ⁇ l/well, followed by incubation at room temperature for 1 hour, exactly.
• a buffer blank control was included to ensure that background binding to plastic was not due to the antibodies used in the assay system.
• a mouse anti-dansyl antibody (DB3, Cytogen Corp.) at 1:1340 dilution in blocking buffer and lOO ⁇ l/well was added followed by incubation at room temperature for 1 hour.
• the plates were then incubated with an anti -mouse ⁇ -HRP conjugated antibody (Southern Biotech 1060-05) at a 1:10,000 dilution in blocking solution, lOO ⁇ l/well for 1 hour at room temperature. Plates were developed using 75 ⁇ l/well Bionostics TMB substrate and incubated for approximately 10 minutes. The developing reaction was stopped using Bionostics Red Stop solution
• ZElan021, full length HAX42 was given the arbitrary value of 1.00 for binding to P100 at a given peptide concentration determined from the signal-to-noise ratio data.
• PAX2 and its derivatives are given as a ratio of HAX42 value to reflect their binding abilities to P100 membrane fractions derived from a Caco-2 cell line as shown in Table 29.
• Table 30 provides a line-up of the PAX2 peptides showing the positive binding peptides in boldface.
• the GST-PAX2 peptide and PAX2 peptide data agree, demonstrating that a binding motif is in the amino acid sequence TNAKHSSHNRRLRTR (GST-106 and ZElan033) .
• PAX2 at value at at (Jackson (Southern peptide 20 ⁇ g/ml at 20 ⁇ g/ml 50/ig/ml 50 ⁇ g/ml Ab) Ab)
• ZElan018 -0.33 1.07 0.95 1.01 ZElan032 1.43 2.87 0.95 1.06 ZElan033 0.35 1.57 0.80 0.66 ZElan035 0.12 0.43 0.81 0.77 ZElan055 0.99 0.73 1.10 0.59 ZElan056 0.00 0.16 0.21 0.21 ZElan057 0.08 0.56 0.25 ZElan058 0.05 0.47 0.16 ZElan073 0.07 -0.11 0.49 0.66 0.49 ZElan074 0.06 0.82 0.52 0.71 0.48 ZElan075 0.13 0.52 0.38 0.47 0.32 ZElan076 0.08 1.00 0.41 0.60 0.42 ZElan077 0.20 0.76 0.54 0.73 0.52 ZElan078 0.11 0.87 0.69 0.68 0.47 ZElan079 0.31 0.97 0.68 0.83 0.53 ZElan080 0.23 0.84 0.45 0.67 0.38 ZElan081 0.01 0.89 0.47 ZElan082 0.00 0.92 0.40 ZElan083 0.43 0.
• PAX2 SEQ ID Peptide Amino acid sequence NO: ZElanOl ⁇ H_N-K (dns) STPPSREAYSRPYSVDSDSDTNAKHSSHNRRLRTRSRPNG -CONE ZElan032 H-N-K (dns) TNAKHSSHNRRLRTRSRPN-CONH 2 ZElan033 H 2 N-K (dns) TNAKHSSHNRRLRTR-CONH 2 ZElan034 H 2 N-K (dns) SSHNRRLRTRSRPN-CONH 2 ZElan035 H 2 N-K (dns) SSHNRRLRTR-CONH j ZElan055 H 2 N-K (dns) TNAKHSSHN-CONH 2 ZElan056 H 2 N-K (dns) RR RTRSRPN-CONH 2 ZElan057 H-N-K(dns) RRLRTRSR-CONH 2 ZElan058 H 2 N-K (
• ZElan021, full length HAX42 was given the arbitrary value of 1.00 for binding to P100 at a given peptide concentration determined from the signal-to-noise ratio data.
• HAX42 and its derivatives are given as a ratio of HAX42 value to reflect their binding abilities to P100 membrane fractions derived from a Caco-2 cell line as shown in Table 31.
• Table 32 provides a line-up of the HAX42 peptides showing the positive binding peptides in boldface.
• a core binding motif appears to lie in the amino acid sequence PGDYNCCGNCNSTG (ZElan091) .
• Solid particles containing a Therapeutic as defined herein are prepared using a coacervation method.
• The are particles are formed from a polymer and have a particle size of between about lOnm and 500 ⁇ m, most preferably 50 to 800 nm.
• the particles contain targeting ligands which are incorporated into the particles using a number of methods .
• the organic phase (B) polymer of the general method given above may be soluble, permeable, impermeable, biodegradable or gastroretentive .
• the polymer may consist of a mixture of polymer or copolymers and may be a natural or synthetic polymer.
• biodegradable polymers include without limitation polyglycolides; polylactides ; poly (lactide-co-glycolides) , including DL, L and D forms; copolyoxalates ; polycaprolactone; polyesteramides; polyorthoesters; polyanhydrides ; polyalkylcyanoacrylates ; polyhydroxybutyrates; polyurethanes; albumin; casein; citosan derivatives; gelatin; acacia; celluloses; polysaccharides ; alginic acid; polypeptides; and the like, copolymers thereof, mixtures thereof and stereoisomers thereof.
• Representative synthetic polymers include alkyl celluloses; hydroxalkyl celluloses; cellulose ethers; cellulose esters; nitrocelluloses ; polymers of acrylic and methacrylic acids and esters thereof; dextrans; polyamides; polycarbonates; polyalkylenes; polyalkylene glycols; polyalkylene oxides; polyalkylene terephthalates ; polyvinyl alcohols; polyvinyl ethers; polyvinyl esters; polyvinyl halides; poyvinylpyrrolidone; polysiloxanes and polyurethanes and co- polymers thereof.
• particles are formed using the following general method:
• the mixture (A) is stirred under low shear conditions at 10- 2000 rpm, preferably 100-600 rpm.
• the pH and/or ionic strength of this solution may be modified using salts, buffers or other modifying agents.
• the viscosity of this solution may be modified using polymers, salts, or other viscosity enhancing or modifying agents.
• a polymer preferably poly (lacide-co-glycolide) , polylactide, polyglycolide or a combination thereof or in any enantiomeric form or a covalent conjugate of the these polymers with a targeting ligand is dissolved in water miscible organic solvents to form organic phase (B) .
• organic phase (B) Most preferably, a combination of acetone and ethanol is used in a range of ratios from 0:100 acetone: ethanol to 100: 0 acetone: ethanol depending upon the polymer used.
• Additional polymer(s), peptide(s) sugars, salts, natural/biological polymers or other agents may also be added to the organic phase (B) to modify the physical and chemical properties of the resultant particle product.
• a drug or bioactive substance may be introduced into either the aqueous phase (A) or the organic phase (B) .
• a targeting ligand may also be introduced into either the aqueous phase (A) or the organic phase (B) at this point.
• the organic phase (B) is added into the stirred aqueous phase (A) at a continuous rate.
• the solvent is evaporated, preferably by a rise in temperature over ambient and/or the use of a vacuum pump.
• the particles are now present as a suspension (C) .
• a targeting ligand may be introduced into the stirred suspension at this point.
• a secondary layer of polymer(s), peptide(s) sugars, salts, natural/biological polymers or other agents may be deposited on to the pre- formed particulate core by any suitable method at this stage.
• the particles (D) are then separated from the suspension (C) using standard colloidal separation techniques, preferably by centrifugation at high x g' force, filtration, gel permeation chromatography, affinity chromatography or charge separation techniques .
• the supernatant is discarded and the particles (D) re-suspended in a washing solution (E) preferably water, salt solution, buffer or organic solvent (s) .
• the particles (D) are separated from the washing liquid in a similar manner as previously described and re-washed, commonly twice.
• a targeting ligand may be dissolved in washing solution (E) at the final washing stage and may be used to wash the particles (D) .
• the particles may then be dried. Particles may then be further processed for example, tabletted, encapsulated or spray dried.
• the release profile of the particles formed above may be varied from immediate to controlled or delayed release dependent upon the formulation used and/or desired.
• Drug loading may be in the range 0-90% w/w.
• Targeting ligand loading may be in the range 0-90% w/w.
• Bovine Insulin (Lot no. 86H0674) lOOmg Peptide: PAX2 (ZElanOl ⁇ ) 10mg/50ml dH 2 0
• the 5% w/v PVA solution was prepared by heating water until near boiling point, adding PVA and stirring until cool.
• the organic phase was prepared by adding acetone, 45ml, and ethanol, 5ml, together.
• the polymer solution was prepared by adding RG504H, 2g, to the organic phase and stirring until dissolved.
• the IKATM reactor vessel was set up, all seals greased and the temperature was set at 25°C.
• Bovine insulin lOOmg
• Bovine insulin lOOmg
• the polymer solution was slowly dripped in the stirring PVA solution with the peristaltic pump set at 40. The solvent was allowed to evaporate by opening the ports and allowing the dispersion to stir overnight at 400 rpm.
• the suspension was centrifuged in a Beckman
• the peptide solution (ZElan018, lOmg in 50ml dH 2 0) was prepared and added to the particles for a final washing stage. The suspended particles were centrifuged as before.
• the supernatant liquid was decanted, the ⁇ cake' broken up, and the particles were dried in the vacuum oven.
• the particles were ground, placed in a securitainer and sent for analysis. The weight of particles recovered was
• Aim To prepare a 2g batch of insulin loaded nanoparticles at a theoretical loading of 50mg/g and with the peptide ZElan018 added at the beginning of manufacture.
• Bovine Insulin (Lot no. 65H0640) lOOmg
• the 5% w/v PVA solution was prepared by heating water until near boiling point, adding PVA and stirring until cool.
• the organic phase was prepared by adding acetone, 45ml, and ethanol, 5ml, together.
• the polymer solution was prepared by adding RG504H (polyactide-co-glycolide, Boehringer Ingelheim) , 2g, to the organic phase prepared in step above and stirring until dissolved.
• the IKATM reactor vessel was set up, all seals greased and the temperature was set at 25°C.
• the PVA solution, 400ml was added into the reactor vessel and stirred at 400 rpm.
• Bovine insulin lOOmg
• PAX2 ZElan018ii, lOmg
• the polymer solution was slowly dripped into the stirring PVA solution with the peristaltic pump set at 40. The solvent was allowed to evaporate by opening the ports and allowing the dispersion to stir overnight at 400 rpm. The suspension was centrifuged in a Beckman UltracentrifugeTM with swing-out rotor at 12,500 rpm, 4°C. The supernatant was decanted and discarded.
• the "cake” of particles was broken up and dH 2 0 (200ml) was added to wash the particles. The centrifugation and washing steps were repeated twice. The 'cake' was broken up and the particles were dried in the vacuum oven.
• the particles were ground, placed in a securitainer and sent for analysis.
• the weight of the particles recovered was 1.6g.
• the potency was 47.3mg/g (94.6% of label claim) .
• Peptide loading was 1.689 ⁇ g/mg (33.8% of label claim).
• Aim To prepare a lg batch of insulin loaded nanoparticles at a theoretical loading of 50mg/g and with the peptide ZElanOl ⁇ added 1 hour before centrifugation.
• Bovine Insulin (Lot no. 65H0640) 50mg Peptide: PAX2 (ZElanOl ⁇ ) 5mg
• the 5% w/v PVA solution was prepared by heating water until near boiling point, adding PVA and stirring until cool.
• the organic phase was prepared by adding acetone, 22.5ml, and ethanol, 2.5ml, together.
• the polymer solution was prepared by adding RG504H, lg, to the organic phase prepared above and stirring until dissolved.
• the IKATM reactor vessel was set up, all seals greased and the temperature was set at 25°C.
• Bovine insulin 50mg was added into the stirring PVA solution. Using clean tubing and a green needle, the polymer solution was slowly dripped in the stirring PVA solution with the peristaltic pump set at 40. The solvent was allowed to evaporate by opening the ports and allowing the dispersion to stir overnight at 400 rpm.
• PAX2 (ZElanOl ⁇ 5mg) was added to the stirring particle suspension. After 1 hr, the suspension was centrifuged in a Beckman UltracentrifugeTM with swing-out rotor at 12,500 rpm, 4°C. The supernatant was decanted and discarded. The "cake" of particles was broken up and dH 2 0 (200ml) was added to wash the particles. The centrifugation and washing steps were repeated twice.
• EXAMPLE 4 Leuprolide acetate loaded nanoparticles Aim: To prepare a 3g batch of leuprolide-acetate loaded nanoparticles at a theoretical loading of 20mg/g and with the peptide ZElan024 added. Formulation Details RG504H (Lot no. 271077) 3. Og Acetone 67.5ml
• Leuprolide acetate (Lot no. V14094) 60mg Peptide: P31 (ZElan024) 15mg/50ml dH 2 0
• the PVA solution was prepared and the organic phase was prepared by adding acetone, 67.5ml, and ethanol, 7.5ml, together.
• the polymer solution was prepared by adding
• RG504H 3g, to the organic phase prepared above and stirring until dissolved.
• the IKATM reactor vessel was set up, all seals greased and the temperature was set at 25°C.
• Leuprolide acetate 60mg was added into the stirring PVA solution. Using clean tubing and a green needle, the polymer solution, was slowly dripped in the stirring PVA solution with the peristaltic pump set at 40. The solvent was allowed to evaporate by opening the ports and allowing the dispersion to stir overnight at 400 rpm. The suspension was centrifuged in a Beckman UltracentrifugeTM with swing-out rotor at 15,000 rpm, 4°C. The supernatant was decanted and retained for analysis. The "cake" of particles was broken up and dH 2 0
• the peptide solution (P31 (SEQ ID NO: 43) , 15mg in 50ml dH 2 0) was prepared and added to the particles for a final washing stage.
• the suspended particles were centrifuged as before.
• the supernatant liquid was decanted, and the particles were dried in the vacuum oven.
• Aim To prepare a 3g batch of insulin loaded 5 nanoparticles at a theoretical loading of 50mg/g and with the polymer-peptide conjugate PLGA-ZElan019 added.
• Bovine Insulin (Lot no. 86H0674) 150mg 15
• the 5% w/v PVA solution was prepared by heating water until near boiling point, adding PVA and stirring until cool.
• the organic phase was prepared by adding acetone, 20 67.5ml, and ethanol, 7.5ml, together.
• the polymer solution was prepared by adding RG504H and the polymer-peptide conjugate to the organic phase and stirring until dissolved.
• the IKATM reactor vessel was set up, all seals greased and the temperature was set at 25°C.
• the suspension was centrifuged in a Beckman
• the ⁇ cake' was broken up and the particles were dried in the vacuum oven.
• the particles were ground, placed 5 in a securitainer and sent for analysis.
• the weight of particles recovered was 2. ⁇ g.
• the potency was 53.1mg/g 106.2% of label claim).
• Peptide loading was 4.02 ⁇ g/mg (80.4% of label claim).
• Wistar rats 300- 350g were fasted for 4 hours and anaesthetized by
• test solution contained either PLGA particles manufactured according to the coacervation procedure given above with or without targeting peptides or by the "spiked" method given above. Insulin (fast-acting bovine; 28.1 iu/mg) was
• Study groups included animals receiving test solutions containing particles coated with the following peptides shown in Table 33. 35 Table 33

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• 1998
  • 1998-05-15 EP EP98922385A patent/EP1019071A4/de not_active Withdrawn
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