MX2008001352A - Tight junction modulating peptide components for enhancing mucosal delivery of therapeutic agents - Google Patents

Abstract

Compounds and components including sequences for mucosal epithelial transport of an active agent are given. Tight junction modulating peptide components are described for use in transport and delivery. Permeability can be enhanced with reversibility. Compounds and components forenhanced delivery may be peptide or protein variants, conjugates, or other analog types and structures.

Description

PEPTIDE COMPONENTS THAT MODULATE THE TIGHT UNION TO IMPROVE THE MUCOSO SUPPLY BACKGROUND OF THE INVENTION A fundamental interest in the treatment of any disease or condition is to ensure the safe and effective delivery of a therapeutic agent drug to the subject. Traditional routes of delivery of therapeutic agents include intravenous injection and oral administration. However, these delivery methods suffer disadvantages and consequently alternative supply systems are needed. A major disadvantage of drug administration by injection is that trained personnel are often needed to administer the drug. In addition, trained personnel are at risk when administering a drug through injection. For self-administered drugs, many patients are reluctant or unable to inject themselves on a regular basis. The injection is also associated with an increased risk of infection. Other disadvantages of drug injection include the variability of delivery results between individuals, as well as intensity and duration of unpredictable drug action. Despite the disadvantages, injection remains the only approved mode of supply for many important therapeutic compounds. These include conventional drugs, as well as a rapidly expanded list of peptide and protein biotherapeutics. The delivery of these compounds via alternative routes of administration, for example, oral, nasal and other mucosal pathways, is desirable, but may provide less bioavailability. For macromolecular species, for example, peptide and protein therapeutic compounds, alternative routes of administration may be limited by susceptibility to inactivation and low absorption through mucosal barriers. Oral administration of some therapeutic agents exhibits very low bioavailability and a considerable time delay of action due to first-pass hepatic metabolism and / or degradation in the gastrointestinal tract. The mucosal administration of therapeutic compounds offers certain advantages over injection and other modes of administration, for example, in terms of convenience and speed of delivery, as well as reduction or elimination of acceptance problems and side effects. However, the mucosal supply of biologically active agents is limited by the functions of the mucosal barrier and other factors. The epithelial cells make up the mucosal barrier and provide a crucial interface between the external environment and the mucosal and submucosal tissues and extracellular compartments. One of the most important functions of mucosal epithelial cells is to determine and regulate mucosal permeability. In this context, epithelial cells create selective permeability barriers between different physiological compartments. The selective permeability is the result of the transport of molecules regulated through the cytoplasm (the transcellular trajectory) and the regulated permeability of the spaces between the cells (the paracellular path). It is known that intercellular junctions between epithelial cells are involved in both the maintenance and regulation of epithelial barrier function, and cell-cell adhesion. Closed junctions (TJ) of epithelial and endothelial cells are particularly important for cell-to-cell junctions that regulate the permeability of the paracellular pathway, and also divide the cell surface into apical and basolateral compartments. The closed junctions form continuous circumferential intercellular contacts between the epithelial cells and create a regulated barrier to the paracellular movement of water, solutes, and immune cells. They also provide a second type of barrier that contributes to the polarity of the cell by limiting the exchange of membrane lipids between the apical and basolateral membrane domains. It is thought that closed junctions are directly involved in the barrier functions and near the epithelial cells creating an intercellular seal to generate a primary barrier against the diffusion of solutes through the paracellular path, and acting as a boundary between the domains of apical and basolateral plasma membrane to create and maintain cellular polarity, respectively. Closed junctions are also involved in the transmigration of leukocytes to reach inflammatory sites. In response to chemo-attractants, leukocytes migrate from the blood crossing the endothelium and, in the case of mucosal infections, crossing the inflamed epithelium. The transmigration occurs first along the paracellular route and seems to be regulated by opening and closing the closed junctions in a highly coordinated and reversible manner. Numerous proteins have been identified in association with TJs, including both integral and peripheral plasma membrane proteins. The current understanding of the complex structure and interactive functions of these proteins remains limited. Among the many proteins associated with epithelial junctions, several categories of membrane proteins have been identified trans-epithelial that can function in the physiological regulation of epithelial junctions. These include a number of "binding adhesion molecules" (JAMs) and other TJ-associated molecules designated as occludins, claudins, and zonulin. The JAMs, occludin, and claudin extend into the paracellular space, and these proteins in particular have been contemplated as candidates for creating an epithelial barrier between adjacent epithelial cells and channels through the epithelial cell layers. In one model, occludin, claudin, and JAM have been proposed to interact as hemophilic binding partners to create a regulated barrier for the paracellular movement of water, solutes, and immune cells between epithelial cells. In the context of drug delivery, the ability of drugs to permeate the epithelial cell layers of mucosal surfaces, unassisted by supply enhancement agents, seems to be related to a number of factors, including molecular size, solubility in lipids, and ionization. In general, small molecules, less than about 300-1,000 daltons, are often able to penetrate mucosal barriers, however, as the molecular size increases, the permeability rapidly decreases. For these reasons, mucosal drug administration requires typically drug amounts greater than administration by injection. Other therapeutic compounds, including large molecule drugs, are often refractory to mucosal delivery. In addition to low intrinsic permeability, large macromolecular drugs are often subject to limited diffusion, as well as luminal and cellular enzymatic degradation and rapid clearance at mucosal sites. Thus, in order to deliver these larger molecules in therapeutically effective amounts, cell permeation enhancement agents are required to aid their passage through those mucosal surfaces and into the systemic circulation where they will be able to act rapidly on the target tissue. Mucous tissues provide a substantial barrier to the free diffusion of macromolecules, while enzymatic activities present in mucosal secretions can severely limit the bioavailability of therapeutic agents, particularly peptides and proteins. In certain mucosal sites, such as the nasal mucosa, the typical residence time of proteins and other macromolecular species delivered is limited, e.g., to about 15-30 minutes or less, due to rapid mucociliary clearance. There has been a long-awaited and unfulfilled need in the technique of pharmaceutical formulations and methods for administering therapeutic compounds that provide improved mucosal delivery, including target tissues and physiological compartments such as in the central nervous system. More specifically, there is a need in the art for safe and reliable methods and compositions for the mucosal delivery of therapeutic compounds for the treatment of diseases and other adverse conditions in mammalian subjects. There is a related need for methods and compositions that will provide efficient delivery of macromolecular drugs by means of one or more mucosal routes in therapeutic amounts, which act rapidly, are easily administered and have limited adverse side effects such as mucosal irritation or tissue damage. There also remains a need in the art for methods and compositions for improving the mucosal delivery of biotherapeutic compounds that will overcome the mechanisms of the mucosal epithelial barrier. The selective permeability of mucosal epithelium has so far presented major obstacles in the mucosal delivery of therapeutic macromolecules, including peptides and biologically active proteins. Accordingly, there remains an unfulfilled need in the art for new methods and tools to facilitate the mucosal delivery of biotherapeutic compounds. In In particular, there is a need in the art for new methods and formulations to facilitate the mucosal delivery of biotherapeutic compounds that have proven to be refractory to supply through mucosal barriers. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates the effects of PN159 on the PTH -3 permeation, using PN159 with additional enhancers (Me-ß-CD, DDPC, EDTA). Figure 2 illustrates the effects of PN159 on PTH permeation? _34, using PN159 without additional enhancers. Figure 3 illustrates the effects of PN159 on the in vivo permeation of peptide YY. Figure 4 illustrates the effects of PN159 on the permeation of an MC-4 agonist receptor. Figure 5 shows the effects of 25-100 μM PN159 on the in vivo permeation of 40 mg / ml Galantamine lactate of an epithelial monolayer. Figure 6 shows the chemical stability of TJM peptide at (A) 5 ° C, (B) 25 ° C, and (C) 40 ° C. The data are plotted for pH 4.0, pH 7.3 and pH 9.0 as filled diamonds, open squares, and filled triangles, respectively. Figure 7 illustrates the permeation kinetics of FITC-dextran M 4000 in the presence of each peptide that modulates the tight union (TJMP). The PYY formulation acted as a positive control and phosphate buffered saline (PBS) was a negative control. The cell permeation was analyzed after a 15-minute cell treatment and also after a 60-minute cell treatment with the TJMP and the FITC dextran M 4000. The graph shows that the permeation depends on the length of the cell. time that the TJMP is in contact with the epithelial cell and that all the TJMPs tested improve the permeation of the FITC-dextran MW4000. Figure 8 illustrates the decrease in transepithelial electrical resistance (TER) after the 1 hour treatment of PN159 and PEG-PN159. Figure 9 illustrates the increase in permeability of FITC-dextran 3000 after treatment with PN159 and PEG-PN159. Figure 10 illustrates the permeation rate of PN159 and PEG-PN159. Figure 11 illustrates that pegylation of PN159 reduces toxicity (LDH analysis). Figure 12 illustrates the average concentration of PYY34-36 in improved plasma after nasal administration with PEGylated peptide PN529 (PEG-PN159). Figure 13 illustrates the mean concentration of PYY34-36 in improved plasma after administration nasal with PEGylated peptide PN529 (PEG-PN159). (Log-Linear Plot) DETAILED DESCRIPTION OF THE INVENTION The present invention meets the above needs and meets additional objectives and advantages by providing new pharmaceutical compositions including the new use of recently discovered closed-junction peptides., to improve the mucosal delivery of the biologically active agent in a mammalian subject. One aspect of the invention is a pharmaceutical formulation comprising a biologically active agent and an effective enhancing amount of the mucosal delivery of a peptide that modulates the tight junction (TJMP) that reversibly improves mucosal epithelial transport of a biologically active agent in a mammalian subject. Preferably, a tight binding modulation component contains a peptide or protein portion consisting of 2-500 amino acid residues, or 2-100 amino acid residues, or 2-50 amino acid residues. The tight binding modulation peptide or protein can be monomeric or oligomeric, e.g., dimeric. The peptide that modulates the tight junction can be produced by means of recombinant or chemical synthesis, consistent with techniques known to those skilled in the appropriate art.

Peptides capable of modulating the function of closed epithelial junctions have been previously described (Johnson, P. H. and S. Quay, Expert, Opin. Drug Deliv. 2: 281-98, 2000). In particular, a peptide that modulates the tight junction (TJMP) new, PN159, showed to reduce the transepithelial electrical resistance (TER) through a tissue barrier and increase the paracellular transport of 3,000 Da MW dextran with low cytotoxicity and high cell viability retention. In preferred embodiments of the invention, the TJMP is selected from a group consisting of: NH2-NH2 KLALKLALKALKAALKLA-amide-amide GWTLNSAGYLLGKINLKALAALAKKIL-NH2-NH2-amide LLETLLKPFQCRICRMNFSTRQARRNHRRRHRR AAVALLPAVLLALLAPRKKRRQRRRPPQ amide NH2-NH2-amide RKKRRQRRRPPQCAAVALLPAVLLALLAP RQIKIWFQNRRMKWKK-amide NH2 KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ-amide NH2-KLWSAWPSLWSSLWKP-amide NH2-RRRQRRKRGGDIMGEWGNEIFGAIAGFLG-amide Maleimide-GLGSLLKKAGKKLKQPKSKRKV-amide NH2-KETWWETWWTEWSQPGRKKRRQRRRRPPQ-amide. In other preferred embodiments of the invention, the TJMP is selected from the group consisting of: CNGRCGGKKKLKLLLKLL LRKLRKRLLRLRKLRKRLLR In one aspect, this invention describes formulations of small molecules, peptides and therapeutic proteins that are suitable for transmucosal delivery, wherein the transmucosal delivery is facilitated by the presence of a peptide that modulates the tight junction, wherein said peptide is conjugated a water soluble polymer. Preferably, the water soluble polymer is a polyalkylene oxide selected from the group consisting of alpha-substituted polyalkylene oxide derivatives, alkyl-capped polyethylene oxides, bis-polyethylene oxides, poly (orthoesters) such as poly (lactico-co) -glycolide) and derivatives thereof, polyethylene glycol homopolymers (PEG) and derivatives thereof, polypropylene glycol homopolymers and derivatives thereof, poly (alkylene oxide) copolymers, poly (alkylene oxide) block copolymers or activated derivatives thereof. Preferably, the polyalkylene oxide has a molecular weight of about 200 to about 50,000. More preferably the polyalkylene oxide has a molecular weight of from about 200 to about 20,000. Especially preferred polyalkylene oxides are polyethylene glycol and polyethylene oxide. TJMP can be conjugated to more than one water-soluble chain. In a preferred embodiment the chain Poly (alkylene oxide) is a polyethylene glycol (PEG) chain, which may have a molecular size between about 0.2 and about 200 Kilodaltons (kDa). The water-soluble polymer can be conjugated to the peptide that modulates the tight junction by means of a separator. This link can be reversible. The water-soluble polymer can be linear or can be branched. In one embodiment, the peptide is covalently linked to a single poly (alkylene oxide) chain. In a related embodiment, the poly (alkylene oxide) has a polydispersity value (Mw / Mn) of less than 2.00, or less than 1.20. The poly (alkylene oxide) chain may be branched or unbranched. The conjugate has been used with water-soluble polymers such as poly (ethylene glycol) (PEG). and PEG derivatives as a strategy for improving the half-life of proteins, in particular for injected dose forms (Calceti, P. and F.M. Veronese, Adv. Drug Deliv. Rev. 55: 1261-77, 2003). Other potential benefits of modifying peptides and proteins with polymers such as PEG include chemical stabilization (DIWAN, M. and TG Park, Int. J. Pharm. 252: 111-22, 2003) and biochemistry (Na, DH, et al. ., J. Pharm, Sci. 93: 256-61, 2004) and attenuation of immunogenicity (Yang, Z., et al., Cancer Res. 64: 6673-78, 2004).

Most examples for the use of protein conjugated PEG are found where the PEG chain has a molecular weight of sufficient length to impart the effect described above. In particular, it has been described that at least 20 kDa MW of PEG is required. For example, Holtsberg et al. (Holtsberg, FW, et al., J. Con trol Rei. 80: 259-71, 2002) showed that for the protein arginine deiminase conjugated to PEG, when PEG was 20 kDa or greater, there was an increase in the pharmacokinetic properties and pharmacodynamics of the formulation when administered intravenously in mice. When PEG MW was lower than 20 kDa, there was little effect. In another example, mono-PEGylation to the salmon calcitonin peptide results in an increase in intranasal bioavailability in rats, the improvement being inversely proportional to the molecular weight (MW) of PEG (Lee, K. C, et al., Calcif. Tissue Int. 73: 545-9, 2033, and Shin, BS, et al., Chem. Pharm. Bull. (Tokyo) 52: 957-60, 2004), incorporated in their entirety by reference herein. . Some preferred poly (alkylene oxides) are selected from the group consisting of alpha-substituted poly (alkylene oxide) derivatives, poly (ethylene glycol) homopolymers (PEG) and derivatives thereof, poly (propylene glycol) homopolymers (PPG) ) and derivatives thereof, poly (ethylene oxide) (PEO) polymers and derivatives of the same, bis-poly (ethylene oxides) and derivatives thereof, copolymers of polyalkylene oxides, and block copolymers of poly (alkylene oxides), poly (lactide-co-glycolide) and derivatives thereof , or activated derivatives thereof. The water-soluble polymer can have a molecular weight of from about 200 to about 40,000 Da, preferably about 200 to about 20,000 Da, or about 200 to 10,000 Da, or about 200 to 5,000 Da. The conjugate between the peptide that modulates the tight junction and the PEG or other water-soluble polymer can be resistant to physiological processes, including proteolysis, enzymatic action or hydrolysis in general. Alternatively, the conjugate can be cleaved by biodegradation processes, for example a pro-drug process. Preferably, the molecule is covalently linked to a single poly (alkylene oxide) chain, which may be branched or unbranched. The conjugation means are generally known to those skilled workers, for example, the U.S. Patent. No. 5,595,732; the Patent of E.U. No. 5,766,897; the Patent of E.U. No. 5,985,265; the Patent of E.U. No. 6,528,485; the Patent of E.U. No. 6,586,398; the Patent of E.U. No. 6,869,932; and the U.S. Patent. No. 6,706,289. In another aspect of the invention, the TJMP decreases the electrical resistance through a barrier of mucosal tissue. In a preferred embodiment, the decrease in electrical resistance is at least 80% of the electrical resistance before applying the permeation enhancer. In a related embodiment, TJMP increases the permeability of the molecule through a mucosal tissue barrier, preferably at least twice. In another embodiment, the increased permeability is paracellular. In another embodiment, the increased permeability results from the modification of closed joints. In an alternative embodiment, the increased permeability is transcellular, or a combination of trans- and paracellular. In another aspect of the invention, the mucosal tissue layer is comprised of a layer of epithelial cells. In a preferred embodiment, the epithelial cell is selected from the group consisting of tracheal, bronchial, alveolar, nasal, pulmonary, gastrointestinal, epidermal or buccal, preferably nasal. In another aspect of the invention an active agent is a peptide or protein. The peptide or protein may have between 2 and 1000 amino acids. In a preferred embodiment, the peptide or protein is comprised between 2 and 50 amino acids. In another embodiment, the peptide or protein is cyclic. In another embodiment, the peptide or protein forms higher order dimers or oligomers by binding physics or chemistry. In a preferred embodiment, the peptide or protein is selected from the group comprising GLP-I, PYY? 34, PTH? _3 and Exendin-4. In another embodiment, the biologically active agent is a protein, preferably selected from the group consisting of interferon-beta, interferon-alpha, insulin, erythropoietin, G-CSF, and GM-CSF, growth hormone, and analogs of any of these . The permeabilization peptides of the invention include PN529, which contains the sequence WEAALAEALAEALAEHLASQPKSKRKV (SEQ ID NO 57). Another aspect of the invention is a method for administering a molecule to an animal comprising the preparation of any of the above formulations, and bringing said formulation into contact with a mucosal surface of such an animal. In a preferred embodiment, the mucosal surface is intranasal. Another aspect of the invention is a dosage form comprising any of the above formulations, in which the dosage form is liquid, preferably in the form of drops. Alternatively, the dosage form may be solid, either to be reconstituted in liquid before administration, to be administered as a powder, or in the form of a capsule, tablet or gel. Another aspect of the invention is a molecule that reversibly enhances mucosal epithelial transport of a biological agent in a mammalian subject, having a peptide component that modulates the tight junction (TJMP), a TJMP analog, a conjugate of a TJMP or a TJMP analog, or complexes thereof . The permeabilization peptides of the invention include PN159, which has the sequence NH2-KLALKLALKALKAALKLA-amide. Analogs of PN159 as described herein, combinations of those analogs, and any derivatives, variants, fragments, mimetics, or fusion molecules of PN159 are included in the invention. The permeabilization agent reversibly enhances paracellular mucosal epithelial transport, typically by modulating tight epithelial binding structures and / or physiology on a mucosal epithelial surface in the subject. This effect typically involves the inhibition by means of the permeabilization agent of the homotypic or heterotypic binding between the adhesive proteins of the epithelial membrane of the neighboring epithelial cells. The target proteins for this homotypic or heterotypic binding block can be selected from several related binding adhesion molecules (JAMs), occludins or claudins. Epithelial Cell Biology A murine-binding adhesion molecule-1 (JAM-1) coding for cDNA has been cloned and corresponds to a predicted type I transmembrane protein (comprising a single transmembrane domain) with a molecular weight of approximately 32-kD (Williams , et al., Molecular Immunology 36: 1175-1188, 1999; Gupta, et al., IUBMB Life 50: 51-56, 2000; Ozaki, et al., J. Immunol., 163: 553-557, 1999; Martin; -Padura, et al., J. Cell Biol. 142: 111-121, 1998). The extracellular segment of the molecule comprises two Ig-like domains described as a "VH-type" amino terminal and a "C2-type" terminal carboxy terminal of β-interspersed sheet (Bazzoni et al., Microcirculation 5: 143-152, 2001). Murine JAM-1 also contains two sites for N-glycosylation, and a cytoplasmic domain. The JAM-1 protein is a member of the immunoglobulin (Ig) superfamily and localizes closed junctions in both epithelial and endothelial cells. Ultra structural studies indicate that JAM-1 is limited to the membrane regions containing occludin fibrils and claudin. Another member of the JAM family, designated "Molecule Associated with the Vascular Endothelial Junction" (VE-JAM), contains two extracellular immunoglobulin-like domains, a transmembrane domain, and a relatively short cytoplasmic flow. VE-JAM is located mainly in the intercellular boundaries of endothelial cells (Palmeri et al., J. Biol. Chem. 275: 19139-19145, 2000). VE-JAM is highly expressed by the endothelial cells of the venules, and is also expressed by the endothelium of other vessels. Another member of the JAM family reported, JAM-3, has a predicted amino acid sequence that exhibits 36% and 32% identity, respectively, to JAM-2 and JAM-1. JAM-3 shows a widely diffused tissue expression with apparent higher levels in the kidney, brain, and placenta. At the cellular level, the transcription of JAM-3 is expressed within the endothelial cells. JAM-3 and JAM-2 have reported being union partners. In particular, the JAM-3 ectodomain according to the report joins the JAM2-Fc. The JAM-3 protein is over-regulated in peripheral blood lymphocytes after activation. (Pia Arrate, et al., J. Biol. Chem. 276: 45826-45832, 2001). Another proposed transmembrane adhesive protein involved in the regulation of the tight epithelial junction is occludin. Occludin is a type II transmembrane protein of approximately 65 kD composed of four transmembrane domains, two extracellular loops, and a large C-terminal cytosolic domain (Furuse et al., J. Cell, Biol. 1231777-1788 (1993); Furuse, et al., J. Cell, Biol. 127: 1617-1626, 1994). When observed by immuno-frozen electron fracture microscopy, occludin is concentrated directly within the fibrils of tight junction (Fujimoto, J. Cell, Sci. 108: 3443-3449, 1995). Two additional integral membrane proteins of the tight junction, claudin-1 and claudin-2, were identified by direct biochemical fragmentation of enriched chicken liver binding membranes (Furuse, et al., J. Cell. Biol. 141: 1539 -1550, 1998). It was found that claudin-1 and claudin-2 are co-purified with occludin as a broad band of gel of approximately 22 kD in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The deduced sequences of two closely related cloned proteins from a mouse cDNA library predicted transmembrane helices, two short extracellular loops, and short cytoplasmic C and N terminals. Despite topologies similar to occludin, they do not share any sequence homology. Subsequently, six more claudin gene products (from claudin-3 to claudin-8) have been cloned and have been shown to be located within the tight-binding fibrils, as determined by frozen gold immunity fracture labeling (Morita, et al. ., Proc. Na ti, Acad. Sci. USA 36: 511-516, 1999). Since the barrier remains in the absence of occludin, it has been considered claudin-1 to claudin-8 as candidates for the primary elements of seal formation of the extracellular space. Other cytoplasmic proteins that have been localized in epithelial junctions include zonulin, symplekina, Cingulin, and 7H6. The reported zonulins are cytoplasmic proteins that bind to the cytoplasmic cauda of occludin. They are representatives of this protein family "ZO-1, ZO-2 and ZO-3". Zonulin is postulated as a human protein analogue of the zonula occludens toxin (ZOT) derived from Vibrio cholerae. Zonulin probably plays a role in regulating the tight junction during developmental, physiological and pathological processes including tissue morphogenesis, fluid movement, macromolecules and leukocytes between the intestinal lumen and the interstitium, and inflammatory / autoimmune disorders (see eg , Wang, et al., J. Cell, Sci. 113: 4435-40, 2000). The expression of zonulin increased in intestinal tissues during the acute phase of celiac disease, a clinical condition in which closed junctions are open and permeability increases. Zonulin induces the disassembly of the tight junction and a subsequent increase in intestinal permeability in intestinal epithelium of non-human primate in Vi tro. Comparison of amino acids in the active fragment V. cholerae ZOT and human zonulin identified a putative receptor binding domain within the N-terminal region of the two proteins. The biologically active ZOT domain increases intestinal permeability interacting with a mammalian cell receptor with the subsequent activation of intracellular signaling leading to the disassembly of the tight intercellular junction. The biologically active ZOT domain has been located towards the carboxyl terminal of the protein and coincides with the predicted cleavage product generated by V. cholerae. This domain shares a putative receptor binding motif with zonulin, the mammalian ZOT analog. The amino acid comparison between the active fragment ZOT and zonulin, combined with site-directed mutagenesis experiments, suggests a binding domain of the octapeptide receptor towards the amino terminus of processed ZOT and the amino terminus of zonulin. (Di Pierro, et al., J. Biol. Chem. 276: 19160-19165, 2001). ZO-1 reported binding with actin, AF-6, ZO-associated kinase (ZAK), fodrin, and a-catenin. Permeabilization peptides for use in the invention include natural or synthetic peptides, therapeutically or prophylactically active (comprised of two or more covalently linked amino acids), proteins, peptide or protein fragments, peptide or protein analogs, peptide or protein mimetics , and derivatives or chemically modified salts of peptides or active proteins. Therefore, as used herein, the term "permeabilization peptide" will often pretend cover all these active species, i. e., peptides and proteins, peptide and protein fragments, peptide and protein analogs, peptide and protein mimetics, and derivatives and chemically modified salts of active peptides or proteins. Often, peptides or permeabilization proteins are muteins that are easily obtained by partial replacement, addition, or deletion of amino acids within a peptide or protein sequence of natural or native origin (eg, wild-type, mutation of natural origin, or allelic variant). In addition, biologically active fragments of peptide or native proteins are included. Such derivatives and mutant fragments retain substantially the desired biological activity of the native peptides or proteins. In the case of peptides or proteins having carbohydrate chains, biologically active variants marked by alterations in these carbohydrate species are also included in the invention. Permeabilization peptides, proteins, analogs and mimetics for use in the methods and compositions of the invention are often formulated in a pharmaceutical composition comprising an effective amount of mucosal delivery or permeabilization of the permeabilization peptide, protein, analog. or mimetic, which reversibly improves paracellular transport epithelial mucosa by modulating the structure and / or physiology of epithelial attachment in a mammalian subject. Biologically Active Agents The methods and compositions of the present invention are directed towards improving the mucosal, e.g., intranasal delivery of a broad spectrum of biologically active agents to achieve desired therapeutic, prophylactic or other physiological results in mammalian subjects. As used herein, the term "biologically active agent" encompasses any substance that produces a physiological response when administered by mucosal means to a mammalian subject in accordance with the methods and compositions herein. Biologically active agents useful in this context include therapeutic or prophylactic agents applied in all major fields of clinical medicine, as well as nutrients, cofactors, enzymes (endogenous or external), antioxidants, and the like. Thus, the biologically active agent can be water soluble or not soluble in water, and can include higher molecular weight proteins, peptides, carbohydrates, glycoproteins, lipids, and / or glycolipids, nucleosides, polynucleotides, and other active agents. Pharmaceutical agents useful in the methods and compositions of the invention include drugs and agents Therapeutic or prophylactic macromolecules encompassing a broad spectrum of compounds, including small molecule drugs, peptides, proteins, and vaccine agents. Exemplary pharmaceutical agents for use in the invention are biologically active for the treatment or prophylaxis of a selected disease or condition in a subject. The biological activity in this context can be determined as any significant (ie, calculable, statistically significant) effect on a physiological parameter, marker, or clinical symptom associated with a disease or condition of the subject, evaluated by an in vi tro or in vitro analysis system. Live appropriate involving real patients, cell cultures, simple analyzes, or acceptable animal models. The methods and compositions of the invention provide unexpected advantages for the treatment of diseases and other conditions in mammalian subjects, whose advantages are mediated, for example, by providing improved speed, duration, fidelity or control of mucosal delivery of therapeutic or prophylactic compounds to reach selected physiological compartments in the subject (eg, within or through the nasal mucosa, in the systemic circulation or the central nervous system (CNS), or to any selected target organ, tissue, fluid, or cellular or extracellular compartment within the subject).

In various exemplary embodiments, the methods and compositions of the invention may incorporate one or more biologically active agent (s) selected from: opiates or opiate antagonists, such as morphine, hydromorphone, oxymorphone, lovorphanol, levallorphan, codeine, nalmefene , nalorphine, nalozone, naltrexone, buprenorphine, butorphanol, and nalbuphine; corticosterones, such as cortisone, hydrocortisone, fludrocortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, dexametoasone, betametho- soone, parametosone, and fluocinolone; other anti-inflammatory drugs, such as colchicine, ibuprofen, indomethacin, and piroxicam; anti viral agents such as acyclovir, ribavarin, trifluorothyridine, Ara-A (Arabinofuranosyladenine), acylguanosine, nordeoxyguanosine, azidothymidine, dideoxydenosine, and dideoxycytidine; antiandrogens such as spironolactone; androgens, such as testosterone; estrogens, such as estradiol; progestins; muscle relaxants, such as papaverine; vasodilators, such as nitroglycerin, vasoactive intestinal peptide and calcitonin-related gene peptide; antihistamines, such as cyproheptadine; agents with histamine receptor site blocking activity, such as doxepin, imipramine, and cimetidine; antitussives, such as dextromethorphan; neuroleptics such as clorazil; antiarrhythmics; antiepileptics; enzymes, such as superoxide dismutase and neuroenkephalinase; anti-fungal agents, such as amphotericin B, griseofulvin, miconazole, ketoconazole, ticonazole, itraconazole, and fluconazole; antibacterials, such as penicillins, cephalosporins, tetracyclines, aminoglycosides, erythromycin, gentamicins, polymyxin B; anticancer agents, 5-fluorouracil, bleomycin, methotrexate, and hydroxyurea, dideoxyinosine, floxuridine, 6-mercaptopurine, doxorubicin, daunorubicin, I-darubicin, taxol and paclitaxel; antioxidants, such as tocopherols, retinoids, carotenoids, ubiquinones, metal chelators, and phytic acid; antiarrhythmic agents, such as quinidine; and antihypertensive agents such as prazosin, verapamil, nifedipine, and diltiazem; analgesics such as acetaminophen and aspirin; monoclonal and polyclonal antibodies, including humanized antibodies, and antibody fragments; antisense oligonucleotides; and RNA, DNA and viral vectors comprising genes encoding therapeutic peptides and proteins. In addition to these exemplary classes and species of active agents, the methods and compositions of the invention encompass any physiologically active agent, as well as any combination of multiple active agents, described above or elsewhere herein or otherwise known in the art. technique, ie individually or combinatorially effective in the methods and compositions of the invention for the treatment or prevention of a selected disease or condition in a mammalian subject (see, Physician's Desk Reference, published by Medical Economics Company, a division of Litton Industries, Inc). Regardless of the class of compound employed, the biologically active agent for use in the invention will be present in the compositions and methods of the invention in an amount sufficient to provide the desired physiological effect without significant unacceptable toxicity or other adverse side effects to the subject. The appropriate dose levels of all biologically active agents will be readily determined without undue experimentation by the skilled artisan. Given the the methods and compositions of the invention provide an improved delivery of the biologically active agent (s), significantly lower dose levels than conventional dose levels can be used. In general, the active substance will be present in the composition in an amount of from about 0.01% to about 50%, often between about 0.1% to about 20%, and commonly between about 1.0% to 5% or 10% by weight of the total intranasal formulation depending on the particular substance employed. As used herein, the terms "peptide" and "protein" biologically active include polypeptides of various sizes, and do not limit the invention to amino acid polymers of any particular size. Peptides from a length as small as a few amino acids, to proteins of any size, as well as peptide-peptide, protein-protein and protein-peptide fusions, are encompassed by the present invention, so long as the protein or peptide is biologically active in the context of obtaining a specific physiological, immunological, therapeutic, or prophylactic effect or response. The present invention provides new formulations and coordinates the administration of methods to improve the mucosal delivery of peptides and proteins biologically active Illustrative examples of peptides and therapeutic proteins for use in this invention include, but are not limited to: plasminogen tissue activator (TPA), epidermal growth factor (EGF), fibroblast growth factor (FGF-acidic or basic), platelet-derived growth factor (PDGF), transforming growth factor (TGF-alpha or beta), vasoactive intestinal peptide, tumor necrosis factor (TNF), hypothalamic releasing factors, prolactin, thyroid stimulating hormone (TSH) , adrenocorticotropic hormone (ACTH), parathyroid hormone (PTH), follicle stimulating hormone (FSF), luteinizing hormone-releasing hormone (LHRH), endorphins, glucagon, calcitonin, oxytocin, carbetocin, aldoetecona, encacaline, somatostin, somatotropin, somatomedin , gonadotropin, estrogen, progesterone, testosterone, alpha-melanocyte stimulating hormone, opiates of non-natural origin, lidocaine, ketoprofen, sufentainil, ter butaline, droperidol, scopolamine, gonadorelin, cyclopirox, buspirone, calcitonin, cromolyn sodium or midazolam, cyclosporin, lisinopril, captopril, delapril, cimetidine, ranitidine, famotidine, superoxide dismutase, asparaginase, arginase, arginine, deaminase, adenosine deaminase ribonuclease, trypsin, chymotrypsin, and papain. Additional examples of useful peptides include, but are not limited to, bombesin, substance P, vasopressin, alpha-globulins, transferin, fibrinogen beta-lipoproteins, beta-globulins, fetuin, alpha-lipoproteins, alpha-globulins, albumin, prealbumin, and other bioactive proteins and recombinant protein products. In more detailed aspects of the invention, methods and compositions are provided for improving the mucosal delivery of biologically active peptide or protein therapeutics specific for the treatment (ie, to eliminate, or reduce the presence or severity of the symptoms of) of a disease or existing condition, or to prevent the outbreak of a disease or condition in a subject identified at risk of disease or subject condition. Peptides or biologically active proteins useful in aspects of the invention include, but are not limited to hematopoietics; anti-infective agents; anti-dementia agents; anti viral agents; anti tumor agents; anti pyrimic; analgesics; anti-inflammatory agents; anti ulcer agents; anti allergic agents; antidepressants; psychotropic agents; cardiotonic; antiarrhythmic agents; vasodilators; anti-hypertensive agents such as hypotensive diuretics; anti diabetic agents; anticoagulants; cholesterol lowering agents; therapeutic agents for osteoporosis; hormones; antibiotics; vaccines; and the similar.

Peptides or biologically active proteins for use in these aspects of the invention include, but are not limited to, cytosines; peptide hormones; growth factors; factors that act on the cardiovascular system; cell adhesion factors; factors that act on the central and peripheral nervous system; factors that act on humoral electrolytes and hemal organic substances; factors that act on the growth or physiology of bones and skeleton; factors that act in the gastrointestinal system; factors that act on the kidney and urinary organs; factors that act on the connective tissue and skin; factors that act on the sensory organs; factors that act on the genital organs; and several enzymes. For example, hormones that can be administered in the methods and compositions of the present invention include androgens, estrogens, prostaglandins, somatotropins, gonadotropins, interleukins, steroids and cytokines. Vaccines that can be administered in the methods and compositions of the present invention include bacterial and viral vaccines, such as vaccines for hepatitis, influenza, respiratory syncytial virus (RSV), parainfluenza virus (PIV), tuberculosis, yellow pox, smallpox, measles , mumps, rubella, pneumonia, and human immunodeficiency syndrome (HIV).

Bacterial toxoids that can be administered in the methods and compositions of the present invention include diphtheria, tetanus, pseudonomas, and mycobacterium tuberculosis. Example of specific cardiovascular or thrombolytic agents for use within the invention include hirugen, hiruli and hirudin. Antibody reagents that are usefully administered with the present invention include monoclonal antibodies, polyclonal antibodies, humanized antibodies, antibody fragments, fusions and multimers, and immunoglobulins. As used herein, the term "amino acid conservative substitution" refers to the general interchangeability of amino acid residues that have similar side chains. For example, a commonly interchangeable group of amino acids having aliphatic side chains is alanine, valine, leucine, and isoleucine; A group of amino acids that has hydroxyl-aliphatic side chains is serine and threonine; A group of amino acids having side chains containing amide is asparagine and glutamine; A group of amino acids that has aromatic side chains is phenylalanine, tyrosine, and tryptophan; an amino acid group having basic side chains is lysine, arginine, and histidine; a group of amino acids that have side chains that contain sulfur is cysteine and methionine. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another. Similarly, the present invention contemplates the substitution of a polar (hydrophilic) residue such as between arginine and lysine, between glutamine and asparagine, and between threonine and serine. In addition, substitution of a basic residue such as lysine, arginine or histidine, or substitution of an acidic residue such as aspartic acid or glutamic acid, is also contemplated. Exemplary conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. The term "biologically active peptide or protein analog" further includes modified forms of native peptide or protein that incorporate stereoisomers (eg, D-amino acids) of the twenty conventional amino acids, or non-natural amino acids such as α, α-disubstituted amino acids, N-alkyl amino acids , lactic acid. These and other non-conventional amino acids can also be substituted or inserted into native peptides or proteins useful within the invention. Examples of non-conventional amino acids include: 4-hydroxyproline, y-carboxyglutamate, e-N, N, N-trimethylisine, e-N-acetylisine, O- phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,? -N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In addition, analogs of biologically active peptides or proteins include single or multiple substitutions, deletions and / or additions of carbohydrates, lipids and / or proteinaceous residues that are of natural or artificial origin as structural components of the peptide or protein in question, or are linked a, or otherwise associated with the peptide or protein. In one aspect, the peptides (including polypeptides) useful within the invention are modified to produce peptide mimetics by replacing one or more naturally occurring side chains of the 20 genetically encoded amino acids (or D amino acids) with other side chains, for example with groups such as alkyl, lower alkyl, 4-, 5-, 6-, 7-membered cyclic alkyl, amide, lower alkyl amide, di (lower alkyl) amide, lower alkoxy, hydroxy, carboxy, and the derivatives of lower ester thereof, and with heterocyclics of 4-, 5-, 6-, to 7 members. For example, proline analogs can be manufactured in which the ring size of the proline residue changes from 5 members to 4, 6, or 7 members. The cyclic groups can be saturated or unsaturated, and if they are unsaturated, they can be aromatic or non-aromatic. Heterocyclic groups can contain one or more nitrogen, oxygen, and / or sulfur heteroatoms. Examples of such groups include furazanyl, furyl, imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (eg, morpholino), oxazolyl, piperazinyl (eg, 1-piperazinyl), piperidyl (eg, 1-piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl (eg, 1-pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (eg, thiomorpholino), triazolyl. These heterocyclic groups can be substituted or unsubstituted. When a group is substituted, the substituent may be alkyl, alkoxy, halogen, oxygen, or substituted or unsubstituted phenyl. Peptides and proteins, as well as peptide and protein analogs or mimetics, may also be covalently linked to one or more of a variety of non-protein polymers, eg, polyethylene glycol, polypropylene glycol, or polyoxyalkenes, in the manner set forth in the US Pat. USA No. 4,640,835; the Patent of E.U. No. 4,496,689; the Patent of E.U. No. 4,301,144; the Patent of E.U. No. 4,670,417; the Patent of E.U. No. 4,791,192; or the U.S. Patent. No. 4,179,337. Other peptide and protein analogs and mimetics within the invention include glycosylation variants, and covalent or conjugated aggregates with other chemical residues. Covalent derivatives can be prepared by linking functionalities to groups found on the amino acid side chains or at the N- or C- terminus, by means well known in the art. These derivatives may include, without limitation, aliphatic esters or amides of the carboxyl terminus, or residues containing carboxyl side chains, 0-acyl derivatives of the hydroxyl group-containing residues and N-acyl derivatives of the amino terminal amino acid or residues that contain an amino group, eg, lysine or arginine. The acyl groups are selected from the group of alkyl residues including C3 * to C18 normal alkyl, thereby forming alkanoyl aroyl species. Covalent attachment to carrier proteins, e.g., immunogenic residues, may also be employed. In addition to these modifications, glycosylation alterations of peptides and biologically active proteins can be carried out, e.g., by modifying the glycosylation patterns of a peptide during its synthesis and processing, or in subsequent processing steps. Particularly preferred means to accomplish this is to expose the peptide to glycosylation enzymes derived from cells that normally provide such processing, e.g., mammalian glycosylation enzymes. Deglycosylation enzymes can also successfully used to produce peptides and modified proteins useful within the invention. Also included are versions of a native primary amino acid sequence having other minor modifications, including phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine, or other residues, including ribosyl groups or crosslinking agents. The peptidomimetics may also have amino acid residues that have been chemically modified by phosphorylation, sulfonation, biotinylation, or the addition or removal of other residues, particularly those that have molecular forms similar to phosphate groups. Active peptides can be cycled for use within the invention, or incorporate a deamino or decarboxy residue at the peptide terminus, such that there is no amino terminal or carboxyl group, which decreases the susceptibility to proteases, or restricts the conformation of the peptide. The C-terminal functional groups among the peptide analogs and mimetics of the present invention include amide, lower alkyl amide, di (lower alkyl) amide, lower alkoxy, hydroxy, and carboxy, and the lower ester derivatives thereof, and the pharmaceutically acceptable salts thereof. A variety of additives, diluents, bases and delivery vehicles are provided within the invention that effectively control the water content to improve the stability of the protein. These reagents and carrier materials effective as an anti-aggregation agent in this regard include, for example, polymers of various functionalities, such as polyethylene glycol, dextran, dextran, diethylaminoethyl, and carboxymethyl cellulose, which significantly increase stability and reduce solid-based aggregation. of peptides and proteins mixed with them or linked thereto. In some examples, the activity or physical stability of proteins can also be improved by various additives to aqueous solutions of the peptide or protein drugs. For example, additives, such as polyols, can be used (including sugars), amino acids, proteins such as collagen and gelatin and various salts. Certain additives, in particular sugars and other polyols, also impart significant physical stability to dry, e.g., lyophilized proteins. These additives can also be used within the invention to protect the proteins against aggregation not only during lyophilization but also during storage in the dry state. For example sucrose and Ficoll 70 (a polymer with sucrose units) exhibit significant protection against aggregation to peptide or protein during solid phase incubation under various conditions. These additives can also improve the stability of the solid proteins embedded within the polymer matrices. Still further additives, for example sucrose, stabilize the proteins against aggregation of solid state in humid atmospheres at elevated temperatures, as may occur in certain sustained release formulations of the invention. Proteins such as gelatin and collagen also serve as stabilizing or bulking agents to reduce the denaturation and aggregation of unstable proteins in this context. These additives can be incorporated in polymer melt processes and in the compositions in the invention. For example, the polypeptide microparticles can be prepared by simple lyophilization or by spray drying a solution containing various stabilizing additives described above. The sustained release of non-aggregated peptides and proteins can thus be obtained over an extended period of time. Several additional preparative components and methods, as well as specific formulation additives, are provided herein that produce formulations for mucosal delivery of peptides and proteins prone to aggregation, wherein the peptide or protein is stabilized in a substantially pure form, not added A range of components and additives are contemplated for their use within these methods and formulations. Examples of these anti-aggregation agents are bound cyclodextrin dimers (CDs), which bind selectively to the hydrophobic side chains of the polypeptides. It has been found that these CD dimers bind to hydrophobic patches of proteins in a manner that significantly inhibit aggregation. This inhibition is selective with respect to both the CD dimer and the protein involved. Such selective inhibition of protein aggregation provides additional advantages in the methods and compositions of intranasal delivery of the invention. Additional agents used in this context include CD tromers and tetramers with variable geometries controlled by the linkages that specifically block the aggregation of peptides and proteins (Breslow et al., J. Am. Chem. Soc. 118: 11678-11681, 1996; Breslow et al., PNAS USA 94: 11156-11158, 1997). Agents and Methods for Load Modification and pH Control To improve the transport characteristics of biologically active agents (eg, macromolecular drugs, peptides or proteins) for improved delivery through hydrophobic mucous membrane barriers, the invention also provides techniques and reagents for load modification of selected biologically active agents or agents supply enhancers described herein. In this regard, the relative permeabilities of macromolecules are generally related to their partition coefficients. The degree of ionization of molecules, which depends on the pKa of the molecule and the pH of the mucous membrane surface, also affects the permeability of the molecules. The permeation and partition of the biologically active agents and permeabilization agents for mucosal delivery can be facilitated by charge alteration or load deployment of the active agent or permeabilization agent, which is achieved, for example, by altering charged functional groups , by modifying the pH of the vehicle or delivery solution in which the active agent is delivered, or by the coordinated administration of a loading reagent or pH-altering agent with the active agent. Condoms Condoms such as chlorobutanol, methyl paraben, propyl paraben, sodium benzoate (0.5%), phenol, cresol, p-chloro-ii-cresol, phenylethyl alcohol, benzyl alcohol, phenylmercuric acetate, phenylmercuric borate, phenylmercuric nitrate, thimerosal sorbic acid, benzethonium chloride or benzilconium chloride may be added to the formulations of the invention to inhibit microbial growth. pH and Damping Systems The pH is generally regulated using a buffer such as a system comprising citric acid and a citrate salt (s), such as sodium citrate. Suitable additional buffer systems include acetic acid and an acetate salt system, succinic acid and a succinate salt system, malic acid and a malic salt system, and gluconic acid and a gluconate salt system. Alternatively, buffer systems may be employed which comprise salt / acid mixture systems, such as an acetic acid and a sodium citrate system, a citrate acid, sodium acetate system, and a citric acid, sodium citrate, system of sodium benzoate. For any buffer system, additional acids, such as hydrochloric acid, and additional bases, such as sodium hydroxide, can be added for the final pH adjustment. Additional Agents to Modulate the Structure and / or Physiology of the Epithelial Junction Closed epithelial junctions are generally impervious to molecules with radii of approximately 15 angstroms, unless treated with physiological binding control agents that stimulate substantial binding opening as provided in the present invention. Among the "secondary" tight junction regulation components that will serve as useful targets for secondary physiological modulation in methods and Compositions of the invention, the heterodimeric complex Z01-Z02 has been shown to be amenable to physiological regulation by exogenous agents that can easily and effectively alter the paracellular permeability in mucosal epithelium. In such an agent that has been extensively studied is the bacterial toxin of Vibrio cholerae known as "zonula occludens toxin" (ZOT). See also, WO 96/37196; US Patents Nos. 5,945,510; 5,948,629; 5,912,323; 5864.014; 5,827,534; 5,665,389; and 5,908,825. Thus, ZOT and other agents that modulate the Z01-Z02 complex will be formulated in combination or administered in coordination with one or more biologically active agents. Formulation and Administration The mucosal delivery formulations of the present invention comprise the biologically active agent that is typically administered in combination with one or more pharmaceutically acceptable carriers and, optionally, other therapeutic ingredients. The vehicle (s) must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the formulation and not emit an unacceptable harmful effect on the subject. Such vehicles are described hereinbefore or otherwise are well known to those skilled in the pharmacology art. Desirably, the formulation should not include substances such as enzymes or oxidizing agents with which it is known that the biologically active agent to be administered is incompatible. The formulations can be prepared by any of the methods well known in the pharmaceutical art. The compositions and methods of the invention can be administered to subjects by a variety of modes of mucosal administration, including oral, rectal, vaginal, intranasal, intrapulmonary, or transdermal delivery, or by topical delivery to the eyes, ears, skin or other mucosal surfaces . The compositions according to the present invention are often administered in an aqueous solution in the form of a nasal or pulmonary spray and can be administered in the form of a spray by a variety of methods known to those skilled in the art. Preferred systems for administering liquids in the form of a nasal spray are described in the U.S. Patent. No. 4,511,069. Such formulations can be conveniently prepared by dissolving the compositions in water according to the present invention to produce an aqueous solution, and sterilizing said solution. The formulations can be presented in multi-dose containers, for example in the sealed distribution system described in the U.S. Patent. No. 4,511,069. Other suitable nasal spray delivery systems have been described in Transdermal Systemic Medicine, Y. W. Chien Ed., Elsevier Publishers, New York, 1985; and in the Patent of E.U. No. 4,778,810. Additional forms of aerosol delivery may include, eg, compressed air, jet, ultrasonic, and piezoelectric nebulizers, which deliver the biologically active agent dissolved or suspended in a pharmaceutical solvent, eg, water, ethanol, or a mixture of same. The nasal and pulmonary spray solutions of the present invention typically comprise the drug or drug to be delivered, optionally formulated with a surface-active agent, such as non-ionic surfactant (eg, polysorbate-80), and one or more buffers, stabilizers, or toners. In some embodiments of the present invention, the nasal spray solution further comprises a propellant. The pH of the nasal spray solution is optionally between about pH 3.0 and 7.2, but when desired the pH is adjusted to optimize the delivery of a charged macromolecular species (eg, a protein or therapeutic peptide) in a substantially un-ionized state . The pharmaceutical solvents used can also be a slightly acidic aqueous buffer (pH 3-6). Shock absorbers suitable for use within these compositions are as described above or otherwise known in the art. Other components may be added to improve or maintain chemical stability, including condoms, surfactants, dispersants, or gases. Suitable preservatives include, but are not limited to, phenol, methyl paraben, paraben m-cresol, thiomersal, benzyl chloride, and the like. Suitable surfactants include, but are not limited to, oleic acid, sorbitan trioleate, polysorbates, lecithin, phosphotidyl cholines, and various long chain diglycerides and phospholipids. Suitable dispersants include, but are not limited to, ethylenediaminetetraacetic acid, and the like. Suitable gases include, but are not limited to, nitrogen, helium, chlorofluorocarbons (CFCs), hydrofluorocarbons (HFCs), carbon dioxide, air, and the like. Suitable stabilizing and toning agents include sugars and other polyols, amino acids, and organic and inorganic salts. The liquid transmucosal formulation can be administered as drops, e.g., installation, or as a drip (spray). The atomizer can be produced by pumps, nebulization, or by other methods described in the art. For pulmonary delivery, liquid droplets for deep lung deposition exhibit a minimum particle size appropriate for deposition within the lung passages which is often approximately less than 10 μm mean mass equivalent aerodynamic diameter (MMEAD), commonly approximately less than 5 μm MMEAD, commonly approximately less than 2 μm MMEAD. For nasal delivery, the liquid droplet particle size is commonly approximately less than 1000 μm MMEAD, commonly less than 100 μm MMEAD. Within alternative embodiments, the mucosal formulations are administered as dry powder formulations comprising the biologically active agent in dry form, usually lyophilized, of an appropriate particle size, or within a range of appropriate particle size, for intranasal delivery . For pulmonary delivery, the powder particle for deep deposition in the lung exhibits a minimum particle size suitable for deposition within the lung passages that is often approximately less than 10 μm in mean mass equivalent aerodynamic diameter (MMEAD ), commonly approximately less than 5 μm MMEAD, commonly approximately less than 2 μm MMEAD. For nasal delivery, the liquid droplet particle size is commonly approximately less than 1000 μm MMEAD, commonly less than 100 μm MMEAD. Breathable powders of intranasal form within this size range can be produced by a variety of conventional techniques, such as jet milling, spray drying, solvent precipitation, supercritical fluid condensation, and the like. These dry MMEAD dry powders can be administered to a patient by means of a conventional dry powder inhaler (DPI) that relies on the patient's breathing, lung or nasal inhalation, to disperse the powder in an aerosolized amount. Alternatively, the dry powder can be administered by means of air assisted devices that use an external powder source to disperse the powder in an aerosolized amount, e.g., a piston pump. The drug powder particles can be formulated in the dry state as agglomerated particles into large particles (> 100 μm MMEAD) comprising a suitable vehicle, such as lactose, wherein the agglomerates of drug particles and carrier particles are broken during the administration of the powder. Dry powder devices typically require a mass of powder in the range from about 1 mg to 20 mg to produce a single aerosolized dose ("puff"). If the required or desired biologically active agent dose is less than this amount, the powdered active agent will typically be combined with a dry pharmaceutical thickening powder to provide the total mass of powder required. Preferred dry thickening powders include sucrose, lactose, dextrose, mannitol, glycine, trehalose, human serum albumin (HSA), and starch. Other suitable dry thickening powders include cellobiose, dextrans, maltotriose, pectin, citrate of sodium, sodium ascorbate, and the like. To formulate compositions for mucosal delivery in the present invention, the biologically active agent can be combined with various pharmaceutically acceptable additives, as well as a base or carrier for the dispersion of the active agent (s). The desired additives include, but are not limited to, pH control agents, such as arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid, etc. In addition, local anesthetics (eg, benzyl alcohol), isotonizing agents (eg, sodium chloride, mannitol, sorbitol), adsorption inhibitors (eg, Tween 20), solubility enhancing agents (eg, cyclodextrins, and derivatives thereof) may be included. , stabilizers (eg, serum albumin), and reducing agents (eg, glutathione). When the composition for the mucosal delivery is a liquid, the tonicity of the formulation, as measured with reference to the 0.9% (w / v) tonicity of physiological saline taken as a unit, is typically adjusted to a value that will not induce damage of substantial irreversible tissue in the nasal mucosa at the site of administration. Generally, the tonicity of the solution is adjusted to a value of about 1/3 to 3, or to 2, or% to 1.7. The biologically active agent may be dispersed in a base or vehicle, which may comprise a compound hydrophilic having the ability to disperse the active agent and any desired additive. The base may be selected from a wide range of suitable vehicles, including but not limited to, polycarboxylic acid copolymers or salts thereof, carboxylic anhydrides (eg, maleic anhydride) with other monomers (eg, methyl (meth) acrylate, acrylic acid, etc.), hydrophilic vinyl polymers such as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone, cellulose derivatives, such as hydroxymethylcellulose, hydroxypropylcellulose, etc., and natural polymers such as cytosan, collagen, sodium alginate, gelatin, hyalurinic acid, and non-toxic metal salts thereof. Often, a biodegradable polymer is selected as a base or carrier, for example, polylactic acid, poly (lactic acid-glycolic acid) copolymer, polyhydroxybutyric acid, poly (hydroxybutyric acid-glycolic acid) copolymer and mixtures thereof. Alternatively or additionally, synthetic adipose acid esters such as polyglycerin adipose esters, sucrose fatty acid esters, etc. may be employed as vehicles. Hydrophilic polymers and other vehicles can be used alone or in combination, and improved structural integrity can be imparted to the vehicle by partial crystallization, ionic bonding, crosslinking and the like. The vehicle can be provided in a variety of forms, including, fluid or viscous solutions, gels, pastes, powders, microspheres and films for direct application to the nasal mucosa. The use of a vehicle selected in this context may result in the promotion of the absorption of the biologically active agent. The biologically active agent can be combined with the base or carrier according to a variety of methods, and the release of the active agent can be by diffusion, vehicle disintegration, or associated formulation of water channels. In some circumstances, the active agent is dispersed in microcapsules (microspheres) or nanocapsules (nanospheres) prepared from a suitable polymer, eg, isobutyl 2-cyanoacrylate (see, eg, Michael, et al., J. Pharmacy Pharmacol. : 1-5, 1991), and dispersed in a biocompatible dispersion medium applied to the nasal mucosa, which produces sustained delivery and biological activity for a prolonged time. To further improve the mucosal delivery of pharmaceutical agents within the invention, formulations comprising the active agent may also contain a low molecular weight hydrophilic compound as a base or excipient. Such hydrophilic low molecular weight compounds provide a means of passage through which a water soluble active agent, such as a physiologically active peptide or protein, can be diffused by means of the base on the body surface where the active agent is absorbed. The low molecular weight hydrophilic compound optionally absorbs moisture from the mucosa or atmosphere of administration and dissolves the water-soluble peptide. The molecular weight of the low molecular weight hydrophilic compound is generally not greater than 10000 and preferably not greater than 3000. Exemplary low molecular weight hydrophilic compounds include polyol compounds, such as oligo-, di-, and monosaccharides such as sucrose, mannitol , lactose, L-arabinose, D-erythrose, D-ribose, D-xylose, D-mannose, D-galactose, lactulose, cellobiose, gentibiose, glycerin and polyethylene glycol. Other examples of low molecular weight hydrophilic compounds very useful as carriers within the invention include N-methylpyrrolidone, and alcohols (e.g., oligovinyl alcohol, ethanol, ethylene glycol, propylene glycol, etc.). These hydrophilic low molecular weight compounds may be used alone or in combination with each other or with other active or inactive components of the intranasal formulation. The compositions of the invention may alternatively contain as many pharmaceutically acceptable carrier substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, eg, sodium acetate. , sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. For solid compositions, conventional non-toxic pharmaceutically acceptable carriers can be used which include, for example, mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like. In certain embodiments of the invention, the biologically active agent is administered in a temporary release formulation, for example in a composition that includes a slow release polymer. The active agent can be prepared with vehicles that protect against rapid release, for example a controlled release vehicle, such as a polymer, a microencapsulated delivery system or a bioadhesive gel. The prolonged supply of the active agent, in various compositions of the invention, can be produced by including in the composition agents that retard absorption, for example, aluminum hydrogels, monostearate and gelatin. The term "subject" as used herein refers to any mammalian patient to whom the compositions of the invention may be administered. Equipment The present invention also includes equipment, multipack packaging and units containing the pharmaceutical compositions described above, active ingredients, and / or means for administering the same for use in the prevention and treatment of diseases and other conditions in mammalian subjects. Briefly, these kits include a package or formulation containing one or more biologically active agents formulated in a pharmaceutical preparation for mucosal delivery. The biologically active agent (s) is optionally contained in a volume administration container or in a unit dose or multi-unit form. Optional administration means may be provided, for example a pulmonary or intranasal spray applicator. The packaging materials optionally include a label or instruction indicating that the pharmaceutical agent packaged therein can be used mucosally, e.g., intranasally, for the treatment or prevention of a specific disease or condition. Polypeptides for Improving Polynucleotide Delivery Within the additional embodiments of the invention, the polynucleotide supply enhancing polypeptide is rationally selected or designed to comprise an amphipathic amino acid sequence. For example, useful polypeptides can be selected to enhance the polynucleotide delivery comprising a a plurality of non-polar or hydrophobic amino acid residues forming a hydrophobic sequence domain or motif, linked to a plurality of charged amino acid residues that form a charged sequence domain or motif, producing an amphipathic peptide. In other embodiments, the polypeptide for enhancing the polynucleotide delivery is selected to comprise a protein transduction domain or motif, and a fusogenic peptide domain or motif. A protein transduction domain is a peptide sequence capable of being inserted into and preferably transiting through the membrane of cells. A fusogenic peptide is a peptide capable of destabilizing a lipid membrane, for example a plasma membrane or membrane surrounding an endosome, which can be improved at low pH. Exemplary fusogenic motifs or domains are found in a wide variety of viral fusion proteins and in other proteins, for example fibroblast growth factor-4 (FGF4). To rationally design the polypeptides to improve the polynucleotide delivery of the invention, a protein transduction domain is employed as a motif that will facilitate entry of the nucleic acid into the cell through the plasma membrane. In certain embodiments, the transported nucleic acid will be encapsulated in an endosome. The interior of the endosomes has a low pH which results in the fusogenic peptide motif destabilizing the endosome membrane. The destabilization and rupture of the endosome membrane allows the release of the siNA into the cytoplasm where the siNA can associate with a RISC complex and target its target mRNA. Examples of protein transduction domains for optional incorporation into polypeptides to enhance the polynucleotide delivery of the invention include: 1. TAT domain of protein transduction (PTD) (SEQ ID NO: 1) KRRQRRR; 2. Penetratin PTD (SEQ ID NO: 2) RQIKIWFQNRRMKWKK; 3. VP22 PTD (SEQ ID NO: 3) DAATATRGRSAASRPTERPRAPARSASRPRRPVD; 4. Kaposi FGF signal sequences (SEQ ID NO: 4) AAVALLPAVLLALLAP, and (SEQ ID NO: 5) AAVLLPVLLPVLLAAP; 5. ß3 human integrin signal sequence (SEQ ID NO: 6) VTVLALGALAGVGVG; 6. Gp41 fusion sequence (SEQ ID NO: 7) GALFLGWLGAAGSTMGA; 7. Crocodyl alligator light chain Ig (v) (SEQ ID NO: 8) MGLGLHLLVLAAALQGA; 8. hCT-derivative peptide (SEQ ID NO: 9) LGTYTQDFNKFHTFPQTAIGVGAP; 9. Transport (SEQ ID NO: 10) GWTLNSAGYLLKINLKALAALAKKIL; 10. Loligomer (SEQ ID NO: 11) TPPKKKRKVEDPKKKK; 11. Arginine peptide (SEQ ID NO: 12) RRRRRRR; and 12. Amphiphilic model peptide (SEQ ID NO: 13) KLALKLALKALKAALKL. Examples of fusogenic domains of viral fusion peptides for optional incorporation into polypeptides to enhance the polynucleotide delivery of the invention include: 1. influenza HA2 (SEQ ID NO: 14) GLFGAIAGFIENGWEG; 2. Sendai Fl (SEQ ID NO: 15) FFGAVIGTIALGVATA; 3. Respiratory Syncytial Virus Fl (SEQ ID NO: 16) FLGFLLGVGSAIASGV; 4. HIV gp41 (SEQ ID NO: 17) GVFVLGFLGFLATAGS; and 5. Ebola GP2 (SEQ ID NO: 18) GAAIGLAWIPYFGPAA. Still within further embodiments of the invention, polypeptides are provided to enhance the delivery of polynucleotide that incorporate a DNA binding domain or motif that facilitates the formation of the siNA-polypeptide complex and / or improves the delivery of siNAs in the methods and compositions of the invention. Exemplary DNA binding domains in this context include several "zinc finger" domains as described for DNA binding regulation proteins and other proteins identified to (See, e.g., Simpson, et al., J. Biol. Chem. 278: 28011-28018, 2003) Table 1: Exemplary motifs of zinc finger of different DNA binding proteins Motive ludiendo, Zinc C2H I. . . . . { . . . . i S65 675 6B5 695 705 715 Spl ACrCEYCKDS EGRSSG DFGK KQBXC mosO? Kwg Wtt? tib ± F? K Sp2? CTCSNCÍ-DG BKHS GEOG8KXHVC HITDCGKTFR KTSS-LRfiHVa igTGSRPFVC Sp3 ACFCPNCKEG GGH? TÍT -i £ ??? ap? c HIGGCGKVYCJ KTSKLE ? HL? L W1ÜSG3RSEVC Sp4 ACSCEHCBEG SGRGSN SFfíKKKCRTC T? IEGCGÜVYG K TT.RflHT.B WHEGS5PFIC DroßBtcl RCTCPHCENE KSGtPPMGP DEHGRKQHIC HIFGCEBI-K- K? SQ IH R SCGEaBFK. DxcsSp TCDCH? CQ EHIG5AGV "HIKKKNIBSC REFGCGKWG KTSEXf» 2LR wü? ÍüRyf c CeTZZCß. S RCTCntcsai ÍÍHG DRGS? H? HLC SV? SCGX? GYK KESK-iHAHLR .Y40B1A.4 HESLKKKIF FFU SHEll - GDGJSIUÍIXC? - NKTYG GEaSHKFSlC Positive pattern C-x (2,4) -C-x (12) -H-x (3) -H * The table demonstrates a conservative zinc finger motif for double-stranded DNA binding characterized by the motif pattern Cx (2, 4) -Cx (12) -Hx (3) -H, which can itself be used for selecting and designing polynucleotide supply enhancement polypeptides additional according to the invention. * The sequences shown in Table 1, for Spl, Sp2, Sp3, Sp4, DrosBtd, DrosSp, CeT22C8.5, and Y4pBlA.4, are assigned herein to SEQ ID Nos 19, 20, 21, 22, 23, 24, 25 and 26, respectively. Alternative DNA binding domains useful for the construction of the polypeptides to enhance the polynucleotide delivery of the invention include, for example, portions of the HIV Tat protein sequence (see, Examples below). Within the exemplary embodiments of the invention described hereinafter, polypeptides for enhancing the polynucleotide delivery can be rationally designed and constructed by combining any of the structural elements, domains or prior motifs in a single polypeptide effective to mediate the improved delivery of siNAs in target cells. For example, a protein transduction domain of the TAT polypeptide was fused to the 20 N-terminal amino acids of the influenza virus hemagglutinin protein, called HA2, to produce an exemplary polypeptide to enhance the polynucleotide delivery herein. Various other constructs of the polypeptide to enhance the polynucleotide delivery are provided in the present disclosure, evidencing that the concepts of the invention are broadly applicable to create and use a diverse assembly of effective polypeptides to improve the polynucleotide delivery to improve the supply of siNa. Even the exemplary additional polypeptides for enhancing the polynucleotide delivery in the invention can be selected from the following peptides: WWETWKPFQCRICMRNFSTRQARRNHRRRHR (SEQ ID NO: 27); GKINLKALAALAKKIL (SEQ ID NO: 28), RVIRVWFQNKRCKDKK (SEQ ID NO: 29), GRKKRRQRRRPPQGRKKRRQRRRPPQGRKKRRQRRRPPQ (SEQ ID NO: 30), GEQIAQLIAGYIDIILKKKKSK (SEQ ID NO: 31), poly lys-trp, 4: 1, MW 20,000-50,000; and poly orn-trp, 4: 1, MW 20,000-50,000. Additional polypeptides to enhance the polynucleotide delivery that are useful in the compositions and methods herein, comprise all or part of the melittin protein sequence. EXAMPLES The invention is illustrated by means of the following examples which do not limit the scope of the invention as described in the claims. EXAMPLE 1 Mucous Supply - Permeation Kinetics and Cytotoxicity Organotropic Model The following methods are generally useful for the evaluation of mucosal delivery parameters, kinetics and side effects for a biologically therapeutic agent. active and an effective amount of mucosal delivery enhancement of a permeabilization peptide that reversibly improves epithelial paracellular transport by modulating the binding structure and / or physiology in a mammalian subject. The EpiAirway ™ system was developed by MatTek Corp. (Ashland, MA) as a model of pseudostratified epithelium that lines the respiratory tract. The epithelial cells grow on porous membrane bottom cell culture inserts at an air-liquid interface, which results in the differentiation of the cells into a highly polarized morphology. The apical surface is ciliated with an ultra microvellose structure and the epithelium produces mucus (the presence of mucin has been confirmed by immunoblotting). The inserts have a diameter of 0.875 cm, providing a surface area of 0.6 cm2. The cells are plated on the inserts at the factory approximately three weeks before shipment. A "team" consists of 24 units. A. Upon arrival, the units are placed on sterile supports in 6-well microplates. Each well receives 5 ml of an appropriate culture medium. This medium based DMEM is free of serum but is supplemented with epidermal growth factor and other factors. The medium is always tested by endogenous levels of any cytosine or growth factor that is considered for intranasal delivery, but that is free of all cytosines and factors studied to date, except insulin. The volume of 5 ml is just enough to provide contact with the bottoms of the units in their bases, but the apical surface of the epithelium is allowed to remain in direct contact with the air. Sterile clamps are used at this stage and in all subsequent stages involving the transfer of units to wells containing liquid to ensure that no trapped air is found between the bottoms of the units and the medium. B. The units in their plates are maintained at 37 ° C in an incubator in a 5% C02 atmosphere in air for 24 hours. At the end of this time, the medium is replaced with fresh medium and the units are returned to the incubator for another 24 hours. Experimental Protocol - Permeation Kinetics A. A 24-unit "team" of EpiAirway ™ can be routinely used to evaluate five different formulations, each of which is applied to wells in quadruplicate. Each well is used for the determination of permeation kinetics (4 time points), transepithelial electrical resistance (TER). An additional set of wells is used as controls, which are treated in a simulated manner during the determination of the permeation kinetics, but, otherwise, they are handled identically to the units containing test sample for transepithelial resistance and viability determinations. B. In all experiments, the mucosal supply formulation to be studied is applied to the apical surface of each unit in a volume of 100 ul, which is sufficient to cover the entire apical surface. An appropriate volume of the test formulation is reserved at the concentration applied to the apical surface (generally no more than 100 ul is required) for the subsequent determination of the concentration of the active material by ELISA or other designated analysis. C. The units are placed in plates of 6 wells without bases for the experiment: each well contains 0.9 ml of medium, which is enough to make contact with the bottom of the porous membrane of the unit, but does not generate any hydrostatic pressure significant ascendant in the unit. D. In order to minimize potential sources of error and avoid any formation of concentration gradients, the units are transferred from a well that contains 0.9 mi to another at each time point in the study, these transfers are made at the following points of time based on a zero time in which a volume of 100 ul of the test material was applied to the apical surface: 15 minutes, 30 minutes, 60 minutes and 120 minutes.

E. Between the time points, the units in their plates are kept in the 37 ° C incubator. Plates containing 0.9 ml of medium per well are also kept in the incubator so that a minimal change in temperature occurs during the short periods when the plates are removed and the units are transferred from one well to another using sterile forceps. F. At the completion of each time point, the medium is removed from the well from which each unit was transferred, and is aliquoted into two tubes (one tube receives 700 ul and the other 200 ul) for the determination of the concentration of the test material permeate and, in the case where the test material is cytotoxic, to release the cytosolic enzyme, lactate dehydrogenase, from the epithelium. These samples are kept in the refrigerator if the analyzes are conducted within 24 hours, or the samples are sub-aliquots and kept frozen at -80 ° C until thawed for analysis once. Repeated freeze-thaw cycles should be avoided. G. In order to minimize errors, all tubes, plates and wells are pre-labeled before starting an experiment. H. At the end of the 120 minute time point, the units are transferred from the last of the wells containing 0.9 ml to the 24 well microplates, containing 0. 3 mi from the middle per well. This volume is again sufficient to make contact with the funds of the units, but not to exert ascending hydrostatic pressure in the units. The units are returned to the incubator prior to the transepithelial resistance measurement. Experimental Protocol - Transepithelial Electrical Resistance A. Respiratory air epithelial cells form closed junctions in vivo as well as in vitro, and therefore restrict the flow of solutes through tissue. These junctions confer a transepithelial resistance of several hundred ohms x cm2 in cut aerial tissues. In EpiAirway ™ MatTek units, it is reported by the manufacturer that transepithelial electrical resistance (TER) is routinely found around 1000 ohms x cm2. The data determined here indicates that the TER of EpiAirway ™ control units that have been exposed in a simulated manner during the sequence of the stages in the permeation study is somewhat lower (700-800 ohms x cm2), but, since the permeation of small molecules is proportional to the inverse of TER, this value is still high enough to provide a substantial barrier to permeation. The porous membrane bottom units without cells, on the other hand, provide only minimal transmembrane resistance (approximately 5-20 ohms x cm2). B. Accurate determinations of TER require that the ohmmeter's electrodes be placed on a significant surface area above and below the membrane, and that the distance of the electrodes from the membrane be controlled in a reproducible manner. The method for the determination of TER recommended by MatTek and used for all experiments in the present, employs an epithelial voltohmometer "EVOM" ™ and a fabric resistance measuring chamber "ENDOHM" ™ from World Precision Instruments, Inc., Sarasota , FL. C. The chamber is initially filled with Dulbecco's phosphate-buffered saline (PBS) for at least 20 minutes prior to TER determinations, in order to balance the electrodes. D. TER determinations are made with 1.5 ml of PBS in the chamber and 350 ul of PBS in the membrane bottom unit being measured. The upper electrode is adjusted to a position just above the membrane of a unit that does not contain cells (but contains 350 ul of PBS) and is then fixed to ensure reproducible placement. The strength of a cell-free unit is typically 5-20 ohms x cm2 ("backup strength"). E. Once the camera has been prepared and the backup resistance recorded, the units in a plate of 24 wells that have been recently used in the permeation determinations, are removed from the incubator and placed individually in the chamber for TER determinations. F. Each unit is first transferred to a petri dish containing PBS to ensure that the bottom of the membrane becomes wet. An aliquot of 350 ul of PBS is added to the unit and then carefully aspirated into a marked tube to rinse the apical surface. A second wash of 350 ul PBS is then applied to the unit and aspirated into the collection tube itself. G. The unit is gently stained free of excess PBS on its outer surface only before being placed in the chamber (containing a fresh aliquot of 1-5 of PBS). An aliquot of 350 ul of PBS is added to the unit before placing the upper electrode in the chamber and the TER is read on an EVOM meter. H. After reading the TER of the unit in the ENDOHM chamber, the unit is removed, the PBS is aspirated and reserved and the unit is returned with an air interface on the apical surface to a 24-well plate containing 0.3 ml of the medium per well. I. The units are read in the following sequence: control treaties in a simulated manner, followed by the samples treated with the formulation, followed by a second reading TER of each of the controls treated in a simulated manner. All TER values are reported as a function of the surface area of the tissue. TER was calculated as: TER = (Ri - Rb) x A Where Rt is the resistance of the insert with a membrane, Rb is the resistance of the insert in the model, and A is the area of the membrane (0.6 cm2). The effect of pharmaceutical formulations comprising intranasal delivery enhancement agents, for example, permeabilization peptides measured by TER through the EpiAirway ™ cell membrane (mucosal epithelial cell layer). The permeabilization peptides are applied to the EpiAirway ™ cell membrane at a concentration of 1.0 mM. A decrease in the value of TER relative to the control value (control = approximately 1000 ohms cm2, normalized to 100), indicates a decrease in the resistance of the cell membrane and an increase in the permeability of the epithelial mucosal cell. Experimental Protocol - LDH Analysis The amount of cell death was analyzed by measuring the loss of lactate dehydrogenase (LDH) from the cells, using a CytoTOx 96 cytotoxicity analysis kit (Promega Corp., Madison, Wl). Fifty microliters of sample were loaded into 96-well analysis plates. HE used as a model a fresh cell-free culture medium. 50 ul of the substrate solution were added to each well and the plates were incubated for 30 minutes at room temperature in the dark. After the incubation, 50 ul of the stop solution was added to each well and the plates were read in an optical density plate reader at 490 nm. Experimental Protocol - EIA Method The EIA equipment (p / n S-1178 (EIAH6101) was purchased from Peninsula Laboratories Inc., (BACHEM Division, San Carlos, CA, 800-922-1516) 17 x polypropylene conical tubes 120 mm (p / n 352997, Falcon, Franklin Lakes, NJ) were used for all sample preparations Eight standards were used for PTH quantitation The rest of the analysis procedure was the same as that of the inserts of the equipment. EXAMPLE 2 Improvement of Epithelial Permeation by PN159 The examples below demonstrate that the permeation enhancement peptides of the invention, exemplified by PN159, improve mucosal permeation to therapeutic peptide drugs, including PTH and YY peptide. of permeation of the peptides of the invention, evidenced for PN159, may be equivalent to, or greater than, the permeation enhancement epithelial achieved through the use of one or multiple small molecule permeation enhancers. The peptide YY3-36 (PYY3-3e) is a peptide of 34 amino acids that has been subjected to numerous clinical tests. The mucosal delivery of this biologically active peptide can be improved in formulations including small molecule permeation enhancers. Accordingly, the present studies established whether the permeation enhancement peptides of the invention, exemplified by PN159, could replace the role of small molecule permeation enhancers to facilitate mucosal delivery of the YY peptide. These studies include the evaluation of in vitro effects of PN159 to decrease transepithelial electrical resistance (TER) and increase the permeation of marker substances, as well as related in vivo studies that proved to be consistent with in vitro results. In the current example, the combination of PN159 with PTH is described. The PTH can be the full length peptide (1-84) or a fragment such as (1-34). The formulation can also be a combination of PTH, a permeabilization peptide and one or more permeation enhancers. The formulation may also contain buffers, toning agents, pH adjusting agents and peptide / protein stabilizers such as amino acids, sugars or polyols, polymers and salts. The present study was designed to evaluate the effect of PN159 itself or in combination with additional permeation enhancers on PTH permeation. The concentrations of PN159 evaluated are 25, 50, and 100 uM. The additional permeation enhancers are 45 mg / ml M-beta-CD, 1 mg / ml DDPC, and 1 mg / ml EDTA. Sorbitol was used as a toniciser (146-190 mM) to adjust the osmolarity of formulations to 220 mOsm / kg. The pH of the formulation was fixed at 4.5. PTH was selected as a peptide model in this example. 2 mg / ml PTH was combined with PN159 with or without additional permeation enhancers. The combination was tested using an in vitro epithelial tissue model to monitor PTH permeation, transepithelial electrical resistance (TER) and cytotoxicity of the formulation by LDH analysis. Transepithelial Electrical Resistance The results of the TER measurements from the present studies show that more than 80% of the TER reduction is caused by PN159. A greater reduction in TER was observed with an increase in the concentration of PN159. The medium applied to the apex side did not reduce TER while the group treated with triton X showed a significant reduction in TER as expected. Cytotoxicity Data for LDH from the present studies showed that no significant cytotoxicity was observed when the cells were treated with 25-100 uM of PN159. The medium applied to the atopic side showed no cytotoxicity while the group treated with Triton X showed significant cytotoxicity as expected. Permeation The PTH permeation data _ _3 for PN159 with and without additional enhancers are shown in Figures 1 and 2, respectively. A significant increase in PTH permeation was observed in the presence of PN159. No significant difference was observed in the permeation% between 25, 50 and 100 uM of PN159. The effect of PN159 on PTH permeation is comparable to 45/1/1 mg / ml of M-beta-CD / DDPC / EDTA. A further increase in PTH permeation was observed with the combination of 45/1/1 mg / ml of M-beta-CD / DDPC / EDTA and PN159. EXAMPLE 3 Improvement of In vivo Permeation by PN159 for a Peptide Hormone Therapeutic Agent Equals or Exceeds the Small Molecule Permeation Enhancers Twenty New Zealand male model rabbits of 3-6 months of age and weighing 2.1-3.0 kg were randomly assigned in one of 5 treatment groups with four animals per group. The test animals were dosed in ul / kg and intranasally by pipette. Table 5 below indicates the composition of five different dose groups. For dosage group 1 (see Table 2) a clinical formulation of PYY including small molecule permeation enhancers was used. The small molecule enhancers in these studies included methyl-b-cyclodextrin, phosphatidylcholine didecanoyl (DDPC), and / or EDTA. Dosage group 2 received PYY dissolved in phosphate buffered saline (PBS). For dosage groups 3-5, various concentrations of PN159 were added to dosage group 2, so that each of the dosage groups 3 to 5 consisted of PYY, PN159 and PBS.

Blood samples were collected in series (approximately 2 ml each) by direct venipuncture of a marginal ear vein in blood collection tubes containing EDTA as an anticoagulant. Blood samples were collected at 0, 2.5, 5, 10, 15, 30, 45, 60, and 120 minutes after dosing. After the blood collection, the tubes were gently oscillated several times for anti-coagulation, and then 50 ul of aprotinin solution was added. The blood was centrifuged at approximately 1600 x g for 15 minutes at approximately 4 ° C, and plasma samples were dised in duplicate aliquots and stored frozen at approximately -70 ° C. By averaging all four animals in a treatment group, the following plasma concentrations of the PYY were measured (Table 3): Table 3 The pharmacokinetic data calculated from previous data are shown below in Table 4: Table 4 The following relative improvement rates were determined compared to the Group 2 formulation (without enhancer). (Table 5): Table 5 The above data are plotted in Figure 3, and demonstrate that the permeabilization peptides of the invention, exemplified by PN159, are capable of improving intranasal permeation in vivo of a Therapeutic of human hormone peptide to an equal or greater degree compared to the small molecule permeation enhancers. The highest effect of the peptide is observed at a concentration of 50 uM. The 100 uM concentration resulted in a somewhat smaller permeation, although both resulted in a higher permeation than the small molecule permeation enhancers. EXAMPLE 4 Improvement of Permeation by PN159 for an Oligopeptide Therapeutic Agent The present example demonstrates the efficacy of an exemplary peptide of the invention, PN159, for improving epithelial permeation for a cyclic apeptide, melanocortin-4 receptor agonist (MC-4RA) , a model oligopeptide agonist for a mammalian cell receptor. In this example, a combination of one or more of the permeabilization peptides with MC-4RA is described. Formulations useful in this context may include a combination of an oligopeptide therapeutics, a permeabilization peptide and one or more different permeation enhancers. The formulation may also contain buffers, toning agents, pH adjusting agents, and peptide / protein stabilizers such as amino acids, sugars or polyols, polymers and salts. The effect of PN159 on the MC-4RA permeation is evaluated in this study. The MC-4RA was a methanesulfonate salt with a molecular weight of approximately 1,100 Da, which modulates the activity of the MC-4 receptor. The concentrations of PN159 evaluated are 5, 25, 50 and 100 uM. 45 mg / ml of M-beta-CD was used as a solubilizer for all formulations to achieve a peptide concentration of 10 mg / ml. The effect of PN159 was established either by itself or in combination with EDTA (1, 2.5, 5 or 10 mg / ml). The pH of the formulation was set at 4 and the osmolarity was found at 220 mOsm / kg. HPLC Method The concentrations of MC-4RA in the basolateral medium were analyzed by RP-HPLC using C18 RP chromatography with a flow rate of 1 ml / minute and a column temperature of 25 ° C. Solvent A: 0.1% TFA in water; Solvent B: 0.1% TFA in ACN Injection Volume: 50 ul Detection: 220 nm OPERATING TIME: 15 minutes The MC-4RA was combined with 5, 25, 50 and 100 uM of PN159, pH 4 and osmolarity -220 mOsm / kg. The combination was tested using an in vitro epithelial tissue model to monitor PTH permeation, transepithelial electrical resistance (TER), and cytotoxicity of the formulation by MTT and LDH analysis. The results of the MC-4RA permeation studies are shown in Figure 4. These studies showed that the PN159In addition to improving mucosal permeation for therapeutic peptide hormone, epithelial permeation for oligopeptide therapeutic agents was also significantly improved. EXAMPLE 5 Improvement of Permeation by PN159 for a Small Molecule Drug The present example demonstrates the efficacy of an exemplary peptide of the invention, PN159, for improving epithelial permeation for a small molecule drug, exemplified by acetylcholine esterase inhibitor galantamine (ACE) ). In this example, a combination of one or more of the permeabilization peptides with a small molecule drug is described. Formulations useful in this context may include a combination of a small molecule drug, a permeabilization peptide, and one or more different permeation enhancers. The formulation may also contain buffers, toning agents, pH adjusting agents, stabilizers and / or preservatives. The present invention combines galantamine with PN159 to improve the permeation of galantamine through the nasal mucosa. This increase in drug permeation it is unexpected because galantamine is a small molecule that can permeate the nasal epithelial membrane independently. Accordingly, the significant improvement of galantamine permeation through the epithelium mediated by the addition of excipients that enhance peptide permeation is surprising, based on the fact that such excipients would not be expected to significantly increase the galantamine permeation through of the epithelial tissue layer. The invention, therefore, will facilitate the nasal delivery of galantamine and other small molecule drugs by increasing their bioavailability. In the present studies, 40 mg / ml of galantamine was combined in the form of lactate salt with 25, 50 and 100 uM of PN159 in solution, pH 5.0 and osmolarity of -270 mOsm. The combination was tested using an in vitro epithelial tissue model to monitor permeation, transepithelial electrical resistance (TER), and cytotoxicity of the formulation by LDH and MTT analysis, as described above. Permeation measurements for galantamine were conducted by standard HPLC analysis, as follows: HPLC analysis The concentration of galantamine in the formulation in the basolateral medium (permeation samples) was determined using an LC isocratic method (Waters Alliance) with UV detection. Column: Waters Symmetry Shield, C18, 5 μm, 25 x 0.46 cm Mobile phase: 5% ACN in 50 mM ammonium format, pH 3.0 Flow rate: 1 ml / min Column temperature: 30 ° C Calibration curve: 0 -400 ug / ml galantamine HBr Detection: UV at 285 nm Based on previous studies, PN159 improves the transmucous supply of small molecules. Galantamine was selected as a model of a low molecular weight drug, and the results for this molecule are considered to be predictive of permeabilization peptide activity for other small molecule drugs. To evaluate the permeabilizing activity in this context, 40 mg / ml galantamine was combined in the form of lactate salt with 25, 50, and 100 uM of PN159 in solution, pH 5.0 and osmolarity of -270 mOsm. The combination was tested using an in vitro epithelial tissue model to monitor galantamine permeation, transepithelial electrical resistance (TER), and cytotoxicity of the formulation by LDH and MTT analysis. In the in vitro tissue model, the addition of PN159 resulted in a dramatic increase in the premeation of the drug through the cell barrier. Specifically, there was an increase of 2.5-3.5 times in the Papp of 40 mg / ml galantamine (Figure 5). PN159 reduced TER in the presence of galantamine just as described in Example II. Cell viability remained high (> 80%) in the presence of galantamine lactate and PN159 at all concentrations tested. In contrast, cytotoxicity was low in the presence of PN159 and galantamine lactate, measured by LDH. Both analyzes suggest that PN159 is non-toxic to the epithelial membrane. Summarizing the above results, PN159 has been shown to surprisingly increase epithelial galantamine permeation as a model of low molecular weight drug model. The addition of PN159 to galantamine in solution significantly improves the permeation of galantamine through the epithelial monolayers. Evidence shows that PN159 temporarily reduces TER through the epithelial membrane without damaging the cells in the membrane, as measured by high cell viability and low cytotoxicity. Consequently PN159 is an exemplary peptide for improving the bioavailability of galantamine and other small molecule drugs in vivo, by the same mechanism demonstrated in this using in vitro models. It is also expected that PN159 will improve the galantamine permeation also at higher concentrations. Chemical Stability The chemical stability of PN159 was determined under therapeutically relevant storage conditions. An HPLC stability indicator method was used. Solutions (50 mM) were stored at various pH conditions (4.0, 7.3 and 9.0) and temperature (5 ° C, 25 ° C, 35 ° C, 40 ° C and 50 ° C). Samples at pH 4 contained 10 mM citrate buffer. Samples at pH 7.3 and 9.0 contained 10 mM phosphate buffer. The representative storage stability data (including the Arrhenius scheme) are shown in Figure 6. As can be seen, PN159 was more chemically stable at low temperature and pH. For example, at 5 ° C and pH 4.0 or pH 7.3, there was essentially a 100% recovery of PN159 during a six month storage. When the storage temperature was increased to 25 ° C, there was a loss of 7% and 26% of natural PN159 for samples at a pH of 4 or pH 7, respectively, after six months. At pH 9 and / or at elevated temperature, e.g., 40 to 50 ° C, a rapid deterioration of PN159 occurred. The pH range of 4.0 to 7.3 and the temperature range of refrigerant to room are the most relevant for intranasal formulations. In consecuense, these data support that PN159 can maintain chemical integrity unstorage conditions relevant for intranasal formulations. There was a marked increase in the permeate drug rate vs. weather. These data were used to calculate the permeability constant (Papp) presented in Table 6. Table 6 Papp Measured Using the In Vitro Tissue Model the pH was 5.0, In the absence of PN159, the Papp for galantamine was approximately 2.1 x 10"6 cm / s In the presence of 25, 50 and 100 mM of PN159, the Papp was 5.1 x 10" 6, 6.2 x 10"6 and 7.2 x 10"6 cm / s, respectively. Therefore, PN159 produced an increase of 2.4 to 3.4 times in the Pap of this model of low molecular weight drug. Having established the utility of PN159 for transmucosal formulations of low molecular weight compounds, it was important to discern whether these observations would be extrapolated to larger molecules, e.g., peptides and therapeutic proteins. For this purpose, in vitro studies were carried out on salmon calcitonin as a therapeutic peptide model in the absence and in the presence of 25, 50, and 100 mM of PN159. In the absence of PN159, the Papp for calcitonin was approximately 1 x 10 ~ 7 cm / s, approximately one orof magnitude less than for galantamine, presumably due to the difference in molecular weight. The data reveal a dramatic increase in the permeation of calcitonin in the presence of PN159, up to 23 to 47 times in Papp compared to the case of calcitonin alone (Table 6). In orto explore the generality of these findings, two additional peptides were examined, namely human parathyroid hormone 1-34 (PTH? _34) and human peptide YY 3-36 (PYY3-36) in the in vitro model in the absence and in presence of PN159 (Papp data presented in Table 6). In the absence of PN159, the Papp of these two peptides was consistent with that of calcitonin. In the case of PTH? _3, the presence of PN159 produced an approximately 3-5 fold increase in Papp- When the PYY3_36 was formulated in the presence of PN159, the Papp was increased approximately 12 to 17 times. These data confirm the generality of our discovery, that PN159 has utility to improve the transmucosal drug supply. EXAMPLE 6 D-amino acid Versions of PN159 The substituted PN159 D-amino acid peptides listed in Table 7 were synthesized and purified and tested for their ability to improve TER and permeability using the methods described in the previous Examples.

Table 7 D-amino acid substitutions PN393 NH2-leucine and 1.06 +/- 0.00 1.02 +/- 0.16 klalklalkalkaalklalysin All D-amide (SEQ ID NO: substituted 35) rich with PN407 NH2- substitutions 1.08 +/- 0.01 1.20 +/- 0.05 LKILKkLlkKLLkLL- D going (SEQ ID NO: 36) PN434 NH2- D-substituted 0.12 +/- 0.01 0.02 +/- 0.00 KlaLKlALkAlkAALkLA-amide (SEQ ID NO: 37) PN408 NH2- PN159 retro- 1.05 +/- 0.01 1.16 +/- 0.07 alklaaklaklalklalk- inverse amide (SEQ ID NO: -3Q \ The PN407 shows a minor but statistically significant improvement in permeability. Both the All D and retro-inverse forms of PN159 show a decrease in the recovery of TER suggesting a greater reduction effect of TER that could be useful for in vivo delivery.The random substitution D (PN434) can cause null activities in both the reduction of TER as in the improvement of permeability EXAMPLE 7 Changes in Length of PN159 The PN159 peptides that have length changes, listed in Table 8, were synthesized and purified, and were tested for their ability to improve TER and permeability, using the methods described in the previous Examples.

Table 8 Different Sizes PN422 NH2- 26 aa 0.47 +/- 0.07 0.07 +/- 0.01 KLALKLALKALKAAL elongated KLALKLALKAL- amide (SEQ ID NO: 44) * average values of multiple repetitions The results show that the lengths of PN159 are important for their reduction of TER and their better permeability activity. The PN159 elongated at 20 aa increased the reduction effect of TER, but reduced the permeability effect. The recovery of TER is slower. The PN159 shortened to 16 aa shows no effect on the reduction of TER, but reduces the permeability effect. The PN159 shortened to 14 aa drastically reduced permeability, suggesting that the length of PN159 is crucial in permeability. Contrary to the permeability effect, the effect of the length of PN159 on the reduction of TER is more gradual. EXAMPLE 8 Tryptophan and Arginine Substitutions in PN159 The PN159 peptides having amino acid substitutions listed in Table 9 were synthesized and purified, and tested for their ability to improve TER and permeability using the methods described in the previous Examples.

Table 9 Amino Acid Substitutions The results show that a major group of arginine guanidinium is more effective than lysine and histidine. Tryptophan is a preferred amino acid at the water-membrane interface 1. PN407 shows a minor but statistically significant improvement in permeability. The replacement of lysine arginine drastically reduces permeability, but has less impact on the reduction of TER, suggesting the importance of lysine in permeability. The unique replacement of alanine in 10 aa with asparagine, ablate permeability, suggesting the importance of helicity alpha for PN159 activities. EXAMPLE 9 Changes in Hydrophobicity in PN159 The PN159 peptides having amino acid substitutions listed in Table 10 were synthesized and purified, and tested for their ability to improve TER and permeability, using the methods described in the previous Examples. Table 10 Hydrophobic facets mean values of multiple repetitions PN159 has 280 degrees of hydrophobic facets. The results show that the reduction of the hydrophobic facets can cause the reduction of PN159 activities. The ampfifficulty of PN159 is also important for its activities. Methods and Protocols in vitro Each TJMP was analyzed by transepithelial electrical resistance (TER), recovery TER, cytotoxicity (LDH), and sample permeation (EIA). The cell culture conditions and the protocols for each analysis are explained below in detail. EXAMPLE 10 Methods and Protocols in vitro The peptides of modulation of tight junction or TJMPs are peptides capable of compromising the integrity of closed junctions with the effect of creating openings between epithelial cells and consequently, reducing the barrier function of an epithelium. The integrity status of the tight junction can be analyzed in vitro by measuring the level of electrical resistance and the degree of sample permeation through a human nasal epithelial tissue model system. A reduction in electrical resistance and an improvement in permeation suggest that closed junctions have been compromised and openings created between the epithelial cells. Indeed, peptides that induce a measured reduction in electrical resistance through a tissue membrane referred to as reduction (TER), and promote improved permeation of a small molecule through a tissue membrane are classified as TJMPs. Additionally, the level of cellular toxicity for TJMPs is also established to determine whether these peptides could function as tight binding modulation peptides in the delivery of the drug through the mucosal surface, for example, intranasal (IN) drug delivery. The analyzes used to visualize the exemplary peptides of the present invention (refer to Table 23 of Example 25) are described in the present example. Analyzes include transepithelial electrical resistance (TER), cytotoxicity (LDH), and sample permeation. The reagents used and the cell culture conditions are also described. Table 11 illustrates the sample reagents used in the subsequent Examples. Table 11 Sample Reagents TC = tissue culture Cell Cultures The EpiAirway ™ system was developed by MatTek Corp. (Ashland, MA) as a model of the pseudostratified epithelium lining the respiratory tract. The epithelial cells grow in porous membrane bottom cell culture inserts at an air-liquid interface, which results in the differentiation of the cells into a highly polarized morphology. The apical surface is ciliated with an ultra microvellose structure and the epithelium produces mucus (the presence of mucin has been confirmed by immunoblotting). The cells are plated on the inserts at the factory approximately three weeks before shipment. The EpiAirway ™ culture membranes were received the day before the start of the experiments. They embark on Dulbecco's modified Eagle's Medium (DMEM) free of phenol red and hydrocortisone free. The cells are ciliated and pseudostratified, grown to confluence in a Millipore Multiscreen Caco-2 96-well analysis system comprised of a polycarbonate filter system. Upon receiving them. The insert system will be stored unopened at 4 ° C and / or cultured in 250 ul of basal medium per well (Dulbecco's Modified Eagle's Medium (DMEM) free of phenol red and hydrocortisone free) at 37 ° C / 5% C02 for 24 hours before use.

This model system was used to evaluate the efficacy of TJMPs to modulate the effect of TER, cytotoxicity and improve the permeation of a monolayer of epithelial cells. The MatTek Corp. cell line, (Ashland, MA) will be the source of normal tracheal / bronchial epithelial cells derived from humans (EpiAirway ™ tissue model). The cells are provided as inserts grown to confluence in Millipore Milicell-CM filters comprised of transparent hydrophilic Teflon (PTFE). Upon receipt, the membranes are cultured in 1 ml of basal medium (Dulbecco's Modified Eagle's Medium (DMEM) free of phenol red and hydrocortisone-free) at 37 ° C / 5% C02 for 24-48 hours before use. The inserts are fed during each day of recovery. Madin-Darbey canine kidney cells (MDCK), human intestinal epithelial cells (Caco-2), and human bronchial epithelial cells (16HBE14o-) were seeded in Millipore 96-well Multi-Screen Caco-2 inserts. These cells grew as a monolayer and under conditions similar to EpiAirway epithelial cells. Peptide Synthesis Peptide synthesis was carried out in a Synthesized Rainin Symphony on a 50 umol scale using NovaBiochem TGR resin. The deprotections are carried out by two 20% piperidine treatments in DMF for 10 minutes. After deprotection the resin was washed once with 10 ml of DMF containing 5% HOBt (30 s) and 4 times with 10 ml of DMF (30 s). the couplings were carried out by supplying a 5-fold excess of amino acid Fmoc in DMF to the reaction vessel followed by the supply of an equal volume of activator solution containing a 6.25-fold excess of N-methylmorpholine and a 5-fold excess of HCTU. A coupling time of 40 minutes was used throughout the synthesis. After the first coupling reaction, the resin was washed twice with 10 ml of DMF (30 s) prior to the start of the second coupling step. For pegylated peptides, upon completion of the peptide synthesis, the N-terminus Fmoc group was removed and 2 equivalents of 0- (N-Fmoc-2-aminoethyl) -O '(2-carboxyethyl) -undecanoethylene glycol in DMF were added manually to the reaction containers. Although in manual mode, 2 equivalents of the activator solution were supplied to the reaction vessel and the coupling was allowed to proceed overnight. Generally, coupling efficiencies greater than 97% were achieved and all unreacted peptide was capped with acetic anhydride. The splitting was carried out in the individual reaction vessels by supplying 10 ml of TFA containing 2.5% TIS, 2.4% water, followed by stirring Gentle with nitrogen for 3 hours. The splitting solution was automatically collected in conical tubes, emptied and the volume reduced by evaporation under reduced pressure. The resulting solution was triturated with an excess of cold ether, filtered and extensively washed with cold ether. After drying, the crude peptide was absorbed in Millipore water and lyophilized to dryness. FITC Permeation Analysis (fluorescence-5-isothiocyanate) -dextran A FITC-labeled dextran with a molecular weight of 3000 (FD3) was used to establish the efficacy of an individual TJMP in the monolayer permeation of epithelial cells. The tissue insert plates were transferred to a 96-well receptor plate containing 200 ul of DPBS ++ as the basal medium. The apical surface of each tissue culture insert was incubated with a 20 ul sample of a single test formulation (refer to Table 24 of Example 25 for details of the test formulations) for one hour at 37 ° C in the darkness in a shaker (-100 rpm). After the 1 hour incubation period, the underlying basal medium samples were taken from each tissue culture insert and stored temporarily in the dark at room temperature until FD3 levels were quantified by fluorescence spectroscopy. For FD3 measurements, they were transferred 150 ul of the basal medium sample to a black 96-well clear bottom plate. Fluorescence emission at 528/20 after excitation at 485/20 was measured using a fluorescence plate reader FL x 800 from Biotek Instruments. The permeation was calculated as: ChxVh% Permeation = jclOO CaxVa Vb Cb Apparent permeability (Papp), cm / sec = --- - SAxCa at The terms of the formula for permeation defined: Cb: Basolateral concentration Ca: Apic concentration Vb: Basolateral volume Va: Apic volume SA: Filter surface area Dt: Elapsed time Each tissue insert will be placed in an individual well containing 1 ml of basal medium MatTek. On the apical surface of the inserts, 25 ul of the test formulation will be applied according to the design of the study, and the samples will be placed on a shaker (-100 rpm) for 1.5 hours at 37 ° C. a dextran solution marked with FITC is added to the inserts in an apical manner and the measurement of fluorescence from the basolateral medium after the incubation period. The concentration of FITC-dextran is expressed as the percentage of the starting material applied to the cells. A dextran labeled with FITC with a molecular weight of 4000 (MW 4000) was used to establish the limitations of the loading size in the permeation of the individual TJMP. Of course, several sizes of dextrans marked with FITC are available to perform size limitation studies. Transepithelial Electrical Resistance (TER) and Recovery TER The TER measurements will be completed using the Endohm-12 tissue resistance measurement chamber connected to the EVOM epithelial voltometer (World Precision Instruments, Sarasota, FL) with the electrode guides. The electrodes and an insert in the tissue culture model will be equilibrated for at least 20 minutes in the MatTek medium with the energy deactivated prior to calibration verification. The backup resistance will be measured with 1.5 ml of the medium in the Endohm tissue chamber and 300 ul of the medium in the model insert. The upper electrode will be adjusted so that it is near, but not in contact with, the upper surface of the insert membrane. The backup resistance of the insert in the model should be approximately 5-20 ohms. For each TER determination 300 ul of the MatTek medium will be added to the insert followed by the placement in the Endohm camera. All TER values are reported as a function of the surface area of the tissue. The TER was calculated as: TER = (Ri - Rb) x A Where Ri is the resistance of the insert with a membrane, Rb is the resistance of the insert in the model, and A is the area of the membrane (0.6 cm2). A decrease in the TER value in relation to the control value (control = approximately 1000 ohms-cm2, normalized to 100), indicates a decrease in the resistance of the cell membrane and an increase in the permeability of the epithelial mucosal cell. For the recovery of TER, the TERs were measured at 1, 3, 5 and 21 hours after treatment. the percentage of TER was calculated as:% TER = (TER TpOSt-treatment / TER To) / (TER Tpost-tratßmiento / TER T0 for medium control). In some modalities, TER measurements were taken using the REMS Autosampler (World Precision Instruments, Sarasota, FL) with the electrode guides. The electrodes and an insert in the tissue culture model will be equilibrated for at least 20 minutes in the Air-100TM MatTek medium with the energy deactivated prior to calibration verification. The backrest resistance of the insert system has been established by multiple measurements of an insert plate in the model and the same value for each test on the platform. The zero time TER (TER 0) was measured before the incubation of the inserts with the test formulation. The upper electrode will be adjusted so that it is close, but not in contact with, the upper surface of the insert membrane. The backup resistance of the insert in the model should be approximately 5-20 ohms. For each TER determination, 100 ul of the Air-100 TM MatTek medium will be added to the insert and 250 ul in the basal well followed by placement in the Endohm chamber. All TER values are reported as a function of the surface area of the tissue. The resistance was expressed as both Ohms * cm2 and in percentage original TER value. The TER values were calculated as: nominal resistance, ohm * cm2 = (TERt - model) * 0.12 "TERt - model TER Relativo,% = TERO -mod elo Terms for the calculation of TER were defined: TERO: measurement of TER at zero time TERt: measurement of TER taken at time t after incubation of the test formulation Model: measurement of backup resistance A decrease in value of TER relative to the control value indicates a decrease in the resistance of cellular membrane and an increase in the permeability of the mucosal epithelial cell. Cytotoxicity (LDH Analysis) The amount of cell death will be analyzed by measuring the loss of lactate dehydrogenase (LDH) from the cells using a CytoTox 96 cytotoxicity analysis kit.

(Promega Corp., Madison, Wl). Fifty microliters of the sample will be loaded into 96-well analysis plates. A fresh cell-free culture medium will be used as in the model. Fifty microliters of the substrate solution will be added to each well and the plates will be incubated for 30 minutes at room temperature in the dark. After incubation, 50 ul of the stop solution will be added to each well and the plates will be read on an optical density plate reader at 490 nm. The measurement of the LDH release in the basolateral medium indicates the relative cytotoxicity of the samples. One hundred percent lysis of the control inserts with 0.3% octylphenolpoly (ethylene glycol ether) x (Triton X-100) allows expressing LDH values as a percentage of total lysis. Alternatively, cytotoxicity can be measured using a WST-1 analysis. The WST-1 analysis measures cell viability based on mitochondrial metabolic activity. The apex side of the cell monolayer was incubated with the reagent WST-1 (Roche) for 4 hours at 37 ° C after of peptide treatment, washing and measurement of TER at 10 minutes after treatment. The apical cell supernatants were measured at OD 450 nm using a microplate reader. % values = sample 0D 450 / medium control OD 450- In some embodiments, the amount of cell death was analyzed by measuring the release of lactate dehydrogenase (LDH) from the cells in the apex medium using a CytoTox 96 cytotoxicity analysis kit. (Promega Corp., Madison, Wl). One percent octylphenolpoly (ethylene glycol ether) x (Triton X-100) diluted in phosphate buffered saline (PBS) causes 100% lysis in the cultured cells and served as a positive control for LDH analysis. After the one hour incubation period with a test formulation (refer to Table 24 of Example 25 for details of the test formulations), the total liquid volume of each insert was brought to a final volume of 200 ul with the culture medium The apex medium was then mixed by pipette four times with a multichannel pipette adjusted to a volume of 100 ul. After mixing, a 100 ul sample was transferred from the apex side of each insert to a new 96 well plate. The samples from the apex medium were sealed with a plate sealer and stored at room temperature on the same day of analysis or stored overnight at 4 ° C for analysis the next day. To measure LDH levels, 5 ul of the 100-ul average-ampule sample were diluted in 45 ul of DPBS in a new 96-well plate. A fresh cell-free culture medium will be used as in the model. Fifty microliters of the substrate solution was added to each well and incubated for 30 minutes at room temperature away from direct light. After the 30 minute incubation, 50 ul of the detention solution was added to each well. The optical density (OD) was measured at 490 nm with an uQuant absorbance plate reader from Biotek Instruments. The measurement of the LDH release in the apex medium indicates the relative cytotoxicity of the samples. The percentage cytotoxicity for each test formulation was calculated by subtracting the measured absorbance of the PBS control (baseline level of LDH release) from the measured absorbance of the individual test formulation and then dividing that value by the absorbance measured for the 1 % of the positive control of Triton X-100, multiplied by 100. The formula used to calculate the percentage cytotoxicity is as follows: Relative cytotoxicity,% = - lOO ODtriton Osmolarity Samples were measured by means of Model 20200 from Advanced Instruments Inc., (Norwood, MA).

EXAMPLE 11 Peptides Modulating Closed Epithelial Unions and Improve the Permeation of the In Vitro Epithelial Cell Layer Table 12 shows the amino acid sequence of 11 peptides that modulate the tight binding proteins and improve the permeation of the in vitro epithelial cell layer measured by the TER analysis and the kinetics of permeation For the purposes of these examples, PN27 was selected to represent both PN27 and PN28 due to their similar activities. Table 12 EXAMPLE 12 Tight Union Modulation Peptides Reduce TER The present example evaluated the efficacy of various peptides to modulate tight binding proteins in an in vitro epithelial cell monolayer analyzed by TER reduction. A summary of TER data obtained from experiments performed on EpiAirway epithelial cells for each TJMP is presented in Table 13. The boxes highlighted in the table represent the highest TER reduction observed for that TJMP within the tested concentration range. Table 13 PN159, PN202, PN27, and PN283 reduced TER in an excess of 90% while PN161, PN250, PN228, PN73 and PN58 reduced TER by 82% to 88%. PN28 is not shown, but is functionally equivalent to PN27. Finally, PN183 had a TER reduction of 55%. These data indicate that all TJMPs tested are capable of compromising closed epithelial cell junctions in vitro. Additionally, a recovery analysis of TER was performed to determine the rate at which the EpiAirway epithelial cell is recovered after treatment with the TJMPs. Surprisingly, the results indicate that PN250, PN202 and PN161 have the fastest recovery time of all the TJMPs tested. These data indicate that the effect of TJMPs on the epithelial cell layer is transient in nature.

EXAMPLE 13 In Vitro Permeation Kinetics of Tight Union Modulation Peptides This example addressed the efficacy of TJMPs in mediating EpiAirway epithelial cell permeation. Table 14 below shows a summary of the permeation kinetics for each TJMP shown in percent permeation. The boxes highlighted in the table represent the highest degree of permeation observed for that TJMP within the tested concentration range. Table 14 These data indicate that all TJMPs tested are able to improve the in vitro permeation of a monolayer of epithelial cells. In general, the degree of permeability correlates with the ability of the peptides to reduce TER. EXAMPLE 14 Tight Union Modulation Peptides Do Not Cause Significant Cytotoxicity The present example evaluated the cytotoxic effect on epithelial cells after exposure to TJMPs. LDG analysis was carried out after a 15 minute and 60 minute treatment with each peptide. In all the examples, after a 15-minute treatment, no release of LDH was observed. After a 60-minute treatment, the cytotoxicity levels varied among the tested peptides but were found to be within acceptable levels indicating that none of the peptides tested causes significant damage to the cell. EXAMPLE 15 The Reduction of TER by Tight Union Modulation Peptides is Consistent Among All Proven Epithelial Cell Types To determine whether the results of TER observed in the EpiAirway epithelial cell culture system were representative of other types of epithelial cell, treated MDCK, Caco-2 and 16HBE14o cells with the TJMPs and analyzed by TER. In all the examples, the TER results observed with these cell types were consistent with the TER results observed with EpiAirway epithelial cells indicating that these TJMPs have the capacity to reduce TER among all epithelial cell types. EXAMPLE 16 Tight Union Modulation Peptides Classified on the Basis of Performance Nine TJMPs were classified into 4 different rows of performance according to their level of permeability., TER values, recovery rate of TER, and cytotoxicity, as shown in Table 15. PN183 and PN28 were not included in Table 15. The following table summarizes each optimal concentration of TJMPs (ie, the highest degree of reduction of TER associated with the highest level of permeability and that did not show any significant cytotoxicity) and the corresponding percentage permeation after a 15-minute treatment of EpiAirway epithelial cells with the peptide and after a 60-minute treatment of the cells epithelial EpiAirway with the peptide. Additionally, the LDH values (cytotoxicity) are shown for a 15 minute and 60 minute treatment for each peptide. The recovery of TER is also shown. The recovery rate of TER directly correlates with the decline value (i.e., the larger decline value is correlated with a faster recovery of TER). Table 15 EXAMPLE 17 Tight-Union Modulation Peptides Enhance FITC-Dextran MW4000 Permeation Through a Monolayer of Epithelial Cells In this example a study was conducted to determine the permeation kinetics of FITC-dextran MW4000 in the presence of each TJMP. This experiment established whether the permeation depended on the incubation time of the peptide with the epithelial cell layer and whether the permeation depends on the size of the load. The cell permeation was analyzed after a 15-minute treatment of the cells and also after a 60-minute treatment of the cells with a TJMP and the FITC-dextran MW4000 (Figure 7). The PYY formulation was used as the positive control and phosphate buffered saline (PBS) was used as a negative control. The peptides were tested at a concentration that demonstrated the highest degree of reduction of TER associated with the highest level of permeability and did not show any significant cytotoxicity. The 60-minute treatment showed a significantly higher degree of permeation than the 15-minute treatment for the same TJMP. Surprisingly, PN161, PN127 and PN228 showed a permeation level equivalent to PN159 (approximately 7.5%). The TJMPs, PN250, PN283, PN202, PN58 achieved approximately 5% permeation after 60 minutes of incubation with the cells, which is just the permeation achieved by PN161, PN127, PN228 and PN159. These data indicate that all TJMPs tested are capable of improving FITC-dextran MW4000 permeation and this improvement depends on how long the peptide is in contact with the epithelial cell layer. The above experiments demonstrate that the TJMPs tested are capable of improving the in vitro permeation of a monolayer of epithelial cells.

EXAMPLE 18 In vitro Enhanced Permeation by a Peptide that Modulates Tight Junction Strongly Correlated with Enhanced Permeation Observed in vivo A linear regression analysis was performed to determine if the permeation kinetics of TJMP observed in the epithelial cell model EpiAirway in vitro correlates with the in vivo pharmacokinetic data observed for that same TJMP. To determine if the in vitro permeation data work as a good indicated for in vivo success, the area under the ultimate curve value (AUC-last) derived from in vivo pharmacokinetic studies performed with PYY and TJMPs was plotted against permeation studies of monolayer of in vitro epithelial cells made with PYY and TJMPs. The in vitro permeation was expressed as a percentage and AUC-last as Min * pg / ml. In vitro and in vivo studies for 10 different TJMPs were plotted and a linear regression was performed. An R2 value of 0.82 was derived (correlation 82%) indicating that there is a strong correlation for the AUC values derived in vivo and the percentage permeability observed in vitro. Surprisingly, when the inter-analysis variability is excluded, an R2 value of 0.996 was derived (essentially 100%) indicating that there is a direct correlation between in vitro permeability and in vivo success.

Therefore, in vitro permeation can be used to predict success in vivo. EXAMPLE 19 Enhancement of In vivo Permeation by means of a TJMP for a Peptide Hormone Therapeutic Agent Equals or Exceeds that of Small Molecule Permeation Enhancers Twenty New Zealand male model rabbits of 3-6 months of age and weighing 2.1-3.0 kg were randomly assigned in one of 5 treatment groups with four animals per group. The test animals were dosed at 15 ul / kg and intranasally by pipette. Table 19 below indicates the composition of five different dose groups. For dosage group 1 (see Table 16) a clinical formulation of PYY including small molecule permeation enhancers was used. The small molecule enhancers in these studies included methyl-b-cyclodextrin, phosphatidylcholine didecanoyl (DDPC), and / or EDTA. Dosage group 2 received PYY dissolved in phosphate buffered saline (PBS). For dosage groups 3-5, various concentrations of PN159 were added to dosage group 2, so that each of the dosage groups 3 to 5 consisted of PYY, PN159 and PBS.

Table 16 Blood samples were collected in series (approximately 2 ml each) by direct venipuncture of a marginal ear vein in blood collection tubes containing EDTA as an anticoagulant. Blood samples were collected at 0, 2.5, 5, 10, 15, 30, 45, 60, and 120 minutes after dosing. After the blood collection, the tubes were gently oscillated several times for anti-coagulation, and then 50 ul of aprotinin solution was added. The blood was centrifuged at approximately 1600 x g for 15 minutes at approximately 4 ° C, and plasma samples were dispensed in duplicate aliquots and stored frozen at approximately -70 ° C.

By averaging all four animals in a treatment group, the following plasma concentrations of the PYY were measured (Table 17): Table 17 The pharmacokinetic data calculated from the above data are shown below in Table 18: Table 18 The following relative improvement rates were determined in comparison with Group 2 (without improver). (Table 19): Table 19 The above data demonstrate that TJMP improves intranasal permeation in vivo of a human hormone peptide therapeutic to an equal or greater degree compared to small molecule permeation enhancers. The highest effect of the peptide is observed at a concentration of 50 uM. The 100 uM concentration resulted in a somewhat smaller permeation, although both resulted in a higher permeation than the small molecule permeation enhancers. EXAMPLE 20 Improvement of Permeation by TJMP for an Oligopeptide Therapeutic Agent The present example demonstrates the efficacy of an exemplary peptide of the invention, PN159, to improve epithelial permeation for a cyclic pentapeptide, melanocortin-4 receptor agonist (MC-4RA) , an agonist oligopeptide model for a mammalian cell receptor. In this example, a combination of one or more of the permeabilization peptides with MC-4RA is described. Formulations useful in this context may include a combination of an oligopeptide therapeutics, a permeabilization peptide and one or more different permeation enhancers. The formulation may also contain buffers, toning agents, pH adjusting agents, and peptide / protein stabilizers such as amino acids, sugars or polyols, polymers and salts. The effect of PN159 on the permeation of MC-4RA was evaluated in this study. MC-4RA was a methanesulfonate salt with a molecular weight of approximately 1,100 Da, which modulates the activity of the MC-4 receptor. The concentrations of PN159 evaluated are 5, 25, 50 and 100 uM. 45 mg / ml of M-beta-CD was used as a solubilizer for all formulations to achieve a peptide concentration of 10 mg / ml. The effect of PN159 was established either by itself or in combination with EDTA (1, 2.5, 5 or 10 mg / ml). The pH of the formulation was set at 4 and the osmolarity was found at 220 mOsm / kg. HPLC Method The concentrations of MC-4RA in the basolateral medium were analyzed by RP-HPLC using C18 RP chromatography with a flow rate of 1 ml / minute and a column temperature of 25 ° C. Solvent A: 0.1% TFA in water; Solvent B: 0.1% TFA in ACN Injection Volume: 50 ul Detection: 220 nm OPERATING TIME: 15 minutes The MC-4RA was combined with 5, 25, 50 and 100 uM of PN159, pH 4 and osmolarity -220 mOsm / kg. The combination was tested using an in vitro epithelial tissue model to monitor PTH permeation, transepithelial electrical resistance (TER), and cytotoxicity of the formulation by MTT and LDH analysis. The results of studies of the permeation of MC-4RA evidenced that TJMP, in addition to improving mucosal permeation for therapeutic peptide hormone, significantly improved epithelial permeation for an oligopeptide therapeutic agent. EXAMPLE 21 Improvement of Permeation by TJMP for a Small Molecule Drug The present example demonstrates the efficacy of an exemplary peptide of the invention, PN159, to improve epithelial permeation for a small molecule drug, exemplified by acetylcholine esterase inhibitor galantamine (ACE) ). In this example, we describe a combination of one or more of the permeabilization peptides with a small molecule drug. Formulations useful in this context may include a combination of a small molecule drug, a permeabilization peptide, and one or more different permeation enhancers. The formulation may also contain buffers, toning agents, pH adjusting agents, stabilizers and / or preservatives. The present invention combines galantamine with PN159 to improve the permeation of galantamine through the nasal mucosa. This increase in drug permeation is unexpected because galantamine is a small molecule that can permeate the nasal epithelial membrane independently. Accordingly, the significant improvement of galantamine permeation through the epithelium mediated by the addition of excipients that enhance peptide permeation is surprising, based on the fact that such excipients would not be expected to significantly increase the galantamine permeation through of the epithelial tissue layer. The invention, therefore, will facilitate the nasal delivery of galantamine and other small molecule drugs by increasing their bioavailability. In the present studies, 40 mg / ml of galantamine was combined in the form of lactate salt with 25, 50 and 100 uM of PN159 in solution, pH 5.0 and osmolarity of -20 mOsm. The The combination was tested using an in vitro epithelial tissue model to monitor permeation, transepithelial electrical resistance (TER), and cytotoxicity of the formulation by MTT and LDH analysis, as described above. the permeation measurements for galantamine were conducted by standard HPLC analysis, as follows: HPLC analysis The concentration of galantamine in the formulation in the basolateral medium (permeation samples) was determined using an LC isocratic method (Waters Alliance) with UV detection. Column: Waters Symmetry Shield, C18, 5 μm, 25 x 0.46 cm Mobile phase: 5% ACN in 50 mM ammonium format, pH 3.0 Flow rate: 1 ml / min Column temperature: 30 ° C Calibration curve: 0 -400 ug / ml galantamine HBr Detection: UV at 285 nm Based on previous studies, PN159 improves the transmucous supply of small molecules. Galantamine was selected as a model of a low molecular weight drug, and the results for this molecule are considered predictive of the activity of the peptide of permeabilization for other small molecule drugs. To evaluate the permeabilizing activity in this context, 40 mg / ml galantamine was combined in the form of lactate salt with 25, 50, and 100 uM of PN159 in solution, pH 5.0 and osmolarity of -270 mOsm. The combination was tested using an in vitro epithelial tissue model to monitor galantamine permeation, transepithelial electrical resistance (TER), and cytotoxicity of the formulation by MTT and LDH analysis. In the in vitro tissue model, the addition of PN159 resulted in a dramatic increase in the premeation of the drug through the cell barrier. Specifically, there was an increase of 2.5 - 3.5 times in the Papp of 40 mg / ml of galantamine. PN159 reduced TER in the presence of galantamine just as described in previous examples. Cell viability remained high (> 80%) in the presence of galantamine lactate and PN159 at all concentrations tested. In contrast, cytotoxicity was low in the presence of PN159 and galantamine lactate, measured by LDH. Both analyzes suggest that PN159 is non-toxic to the epithelial membrane. In the absence of PN159, the Papp for galantamine was approximately 2.1 x 10"6 cm / s In the presence of 25, 50 and 100 mM of PN159, the Papp was 5.1 x 10" 6, 6.2 x 10"6 and 7.2 x ~ 6 cm / s, respectively. Therefore, PN159 produced a 3-4 fold increase in the Pap of this model of low molecular weight drug. The TJMP surprisingly increased the epithelial permeation of Galantamine as a model of low molecular weight drug. The addition of PN159 to galantamine in solution significantly improved the galantamine permeation through the epithelial monolayers. Evidence shows that PN159 temporarily reduced TER through the epithelial membrane without damaging the cells in the membrane, as measured by high cell viability and low cytotoxicity. TJMP improved the bioavailability of galantamine and other small molecule drugs in vivo through the same mechanism demonstrated in the present using in vitro models. It is also expected that the TJMP will improve the galantamine permeation also at higher concentrations. EXAMPLE 22 Improvement of Permeation by TJMP for Proteins Having established the utility of PN159 for transmucosal formulations of low molecular weight compounds, it was important to discern whether these observations would be extrapolated to larger molecules, e.g .. peptides and therapeutic proteins. For this purpose, in vitro studies were carried out on salmon calcitonin as a therapeutic peptide model in the absence and in the presence of 25, 50, and 100 mM of PN159. In the absence of PN159, the Papp for calcitonin was approximately 1 x 10"7 cm / s, approximately one order of magnitude less than for galantamine, presumably due to the difference in molecular weight.The data reveal a dramatic increase in permeation of calcitonin in the presence of PN159, up to 23 to 47 times in Papp compared to the case of calcitonin alone (Table 20) Table 20 Papp Measured Using the In Vitro Tissue Model at pH was 5.0. In order to explore the generality of these findings, two additional peptides were examined, namely human parathyroid hormone 1-34 (PTH? -3) and human peptide YY 3-36 (PYY3-36) in the in vitro model in the absence and in the presence of PN159 (Papp data presented in Table 20). In the absence of PN159, the Papp of these two peptides was consistent with that of calcitonin. In the case of PTH? _34, the presence of PN159 produced an approximately 3-5-fold increase in Papp- When the PYY3.36 was formulated in the presence of PN159, the Papp was increased approximately 12 to 17 times. These data confirm the generality of our discovery, that TJMP improved transmucosal drug supply for small molecules and proteins. EXAMPLE 23 Chemical Stability of TJMP The chemical stability of PN159 was determined under therapeutically relevant storage conditions. An HPLC stability indicator method was used. Solutions (50 mM) were stored at various pH (4.0, 7.3 and 9.0) and temperature conditions (5 ° C, 25 ° C, 35 ° C, 40 ° C and 50 ° C). Samples at pH 4 contained 10 mM citrate buffer. Samples at pH 7.3 and 9.0 contained 10 mM phosphate buffer. The storage stability results (including the Arrhenius scheme) show that the PN159 was more thermally stable at low temperature and pH. For example, at 5 ° C and pH 4.0 or pH 7.3, there was essentially a 100% recovery of PN159 during a six month storage. When the storage temperature was increased to 25 ° C, there was a loss of 7% and 26% of natural PN159 for samples at a pH of 4 or pH 7, respectively, after six months. At pH 9 and / or at elevated temperature, e.g., 40 to 50 ° C, a rapid deterioration of PN159 occurred. The pH range of 4.0 to 7.3 and the temperature range of refrigerant to room are the most relevant for intranasal formulations. Accordingly, these data support that the TJMP can maintain chemical integrity under storage conditions relevant to intranasal formulations. EXAMPLE 24 In Vivo Evaluation of Tight-Union Modulation Peptides in Rabbits by Intranasal Administration A pharmacokinetic (PK) study in rabbits was carried out to evaluate the plasma pharmacokinetic properties of peptide YY (PYY) with various tight-binding modulation peptides. (TJMPs) administered by supply intranasal (IN). Animal Model In this study, New Zealand model rabbits (Hra: (NZW) SPF) were used as test subjects to evaluate the plasma pharmacokinetics of MC-4RA by intranasal administration and intravenous infusion. The treatment of the animals was in accordance with the regulations set forth in the USDA Animal Welfare Act (9 CFR Parts 1, 2 and 3) and the conditions specified in the Guide for the Care and Use of Laboratory Animáis (ILAR publication, 1996, National Academy Press). Rabbits were selected as animal subjects for this study because the pharmacokinetic profile derived from a drug administered to rabbits closely resembles the PK profile for the same drug in humans. Dosage Management The experimental design and dose regime for the 9 TJMPs tested is summarized in Table 21. All experimental groups were given 205 ug / kg of PYY (3-36) in combination with an individual TJMP or buffered saline with phosphate (PBS: negative control) by intranasal administration (IN). Each formulation was administered once in the left nostrils using a pipette and a disposable plastic swab. The head of the animal was tilted back and the dose was administered at the time of inhalation of the animal in order to allow the capillary action to attract the solution in the nostrils. After the IN administration, the head of the animal was kept in the tilted back position for approximately 15 seconds to avoid loss of the administered dose. During the procedure, extreme care was taken to avoid any tissue damage potentially resulting from contact with the intranasal mucosa. Table 21 PN556 has the same primary sequence as PN283, but has no maleimide modification at the N-terminus of the peptide. Collection of Blood and Plasma Samples After administration of the IN dose, serial blood samples were taken from each animal by direct venipuncture of a marginal ear vein. Blood samples were collected prior to predose, 5, 10, 15, 20, 30, 45, 60, 90, 120 and 180 minutes after the dose. Samples were collected in tubes containing EDTA dipotassium as the anticoagulant. The tubes were cooled until centrifuged. All samples were centrifuged within 1 hour of collection. The plasma was harvested and transferred into pre-labeled plastic vials, frozen in a dry ice / acetone bath, and then stored at about -70 ° C until a pharmacokinetic analysis was performed. Clinical observations were made at each time of the blood sampling and the examination of both nostrils was carried out for all animals in the IN administration test groups, just before 5 minutes and 1 hour after dosing. intranasal Analytical Method Samples of each animal in all study groups were analyzed by levels of PYY (3-36) using ELISA.

The test items before and after dosing were operated on HPLC for quality control. The aliquots of plasma (0.1 ml) were precipitated in protein with acetonitrile after adding an internal analytical standard. The supernatant was dried with nitrogen, reconstituted in HPLC buffer and then injected onto an HPLC system. The effluent is detected by a triple quadruple mass spectrometer in series of positive ion electrospray ionization. The PK data was analyzed by WinNonlin (Pharsight Corp., Mountain View). Results The average plasma PK parameters for each test group are summarized in Table 22. No adverse clinical signs were observed after the administration of any of the formulations. The post-intranasal examination of both nostrils of the animals administered with formulations via IN, revealed neither redness nor swelling. The study of PK evaluated the Cmax (maximum concentration observed), the Tmax (time of maximum concentration) and AUC (area under the curve) last and infinite (inf) Eight TJMPs were classified and categorized into 4 different rows of performance according to their level of in vivo permeability containing the Row I TJMPs with the highest level of permeability in vivo and containing each row TJMPs with progressively decreased levels of permeability in vivo. Table 22 These data show that the observed in vivo permeability for both PN161 and PN27 is comparable with PN159; and the remaining TJMPs at the tested concentrations reached a level of in vivo permeability below that of PN159. EXAMPLE 25 Tight Union Modulation Peptides that Improve the Permeation of the Epithelial Cell Layer in vitro The present example describes the exemplary peptides PN679 and PN745 of the present invention (shown in Table 23) and the test formulation for each peptide (shown in Table 24) displayed to determine the effective concentration range of each peptide for improvement. of the monolayer permeation of epithelial cells. Table 23 Tight Union Modulation Peptides Table 24 below describes the individual test formulations containing an exemplary peptide (column "Active Agent" in Table 24) of the present invention and the test formulations that served as test formulation controls whether positive or negative that were examined by TER, LDH (cytotoxicity) and sample permeation improvement analysis. each peptide was tested at concentrations of 25 uM, 100 uM, 250 uM and 100 uM. PN159 (test formulation # 11) in the present served as a TJMP positive control and had previously demonstrated the ability to effectively reduce TER and improve sample permeation at 25 uM. One percent of Triton X-100 ™ (test formulation # 14) worked as a positive control for both the cytotoxicity (LDH) analysis and the TER reduction analysis. "Special sauce" (SS) served in the present as an enhancer of small molecule permeation. DPBS ++ served as a negative control. Each test formulation had a final volume of 300 ul and an objective pH of 7, except for test formulation # 12, which had a target pH of 5. One percent Triton X-100 ™ (test formulation # 14) It worked as a positive control for the analysis of cytotoxicity (LDH). From the total volume of 300 ul for each test formulation, only a 20 ul sample was applied to human-derived tracheal / bronchial epithelial cells (EpiAirway ™ tissue model system) in order to achieve the effect of each formulation of test in TER, LDH and sample permeation.

Table 24 Test Formulations S 'special sauce' EXAMPLE 26 PN679 and PN745 Modulate Tight Union Proteins in Vitro The present example demonstrates that exemplary peptides PN679 and PN745 effectively reduced TER and significantly improved sample permeation in a dose-dependent manner without causing significant cell toxicity indicating that these peptides are Effective TJMPs. Table 25 summarizes the TER, LDH and sample permeation data (FD3) for the test formulations described in Table 24 of Example 25. Test formulation # 1 for PN679 and test formulation # 6 for PN745 were analyzed twice. The results of the additional analysis for TER, LDH and sample permeations are shown in parentheses. Table 25 Summary of TER, LDH and Sample Permeation Improvement Data SS = "special sauce" The test formulations including 100 uM, 250 uM, 500 uM and 1000 uM of any of the exemplary peptides PN679 (test formulations # 1, # 2, # 3 and # 4) or PN745 (formulations of test # 6, # 7, # 8 and # 9) of the present invention, reduced TER to a degree equivalent to the "special sauce" and significantly below the control of the established TJMP PN159. As expected, the negative control DPBS ++ did not reduce TER significantly. The ability of both of these peptides to reduce TER correlated strongly with their ability to improve the permeation of the FD3 molecule. The dose of 100 uM for both PN679 (test formulation # 4) as for PN745 (test formulation # 9), exhibited a percentage permeation similar to TJMP PN159 but with less cytotoxicity (lower% LDH release). Higher concentrations of any of the peptides resulted in an increase in FD3 permeation levels above those of PN159, but also an increase in the release of LDH levels indicating an increase in cytotoxicity. As expected, the control of DPBS ++ did not induce a calculable LDH release. Based on the observed reduction in TER, the sample permeation and cytotoxicity (LDH release), a dose of 100 uM for any of the exemplary peptides of PN679 and PN745, seemed optimal for further analysis for these two TJMPs. The above data show the unexpected discovery that exemplary peptides PN679 and PN745 reduce TER and improve small molecule permeation without significant toxicity of a human epithelial cell monolayer in vitro. These data indicate that these tight-binding modulation peptides (TJMPs) are excellent candidates for use in the delivery of drugs through a mucosal surface, for example, intranasal drug (IN) delivery. EXAMPLE 27 Improved Permeation in vitro by a Peptide that modulates the tight junction correlates with Enhanced Permeation Observed in vivo A linear regression analysis was performed to determine if the permeation kinetics of TJMP observed in the EpiAirway epithelial cell model system in vitro correlates with pharmacokinetic data in vivo observed for the same TJMP. To determine whether in vitro permeation data work as a good indicator for in vivo success, the value of the area under the last curve (AUC last) derived from in vivo pharmacokinetic studies performed with PYY and TJMPs was plotted against permeation studies of monolayer of in vitro epithelial cells made with PYY and TJMPs. The in vitro permeation was expressed as a percentage and AUC last as min * pg / ml. In vitro and in vivo studies for ten different TJMPs were plotted and linear regression was carried out. An R2 value of 0.82 was derived (82% correlation) indicating that there is a strong correlation for the AUC values derived in vivo and the percentage permeability observed in vitro. Surprisingly, when the inter-analysis variability is excluded, an R2 value of 0.996 (essentially 100%) was derived indicating that there is a direct correlation between in vitro permeability and in vivo success. Therefore, in vitro permeation can be used to predict success in vivo.

EXAMPLE 28 Improvement of Permeation in vivo by a TJMP for a Peptide Hormone Therapeutic Agent Iguala or Exceeds the Small Molecule Permeation Enhancers Twenty New Zealand male model rabbits 3-6 months old and weighing 2.1-3.0 kg were randomly assigned in one of 5 treatment groups with four animals per group. The test animals were dosed at 15 ul / kg and intranasally by pipette. Table 26 below indicates the composition of five different dose groups. For dosage group 1 (see Table 26) a clinical formulation of PYY including small molecule permeation enhancers was used. The small molecule enhancers in these studies included methyl-b-cyclodextrin, phosphatidylcholine didecanoyl (DDPC), and / or EDTA. Dosage group 2 received PYY dissolved in phosphate buffered saline (PBS). For dosage groups 3-5, various concentrations of PN159 were added to dosage group 2, so that each of the dosage groups 3 to 5 consisted of PYY, PN159 and PBS.

Table 26 Dosing groups Blood samples were collected in series (approximately 2 ml each) by direct venipuncture of a marginal ear vein in blood collection tubes containing EDTA as an anticoagulant. Blood samples were collected at 0, 2.5, 5, 10, 15, 30, 45, 60, and 120 minutes after dosing. After the blood collection, the tubes were gently oscillated several times for anti-coagulation, and then 50 ul of aprotinin solution was added. The blood was centrifuged at approximately 1600 x g for 15 minutes at approximately 4 ° C, and plasma samples were dispensed in aliquots in duplicate and stored frozen approximately -70 ° C. By averaging all four animals in a treatment group, the following plasma concentrations of PYY were measured (Table 27): Table 27 Summary of Plasma Concentrations of PYY for Test Groups The pharmacokinetic data calculated from previous data are shown below in Table 28: Table 28 Summary of Pharmacokinetic Data The following relative improvement rates were determined in comparison with Group 2 (without improver). (Table 29): Table 29 Relative Improvement Relations The above data demonstrate that TJMP improves intranasal permeation in vivo of a human hormone peptide therapeutic to an equal or greater degree compared to small molecule permeation enhancers. The highest effect of the peptide is observed at a concentration of 50 uM. The concentration of 100 uM resulted in a somewhat lower permeation, although both resulted in a higher permeation than the small molecule permeation enhancers. EXAMPLE 29 Enhancement of Permeation by TJMP for an Oligopeptide Therapeutic Agent The present example demonstrates the efficacy of an exemplary peptide of the invention, PN159, to improve the epithelial permeation for a cyclic pentapeptide, melanocortin-4 receptor agonist (MC-4RA), a model oligopeptide agonist for a mammalian cell receptor. In this example, a combination of one or more of the permeabilization peptides with MC-4RA is described. Formulations useful in this context may include a combination of an oligopeptide therapeutics, a permeabilization peptide and one or more different permeation enhancers. The formulation may also contain buffers, toning agents, pH adjusting agents, and peptide / protein stabilizers such as amino acids, sugars or polyols, polymers and salts. The effect of PN159 on the permeation of MC-4RA was evaluated in this study. MC-4RA was a methanesulfonate salt with a molecular weight of approximately 1,100 Da, which modulates the activity of the MC-4 receptor. The concentrations of PN159 evaluated are 5, 25, 50 and 100 uM. 45 mg / ml of M-beta-CD was used as a solubilizer for all formulations to achieve a peptide concentration of 10 mg / ml. The effect of PN159 was established either by itself or in combination with EDTA (1, 2.5, 5 or 10 mg / ml). The pH of the formulation was set at 4 and the osmolarity was found at 220 mOsm / kg. HPLC method Concentrations of MC-4RA in the medium Basolateral samples were analyzed by RP-HPLC using C18 RP chromatography with a flow rate of 1 ml / minute and a column temperature of 25 ° C. Solvent A: 0.1% TFA in water; Solvent B: 0.1% TFA in ACN Injection Volume: 50 ul Detection: 220 nm OPERATING TIME: 15 minutes The MC-4RA was combined with 5, 25, 50 and 100 uM of PN159, pH 4 and osmolarity -220 mOsm / kg. The combination was tested using an in vitro epithelial tissue model to monitor PTH permeation, transepithelial electrical resistance (TER), and cytotoxicity of the formulation by MTT and LDH analysis. The results of studies of the permeation of MC-4RA evidenced that TJMP, in addition to improving mucosal permeation for therapeutic peptide hormone, significantly improved epithelial permeation for an oligopeptide therapeutic agent. EXAMPLE 30 Improvement of Permeation by TJMP for a Small Molecule Drug The present example demonstrates the efficacy of an exemplary peptide of the invention, PN159, for improving epithelial permeation for a small molecule drug, exemplified by galantamine acetylcholine esterase inhibitor (ACE). In this example, a combination of one or more of the permeabilization peptides with a small molecule drug is described. Formulations useful in this context may include a combination of a small molecule drug, a permeabilization peptide, and one or more different permeation enhancers. The formulation may also contain buffers, toning agents, pH adjusting agents, stabilizers and / or preservatives. The present invention combines galantamine with PN159 to improve the permeation of galantamine through the nasal mucosa. This increase in drug permeation is unexpected because galantamine is a small molecule that can permeate the nasal epithelial membrane independently. Accordingly, the significant improvement of galantamine permeation through the epithelium mediated by the addition of excipients that enhance peptide permeation is surprising, based on the fact that such excipients would not be expected to significantly increase the galantamine permeation through of the epithelial tissue layer. The invention, therefore, will facilitate the nasal delivery of galantamine and other small molecule drugs by increasing their bioavailability. In the present studies, 40 mg / ml was combined of galantamine in the form of a lactate salt with 25, 50 and 100 uM of PN159 in solution, pH 5.0 and osmolarity of -270 mOsm. The combination was tested using an in vitro epithelial tissue model to monitor the permeation, the transepithelial electrical resistance (TER), and the cytotoxicity of the formulation by MTT and LDH analysis, as described above. The permeation measurements for galantamine were conducted by standard HPLC analysis, as follows: HPLC Analysis The concentration of galantamine in the formulation in the basolateral medium (permeation samples) was determined using an LC isocratic method (Waters Alliance) with UV detection. Column: Waters Symmetry Shield, C18, 5 μm, 25 x 0.46 cm Mobile phase: 5% ACN in 50 mM ammonium format, pH 3.0 Flow rate: 1 ml / min Column temperature: 30 ° C Calibration curve: 0 -400 ug / ml galantamine HBr Detection: UV at 285 nm Based on previous studies, PN159 improves the transmucous supply of small molecules. Galantamine was selected as a model of a low-dose drug molecular weight, and the results for this molecule are considered predictive of permeabilization peptide activity for other small molecule drugs. To evaluate the permeabilizing activity in this context, 40 mg / ml galantamine was combined in the form of lactate salt with 25, 50, and 100 uM of PN159 in solution, pH 5.0 and osmolarity of -270 mOsm. The combination was tested using an in vitro epithelial tissue model to monitor galantamine permeation, transepithelial electrical resistance (TER), and cytotoxicity of the formulation by LDH and MTT analysis. In the in vitro tissue model, the addition of PN159 resulted in a dramatic increase in the premeation of the drug through the cell barrier. Specifically, there was an increase of 2.5 - 3.5 times in the Papp of 40 mg / ml of galantamine. PN159 reduced TER in the presence of galantamine just as described in previous examples. Cell viability remained high (> 80%) in the presence of galantamine lactate and PN159 at all concentrations tested. In contrast, cytotoxicity was low in the presence of PN159 and galantamine lactate, measured by LDH. Both analyzes suggest that PN159 is non-toxic to the epithelial membrane. In the absence of PN159, the Papp for galantamine was of approximately 2.1 x 10"6 cm / s In the presence of 25, 50 and 100 mM of PN159, the Papp was 5.1 x 10 ~ 6, 6.2 x 10" 6 and 7.2 x 10 ~ 6 cm / s, respectively. Therefore, PN159 produced an increase of 2.4-3.4 times in the Papp of this model of low molecular weight drug. The TJMP surprisingly increased the epithelial permeation of Galantamine as a model of low molecular weight drug. The addition of PN159 to galantamine in solution significantly improved the galantamine permeation through the epithelial monolayers. Evidence shows that PN159 temporarily reduced TER through the epithelial membrane without damaging the cells in the membrane, as measured by high cell viability and low cytotoxicity. TJMP improved the bioavailability of galantamine and other small molecule drugs in vivo through the same mechanism demonstrated in the present using in vitro models. It is also expected that the TJMP will improve the galantamine permeation also at higher concentrations. EXAMPLE 31 Improvement of Permeation by TJMP for Proteins Having established the utility of PN159 for transmucosal formulations of low molecular weight compounds, it was important to discern if these observations would be extrapolated to larger molecules, e.g .. peptides and therapeutic proteins. For this purpose, they took performed in vitro studies on salmon calcitonin as a therapeutic peptide model in the absence and in the presence of 25, 50, and 100 mM of PN159. In the absence of PN159, the Papp for calcitonin was approximately 1 x 10 ~ 7 cm / s, approximately one order of magnitude less than for galantamine, presumably due to the difference in molecular weight. The data reveal a dramatic increase in the permeation of calcitonin in the presence of PN159, up to 23 to 47 times in Papp compared to the case of calcitonin alone (Table 30). Table 30 Papp Measured Using the In Vitro Fabric Model at pH was 5.0. In order to explore the generality of these findings, two additional peptides were examined, namely human parathyroid hormone 1-34 (PTH? -34) and human peptide YY 3-36 (PYY3-36) in the in vitro model in the absence and in the presence of PN159 (Papp data presented in Table 30). In the absence of PN159, the Papp of these two peptides was consistent with that of calcitonin. In the case of PTH? _34, the presence of PN159 produced approximately an increase in 3-5 times in the app- When the PYY3_36 was formulated in the presence of PN159, the Papp was increased approximately 12 to 17 times. These data confirm the generality of our discovery, that TJMP improved transmucosal drug supply for small molecules and proteins. EXAMPLE 32 Chemical Stability of TJMP Chemical stability of PN159 was determined under Therapeutically relevant storage conditions. An HPLC stability indicator method was used. Solutions (50 mM) were stored at various pH (4.0, 7.3 and 9.0) and temperature conditions (5 ° C, 25 ° C, 35 ° C, 40 ° C and 50 ° C). Samples at pH 4 contained 10 mM citrate buffer. Samples at pH 7.3 and 9.0 contained 10 mM phosphate buffer. The storage stability results (including the Arrhenius scheme) show that the PN159 was more thermally stable at low temperature and pH. For example, at 5 ° C and pH 4.0 or pH 7.3, there was essentially a 100% recovery of PN159 during a six month storage. When the storage temperature was increased to 25 ° C, there was a loss of 7% and 26% of natural PN159 for samples at a pH of 4 or pH 7, respectively, after six months. At pH 9 and / or at elevated temperature, e.g., 40 to 50 ° C, a rapid deterioration of PN159 occurred. The pH range of 4.0 to 7.3 and the temperature range of refrigerant to room are the most relevant for intranasal formulations. Accordingly, these data support that the TJMP can maintain chemical integrity under storage conditions relevant to intranasal formulations. EXAMPLE 33 In vivo Evaluation of Tightly Modulating Peptides in Rabbits by Intranasal Administration A pharmacokinetic study (PK) in rabbits was carried out to evaluate the plasma pharmacokinetic properties of peptide YY (PYY) with various tight binding modulation peptides (TJMPs) administered by intranasal (IN) delivery. Animal Model In this study, New Zealand model rabbits (Hra: (NZW) SPF) were used as test subjects to evaluate the plasma pharmacokinetics of MC-4RA by intranasal administration and intravenous infusion. The treatment of the animals was in accordance with the regulations set forth in the USDA Animal Welfare Act (9 CFR Parts 1, 2 and 3) and the conditions specified in the Guide for the Care and Use of Laboratory Animáis (ILAR publication, 1996, National Academy Press). Rabbits were selected as animal subjects for this study because the pharmacokinetic profile derived from a drug administered to rabbits closely resembles the PK profile for the same drug in humans. Dosage Management The experimental design and dose regimen for the 9 TJMPs tested is summarized in Table 31. All experimental groups were given 205 ug / kg of PYY (3-36) in combination with an individual TJMP or buffered saline with phosphate (PBS: negative control) by intranasal administration (IN). Each formulation was administered once in the left nostrils using a pipette and a disposable plastic swab. The head of the animal was tilted back and the dose was administered at the time of inhalation of the animal in order to allow the capillary action to attract the solution in the nostrils. After the IN administration, the head of the animal was kept in the tilted back position for approximately 15 seconds to avoid loss of the administered dose. During the procedure, extreme care was taken to avoid any tissue damage potentially resulting from contact with the intranasal mucosa. Table 31 Summary of the Test Groups PN556 has the same primary sequence as PN283, but has no maleimide modification at the N-terminus of the peptide. Collection of Blood and Plasma Samples After administration of the IN dose, serial blood samples were taken from each animal by direct venipuncture of a marginal ear vein. Blood samples were collected prior to predose, 5, 10, 15, 20, 30, 45, 60, 90, 120 and 180 minutes after the dose. Samples were collected in tubes containing EDTA dipotassium as the anticoagulant. The tubes were cooled until centrifuged. All samples were centrifuged within 1 hour of collection. The plasma was harvested and transferred into pre-labeled plastic vials, frozen in a dry ice / acetone bath, and then stored at about -70 ° C until a pharmacokinetic analysis was performed. Clinical observations were made at each moment of the blood sampling and the examination of both nostrils was carried out for all animals in the IN administration test groups, just before 5 minutes and 1 hour after intranasal dosing. Analytical Method Samples of each animal in all study groups were analyzed by levels of PYY (3-36) using ELISA. The test items before and after dosing were operated on HPLC for quality control. The aliquots of plasma (0.1 ml) were precipitated in protein with acetonitrile after adding an internal analytical standard. The supernatant was dried with nitrogen, reconstituted in HPLC buffer and then injected onto an HPLC system. The effluent is detected by a triple quadruple mass spectrometer in series of positive ion electrospray ionization. The PK data was analyzed by WinNonlin (Pharsight Corp., Mountain View). Results The average plasma PK parameters for each test group are summarized in Table 32. No adverse clinical signs were observed after the administration of any of the formulations. The post-intranasal examination of both nostrils of the animals administered with formulations via IN, revealed neither redness nor swelling. The PK study evaluated the Cmax (maximum concentration observed), the Tmax (maximum time concentration) and AUC (area under the curve) last and infinite (inf). Eight TJMPs were classified and categorized into 4 different rows of performance according to their in vivo permeability level containing the Fila I TJMPs with the highest level of permeability in vivo and each row containing TJMPs with progressively decreased levels of permeability in vivo. Table 32 EXAMPLE 34 Purification The following pegylated PN159 peptides have been synthesized (Table 33): Table 33 List of Peptides PN159 Pegylated Synthesized Table 33 List of Peptides PN159 Pegylated Synthesized PN526 (SEQ ID NO 58) PEGl-KLALKLALKALKAALKLA-amide PN537 (SEQ ID NO 59) PEG (5000Da) -KLALKLALKALKAALKLA-amide PN570 (SEQ ID NO 60) NH2-KLALKLALKALKAALKLA-PEG1-amide PN571 (SEQ ID NO 61) PEG1-KLALKLKLALKAALKLA-PEG1-amide PN572 (SEQ ID NO 62) PEG3-KLALKLALKALKAALKLA-amide An amount of 150 mg of crude peptide was absorbed in 15 ml of water containing 0.1% TFA and 3 ml of acetic acid. After stirring and sonication, the mixture was transferred to 1.5 ml Eppendorf tubes and centrifuged at 13,000 rpm. The supernatant was collected and filtered through a Millex GV syringe filter of 0.22 um. This solution was loaded on a Zorbax 300SB C18 column (21.2 mm ID x 250 mm, particle size 7 um) through a 5 ml injection loop at a flow rate of 5 ml / min. The purification was completed by operating a linear AB gradient of 0.2% B / min where solvent A is 0.1% TFA in water and solvent B is 0.1% TFA in acetonitrile. Under these conditions the peptide eluted over a range of 15-17% B.

EXAMPLE 35 Cells EpiAirway ™ cells (in a 96-well format (Air-196-HTS) or 24-well individual insert (Air-100), a human tracheal / bronchial tissue model, were purchased from MatTek Corporation (Ashland, MA) to be visualized by tight binding modulation peptides (TJMPs), based on its effect on transepithelial electrical resistance (TER) and permeability.The cultured tissue was from a single donor and was visualized as negative to HIV, hepatitis B, hepatitis C, mycoplasma, bacteria, yeast and fungi EpiAirway tissues were transported cold on agarose gels supplemented with medium EpiAirway tissues were recovered at 37 ° C for 24 hours with a medium provided by the manufacturer. CM) for the EpiAirway models contained DMEM, EFG and other factors, gentamicin (5 ug / ml), Amphotericin B (0.25 ug / ml) and phenol red as pH indicator EXAMPLE 36 Determination of TER The measurement for Air-196 -HTS was carried out using Automated Tissue Resistance System (REMS) (World Precession Instrument (WPI), Inc., (Sarasota, Florida). To monitor TER in 96-well HTS format, Endhom-Multi (STX) was used on the hook of the tissue culture to avoid contamination. In inserts recovered during the night 100 ul were used on the apex side and 250 ul in the basal chamber. The background TER was measured with a model insert (Millipore) and subtracted from the tissue inserts. The medium was decanted by inverting the insert on a paper towel. The insert was gently tapped on the paper towel to ensure maximum removal of the apex medium. For other time points in the measurement of TER, immediately after the treatments, the inserts were rinsed gently with 150 ul of Epi-CM three times and drained completely before the measurement of TER. The results (Figure 8) demonstrate that both the tight binding modulator peptide PN159 and the pegylated PN159 version of the invention tested on monolayer epithelial cells possess strong reversible effects to improve epithelial permeability. The effects observed with both are presented in a predictable manner. In addition, the results show that PEG-159 significantly improves ion permeability (decreases TER) over PN159 alone. The maximum difference in TER between PEG-PN159 and 159 is a PEG-PN159 of 50 uM. EXAMPLE 37 Permeability Analysis Dextrin (MW 3,000) labeled with fluorescence isothiocyanate (FITC) was added to the mixture. treatment at 0.1-1 mg / ml. The treatment mixture was added to the side of the apex wall and the plates were incubated at 37 ° C in an orbital shaker (New Brunswick Scientific, Edison, NJ) for the designated time at 100 rpm. At the end of the incubation, triplicates of 200 ul of the basal medium were transferred to a dark-walled fluorescent reading plate. Fluorescent intensity was measured at a wavelength of 470 nm by means of a FL microplate fluorescence reader, 800 (BIO-TEK INSTRUMENTS, INC., Winooski, VT). Standard dilutions of the standard were used to obtain a standard curve and calculate the concentration. Permeability was measured in two ways, such as the ratio of the donor mass (the epic chamber) or as the mass ratio receptor (the basal chamber), expressed as a percentage. A significant increase in PTH permeation was observed in the presence of both PN159 and PEG-PN159 of the invention (Figure 9). The effects observed with both in some way depend on the concentration between 1 ° uM. In addition, the results show that PEG-PN159 significantly decreases molecular permeability over PN159. When comparing the increase in permeability of PEG-PN159 with PN159 (plotted in Figure 10 as the relationship between the two values), the maximum differences of permeation increases are found at a concentration of 50 uM. EXAMPLE 38 Cytotoxicity Analysis An LDH analysis was used to establish the cytotoxicity of the treatments. The level of LDH by CytoTox96 Non-Radioactive Cytotoxic Assay (Promega, Madison, Wl) following the manufacturer's protocol. for lateral basal LDH levels, triplicates of 50 ul of the basal medium were used to determine the LDH level. For the APH LDH level, 150 ul of the diluted aseptic sample was removed by adding 150 ul of Epi-CM to the aseptic chamber, the medium was pipetted up and down, and 150 ul of the medium was removed and diluted. x (for a final dilution of 8 times) for analysis in triplicates of 50 ul. The total LDH level was determined using the cells in a final concentration of 0.9% Triton X-100 The LDH level in each sample was expressed as a percentage of cell lysis of Triton C-100. The results (Figure 11) show that PEG-PN159 has lower toxicity than PN159. EXAMPLE 39 Pharmacokinetic Data in Rabbits Twenty-five New Zealand male model rabbits of approximately 3 months of age were used in this study. The rabbits received only one administration intranasal, a dose of a tight binding peptide (TJ) and of PYY 3-36 in a nasal orifice, using a pipette and a disposable plastic swab. The rabbits were dosed according to the TJ peptide and control group shown in Table 34. The TJ peptides (PN407, PN408, PN526 (PEG-PN159) and PN159) are all found in 0.75 x DPBS with calcium and magnesium . The negative control is 0.75 x DPBS containing only calcium and magnesium (PBS). A PYY 3-36 positive control formulation without TJ peptide containing DDPC, EDTA, and MbCD in citrate buffer was used for comparison (PDF). The head of the animal was tilted back slightly as the dose was delivered. After dosing, the head of the animal was held in a backward inclined position for approximately 15 seconds. Blood samples were collected in series (approximately 1.5 ml each) by means of direct venipuncture of the marginal vein of the ear in blood collection tubes containing EDTA as an anticoagulant. Blood samples were collected at 0 (predose), 5, 10, 15, 30, 45, 60, 120 and 240 minutes after dosing for the intranasal groups. After collection, the tubes were inverted several times for anticoagulation. Then aprotinin was added at 50 ul to the collection tubes and mixed gently but completely. The samples mixed were placed in freezing packages until centrifuged at approximately 1,600 X g for 15 minutes at approximately 4 ° C. The plasma was divided into duplicate aliquots (approximately 0.35 ml each) and then stored at approximately -70 ° C. Table 34 Dosage Groups for the Pharmacokinetic Study in Rabbits The bioanalytical analysis of PYY 3-36 in rabbit plasma was carried out with conventional ELISA equipment ("Active Total Peptide Yy (PYY) ELISA", Cat. No. DSL-10-33600, Diagnostic Systems Laboratories, Inc., Webster, TX). The analysis is an enzyme immunoassay type intercalated enzymatically amplified in "one stage". In this assay, calibrators, controls and unknown samples are incubated with an anti-PYY antibody in microtiter wells that have been coated with another anti-PYY antibody. After incubation and washing the wells are incubated with the chromogenic substrate, tetramethylbenzidine. An acidic stop solution is then added and the degree of enzymatic inversion of the substrate is determined by measurement of dual wavelength absorbance at 450 and 620 nm. The absorbance measured is proportional to the concentration of PYY present. A logistic method of five-parameter data reduction is applied to the calibrator results to generate a calibration curve for each analysis. The calibration curve is used to interpolate the PYY concentration values of unknown samples from their absorbance results. The components of the equipment were used for all the stages of the analysis with the following exceptions: the reference material PYY3_36 was used to generate the calibrators and controls; the calibrators and controls are prepared with deposited rabbit plasma cut into strips (phase extraction column solid C18) as a diluent; and the unknown samples were diluted, when necessary, in rabbit plasma deposited cut into strips. The combination of the antibody with this equipment was optimized to detect intact human PYY? _36 and is fully cross-reactive with the mouse PYY? _36 and the human PYY3_36. The average pharmacokinetic (PK) data and the standard deviations (SD) are presented in Table 35 for the controls (PBS and PDF) and the TJ peptide formulations (PN159, PN407, PN408 and PN526). The relative bioavailability (% BA) for each tight binding modulator and each control is presented in Table 36. The percentage coefficient of variation for pharmacokinetic variables is presented in Table 37. Table 35 Mean PK Parameters and Standard Deviations (SD) for PYY3_36 in Rabbits Table 36% of Bioavailability of Tight Union Modulators Table 37% of the Coefficient of Variation for Pharmacokinetic Parameters It was considered that the lower limit of quantification (LLOQ) is 15.8 pg / ml. All gross data value that was < NUMBER, was adjusted to 7.9 pg / ml for analysis. The mean concentrations of PYY3_36 in plasma after nasal administration are shown in a linear line in the Figure 12, and a log-linear trace in Figure 13. Mean serum concentrations of PYY3_36 for animals administered with the nasal dose indicated peak concentrations (Tmax) between 15-34 minutes after dosing for all groups. The average Cmax for nasal PBS; PDF; PN159; PN407; PN408 and PN526 at a dose level of 205 ug / kg was 2,646.25; 19.004.40; 18,346.60; 13,980.20; 15,420.00 and 36.066.20 pg / ml, respectively. The AUCúitima media for the nasal PBS; PDF; PN159; PN407; PN408 and PN526 was 118,438.13; 1,289,219.50; 973,038.80; 725,950.50; 721,601.50 and 1,786,973.50 min * pg / ml, respectively. The AUC is average for the nasal PBS; PDF; PN159; PN407; PN408 and PN526 was 147,625.18; 1,319,034.73; 985,572.89; 753,080.86; 758,951.24 and 1,819,888.30 min * pg / ml, respectively. The t was approximately 35-48 minutes for all nasal formulations; however, the PBS was 83 minutes. See Table 35 for a complete list of all pharmacokinetic parameters including standard deviations. The% BA based on AUCuitima for the narrow-binding modulators against the PDF formulation was 75, 56, 56 and 139% for PN159, PN407, PN408 and PN526, respectively. The PBS% availability was only 9% compared to the PDF. The coefficient of variation was also compared (Table 37). All the tight binding modulators had a similar variation when comparing the pharmacokinetic parameters through the formulations for Cmax and AUC. The pharmacokinetic variable across all five formulation groups was analyzed using the one-way analysis of the variation model and it was found that the PBS formulation was significantly lower than the PN526 for Cmax, AUCúitima AUC? Nf? N? Ta (Tmax : p = 0.27, Cmax: p = 0.009, AUC last, p = 0.008, AÜCmfimta, P = 0.0097.

Comparing the Cmax, the PN526 pegylated tight union modulator was 1.9 times larger than the PDF and 13.6, 2.6 and 2.3 times larger than the PBS, PN407 and PN408, respectively. Comparing the AUCúititimar PN526 pegylated tight union modulator was 1.4 times higher than the PDF and 15.1, 2.5 and 2.5 times higher than the PN407 and PN408, respectively. The t. It was around 40 minutes for all groups, except for the PBS at 80 minutes. There was a significant difference between the PN526 and the formulation when comparing the pharmacokinetic parameters, C the pegylated tight peg modulator PN526max and AUC; however, there was no significance between the tight union modulators. The bioavailability was increased with PN526 compared to all other tight binding modulators and the pharmacokinetic parameters were statistically significant in comparison to the control formulation of PBS. These data show that the pegylated peptide formulation, PN526, had% BA increased above the formulations without pegylated peptide, PN159, PN407, PN408, and PBS. In addition, the% of BA for PN526 was also greater than that of the positive control without pegylated PDF peptide. The examples given herein are for illustrative purposes only and are not intended to limit the scope of the invention as described in vindication Although specific terms and values have been employed herein, such terms and values will be construed as exemplary and not limiting of the scope of the invention. All publications and references cited in this description are incorporated herein by reference in their entirety for all purposes.

Claims (78)

1. CLAIMS 1. A peptide-containing compound having mucosal activity in a mammal to improve mucosal epithelial transport of an active agent by modulating mucosal permeability, or a pharmaceutically acceptable salt thereof.
2. 2. The compound of claim 1, wherein the peptide has a molecular mass of less than 10 kilodaltons.
3. 3. The compound of claim 1, wherein the peptide is a peptide that modulates the tight junction.
4. 4. The compound of claim 1, wherein the compound contains a protein transduction domain, a DNA binding domain or a fusogenic domain.
5. 5. The compound of claim 1, wherein the compound contains a zinc indicator domain.
6. 6. The compound of claim 1, wherein the compound contains at least 60% licina, leucine and / or alanine residues.
7. 7. The compound of claim 1, wherein the compound forms an alpha helix.
8. 8. The compound of claim 1, wherein the permeability is in vitro or in vivo.
9. 9. The compound of claim 1, wherein the permeability is reversibly improved.
10. 10. The compound of claim 1, wherein the permeability is substantially a period of less than about ninety minutes.
11. 11. The compound of claim 1, wherein the permeability is substantially improved over a period of less than about sixty minutes.
12. The compound of claim 1, wherein the permeability improves without inducing substantial cytolysis in the mucosa.
13. The compound of claim 1, wherein the permeability improves while retaining cellular viability in the mucosa.
14. The compound of claim 1, wherein the permeability improves by increasing the fluidity of mucosal lipids.
15. 15. The compound of claim 1, wherein the compound is peptide PN159.
16. 16. The compound of claim 1, wherein the compound is an analog, conjugate, derivative, variant, fragment, mimetic, fusion molecule or PN159 complex.
17. 17. The compound of claim 1, wherein the peptide is selected from the group consisting of SEQ. ID. Nos 1-72
18. 18. The compound of claim 1, wherein the peptide is selected from the group consisting of SEQ ID Nos. 32-35, 38, 46-49, 53 and 55.
19. 19. The compound of any of claims 1-18, wherein the compound is covalently linked to a water-soluble chain.
20. The compound of claim 19, wherein the compound has a molecular mass of less than about 300 kilodaltons.
21. 21. The compound of claim 19, wherein the compound has a molecular mass of less than about 200 kilodaltons.
22. 22. The compound of any of claims 1-21, wherein the compound is covalently linked to a poly (alkylene oxide) chain.
23. 23. The compound of claim 22, wherein the poly (alkylene oxide) chain is branched or unbranched.
24. 24. The compound of claim 22, wherein the poly (alkylene oxide) chain is a polyethylene glycol (PEG) chain.
25. 25. The compound of claim 24, wherein the PEG has a molecular size between about 0.2 and about 200 kilodaltons (kDa).
26. 26. The compound of claim 24, wherein the PEG is less than 40 kDa in size.
27. 27. The compound of claim 24, wherein the PEG has a size less than 20 kDa.
28. 28. The compound of claim 24, wherein the PEG has a size less than 10 kDa.
29. 29. The compound of claim 24, wherein the PEG has a size less than 5 kDa.
30. 30. The compound of claim 24, wherein the PEG has a size less than 2 kDa.
31. The compound of claim 22, wherein the poly (alkylene oxide) has a polydispersity value (Mw / Mn) of less than 2.00.
32. 32. The compound of claim 22, wherein the poly (alkylene oxide) has a polydispersity value (Mw / Mn) of less than 1.20.
33. 33. The compound of claim 22, wherein the compound and the poly (alkylene oxide) are conjugated through a separating residue.
34. 34. A pharmaceutical formulation comprising an effective amount that improves mucosal epithelial transport of a compound of any of claims 1-33 and a therapeutically effective amount of an active agent.
35. 35. The formulation of claim 34, wherein the formulation decreases electrical resistance through a mucosal tissue barrier.
36. 36. The formulation of claim 35, in where the decrease in electrical resistance is at least 80%.
37. 37. The formulation of claim 34, wherein the formulation increases the permeability of the active agent through a mucosal tissue barrier relative to a similar formulation that does not contain the compound of any of claims 1-33.
38. 38. The formulation of claim 37, wherein the increase in permeability is at least two times.
39. 39. The formulation of claim 37, wherein the permeability is paracellular.
40. 40. The formulation of claim 37, wherein the increased permeability results from the modulation of a tight joint.
41. 41. The formulation of claim 37, wherein the permeability is transcellular or a mixture of trans and paracellular.
42. 42. The formulation of claim 37, wherein the mucosal tissue barrier is a layer of epithelial cells.
43. 43. The formulation of claim 42, wherein the epithelial cell is selected from the group consisting of tracheal, bronchial, alveolar, nasal, pulmonary, gastrointestinal, epidermal, and buccal.
44. 44. The formulation of claim 42, wherein the epithelial cell is nasal.
45. 45. The formulation of claim 34, wherein the active agent is a peptide, protein or nucleic acid.
46. 46. The formulation of claim 45, wherein the peptide or protein is comprised of from 2 to 1000 amino acids.
47. 47. The formulation of claim 45, wherein the peptide or protein is comprised between 2 and 50 amino acids.
48. 48. The formulation of claim 45, wherein the peptide or protein is cyclic.
49. 49. The formulation of claim 45, wherein the peptide or protein is a dimer or oligomer.
50. 50. The formulation of claim 45, wherein the peptide or protein is selected from the group consisting of GLP-1, PYY 3-36, PTH 1-34 and Exendin-4.
51. 51. The formulation of claim 45, wherein the protein is selected from the group consisting of beta-interferon, alpha-interferon, insulin, erythropoietin, G-CSF, GM-CSF, growth hormone, and their analogues.
52. 52. A dosage form comprising the formulation of any of claims 34-51, wherein the dosage form is liquid.
53. 53. The dosage form of claim 52, wherein the liquid is in the form of drops.
54. 54. The dosage form of claim 52, wherein the liquid is in the form of an aerosol.
55. 55. A dosage form comprising the formulation of any of claims 34-51, wherein the dosage form is solid.
56. 56. The dosage form of claim 55, wherein the solid is reconstituted in liquid prior to administration.
57. 57. The dosage form of claim 55, wherein the solid is administered as a powder.
58. 58. The dosage form of claim 55, wherein the solid is in the form of a capsule, tablet or gel.
59. 59. A method for administering a molecule to an animal, comprising providing a formulation as in any of claims 34-58 and contacting the formulation with a mucosal surface of the animal.
60. 60. The method of claim 59, wherein the mucosal surface is intranasal.
61. 61. A method for increasing the bioavailability of an active agent administered intranasally in a mammal, comprising providing a formulation as in any of claims 34-58 and administering the formulation to the mammal.
62. 62. The compound of claim 1, wherein the active agent is an siRNA.
63. 63. The compound of claim 1, wherein the active agent is a dsDNA.
64. 64. The compound of claim 1, wherein the active agent is a hematopoietic, an anti-infective, an anti-dementia, an antiviral, an anti-tumor, an antipyretic, an analgesic, an anti-inflammatory, an anti-ulcer, an antiallergenic, an antidepressant, a psychotropic, a cardiotonic, an anti-arrhythmic, a vasodilator, an anti-hypertensive, a hypotensive diuretic, an antidiabetic, an anticoagulant, a cholesterol lowering agent, a therapeutic for osteoporosis, a hormone, an antibiotic or a vaccine.
65. 65. The compound of claim 1, wherein the active agent is a cytosine, a peptide, a hormone, a growth factor, a cardiovascular factor, a cell adhesion factor, a central or peripheral nervous system factor, a factor of humoral electrolyte, an organic hemal substance, a bone growth factor, a gastrointestinal factor, a renal factor, a connective tissue factor, a sensory organ factor, a factor of the immune system, a factor of the respiratory system, or a genital organ factor.
66. 66. The compound of claim 1 wherein the active agent is an androgen, an estrogen, a prostaglandin, a somatotropin, a gonadotropin, an interleukin, a steroid or a cytosine.
67. 67. The compound of claim 1, wherein the active agent is a vaccine for hepatitis, influenza, respiratory syncytial virus (RSV), parainfluenza virus (PIV), tuberculosis, yellowpox, smallpox, measles, mumps, rubella, pneumonia. , or human immunodeficiency virus (HIV).
68. 68. The compound of claim 1, wherein the active agent is a bacterial toxoid for diphtheria, tetanus, pseudomonas or tuberculosis mycobacteria.
69. 69. The compound of claim 1, wherein the active agent is hirugen, hiru or hirudin.
70. 70. The compound of claim 1, wherein the active agent is a monoclonal antibody, a polyclonal antibody, a humanized antibody, an antibody fragment or an immunoglobulin.
71. 71. The compound of claim 1, wherein the active agent is morphine, hydromorphone, oxymorphone, lovorphanol, levalorfan, codeine, nalmefene, nalorphine, nalozone, naltrexone, buprenorphine, butorphanol or nalbuphine.
72. 72. The compound of claim 1, wherein The active agent is cortisone, hydrocortisone, fludrocortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, dexametho- saone, betametho- soone, parametosone or fluocinoclone.
73. 73. The compound of claim 1, wherein the active agent is colchicine, acetaminophen, aspirin, ibuprofen, ketoprofen, indomethacin, naproxen, meloxicam or piroxicam.
74. 74. The compound of claim 1, wherein the active agent is acyclovir, ribavarin, trifluorothyridine, Ara-A (Arabinofuranosiladenina), acilguanosina, nordeoxiguanosina, azidotimidina, dideoxiadenosina or dideoxicitidina.
75. 75. The compound of claim 1, wherein the active agent is spironolactone, testosterone, estradiol, progestin, gonadotropin, estrogen or progesterone.
76. 76. The compound of claim 1, wherein the active agent is papaverine, nitroglycerin, vasoactive intestinal peptide, calcitonin-related gene peptide, cyproheptadine, doxepin, imipramine, cimetidine, dextromethorphan, clozaril, superoxide dismutase, neuroenkephalinase, amphotericin B, griseofluvin, miconazole, ketoconazole, thioconazole, itraconazole, fluconazole, cephalosporin, tetracycline, aminoglycoside, erythromycin, gentamicin, polymyxin B, 5-fluorouracil, bleomycin, methotrexate and hydroxyurea, dideoxyinosine, floxuridine, 6-mercaptopurine, doxorubicin, daunorubicin, I-darubicin, taxol, paclitaxel, tocopherol, quinidine, prazosin, verapamil, nifedipine or diltiazem.
77. 77. The compound of claim 1, wherein the active agent is a tissue plasminogen activator (TPA), epidermal growth factor (EGF), fibroblast growth factor (FGF-acidic or basic), derived growth factor. of platelets (PDGF), transforming growth factor (TGF-alpha or beta), vasoactive intestinal peptide, tumor necrosis factor (TNF), hypothalamic release factor, prolactin, thyroid stimulating hormone (TSH), adrenocorticotropic hormone ( ACTH), parathyroid hormone (PTH), follicle stimulating hormone (FSF), luteinizing hormone releasing hormone, (LHRH), endorphin, glucagon, calcitonin, oxytocin, carbetocin, aldoetecona, encafaline, somatostin, somatotropin, somatomedin, stimulating hormone of alpha-melanocyte, lidocaine, sufentainil, terbutaline, droperidol, scopolamine, gonadorelin, ciclopirox, buspirone, calcitonin, cromolyn sodium, or midazolam, cyclosporine, lisinopril, c eligpril, delapril, ranitidine, famotidine, superoxide dismutase, asparaginase, arginase, arginine deaminase, adenosine deaminase ribonuclease, trypsin, chymotrypsin, papain, bombesin, substance P, vasopressin, alpha-globulins, transferrin, fibrinogen beta-lipoprotein, beta-globulin, prothrombin, ceruloplasmin, alpha 2-glycoprotein, alpha 2-globulin, fetuin, alpha 1-lipoprotein, alpha 1-globulin, albumin or prealbumin.
78. 78. A pharmaceutical product comprising a solution containing a compound of claim 1 and an actuator for a mucosal, intranasal or pulmonary spray.