MX2008007075A - Pharmaceutical formulation for increased epithelial permeability of glucose-regulating peptide. - Google Patents

Abstract

What is described is a pharmaceutical formulation comprising a mixture of a pharmaceutically effective amount of glucose-regulating peptide (GRP) and enhancers, wherein the pharmaceutical formulation is used in the treatment of a metabolic syndrome.

Description

PHARMACEUTICAL FORMULATION FOR INCREASED PEPTIDE PERMEABILITY THAT REGULATES GLUCOSE BACKGROUND OF THE INVENTION Peptides that regulate glucose ("GRP") are a class of peptides that have therapeutic potential in the treatment of insulin-dependent diabetes mellitus (IDD), gestational diabetes or non-insulin-dependent diabetes mellitus ( NIDDM); the treatment of obesity, and the treatment of dyslipidemia. See U.S. Patent No. 6,5006,724, U.S. Patent Application Publication No. 20030036504A1; European Patent No. EP1083924B1; International Patent Application Publication No. O 98 / 30231A1; and International Patent Application No. WO 00 / 73331A2. These GRPs include glucagon-like peptide (GLP), for example, GLP-1; the exendins, especially exendin-4, also known as exenatide; and amylin peptides and amylin analogs, such as pramlintide. However, to date, these GRPs have only been administered to humans by injection. A major disadvantage of administering the drug by injection is that trained personnel are often required to administer the drug. Additionally, trained personnel are placed in harmful form when a drug is administered by injection. For self-medication When administered, many patients are reluctant or unable to give themselves the injections on a regular basis. Injection is also associated with increased risks of infection. Other disadvantages of drug injection include variability of delivery results between individuals, as well as unpredictable intensity and duration of drug action. Oral administration is available as an alternative; however, certain therapeutic agents exhibit very low bioavailability and considerable time lag in action when given by this route, due to first pass hepatic metabolism and degradation in the gastrointestinal tract. Thus, there is a need to develop different modes of administration of GRP by injection and / or oral administration. The mucosal administration of therapeutic compounds offers certain advantages over injection and other modes of administration, for example, convenience and speed of delivery, as well as reduction or elimination of compliance problems and side effects that the supply achieves. However, the mucosal supply of biologically active agents is limited by mucosal barrier functions and other factors. The epithelial cells make the mucosal barrier and provide a crucial interface between the external environment and 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 barriers of selective permeability between different physiological compartments. The selective permeability is the result of the regulated transport of molecules through the cytoplasm (the transcellular path) and the regulated permeability of the spaces between the cells (the paracellular path). The intercellular junctions between the epithelial cells are known to be involved in both maintenance as regulation of the epithelial barrier function and cell-cell adhesion. The tight junctions (TJ) of epithelial and endothelial cells are particularly important for the cell-cell junctions that regulate the permeability of the paracellular pathway, and also divide the cell surface into apical and basolateral compartments. The tight junctions form continuous circumferential intercellular contacts between 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 cell polarity by limiting the exchange of membrane lipids between the apical membrane domains and basolateral. In the context of drug delivery, the ability of drugs to permeate the epithelial cell layers of mucosal surfaces, unassisted by agents that improve delivery, seems to be related to a number of factors, including molecular size, lipid solubility and ionization. . In general, small molecules, of less than about 300-1,000 daltons, are often able to penetrate mucosal barriers, however, as molecular size increases, permeability rapidly decreases. The transdermal drug delivery allows the permeation of larger molecules through the layers of skin epithelial cells. Transdermal administration, such as transdermal patch, is another alternative delivery route for larger macromolecular drugs. However, the transdermal delivery may still present more size limitations than the injection. For these reasons, administration of mucosal and epidermal drug typically requires larger amounts of drug than administration by injection. Other therapeutic compounds, including large molecule drugs, are often refractory to mucosal delivery. In addition to the low intrinsic permeability, large macromolecular drugs are often subject to limited diffusion, as well as Lumenal and cellular enzymatic degradation and rapid separation at mucosal sites. Thus, to deliver these larger molecules in therapeutically effective amounts, agents that improve cell permeation are required to help their passage through these mucosal and dermal surfaces and into the systemic circulation where they can act rapidly in the tissue objective .

BRIEF DESCRIPTION OF THE FIGURES FIGURE 1: Reduction in blood glucose concentration after IN administration of GLP-1 compared with Exenatide (SQ) and Saline Control (IN) (corrected for endogenous glucose) in rats. FIGURE 2: Insulin response after intranasal administration of GLP-1 in rats. FIGURE 3: Gastric emptying after intranasal administration of GLP-1 in rats. FIGURE 4: Improved pharmacokinetics of Exendin-4 administered IN with 2X intensifiers, IN with 2X + gelatin enhancers, and IN with IX + gelatin enhancers compared with IN and IV control in rabbits.

DETAILED DESCRIPTION OF THE INVENTION One aspect of the present invention includes the Therapeutic utility of pharmaceutical formulations for the delivery of GRPs, GRP analogs, GRP fragments, and functional derivatives of GRP through an epithelial surface for use in the treatment of human diseases including obesity and diabetes. The present invention fully meets the needs mentioned above and satisfies additional objects and advantages by providing new, effective methods, uses and compositions for transepithelial delivery, especially transmucosal GRP such as GLP and GLP analogs, amylin and amylin analogues, and exendins and analogues of exendin, to treat insulin-dependent diabetes mellitus (IDD), gestational diabetes or non-insulin-dependent diabetes mellitus (NIDDM), dyslipidemia, hyperglycemia, obesity, to induce satiety in an individual, and promote weight loss in an individual. In exemplary embodiments, the intensified delivery methods and compositions of the present invention provide therapeutically effective delivery of the GRP agonists through a biological cell layer for the prevention or treatment of obesity and eating disorders in mammalian subjects. In one aspect of the invention, pharmaceutical formulations suitable for epithelial administration are provided comprising a therapeutically effective amount of a GRP and one or more epithelial supply enhancing agents as described herein, wherein, the formulations are effective in an epithelial delivery method of the invention for preventing the onset or progression of obesity or eating disorders in a mammalian subject. The transepithelial delivery of a therapeutically effective amount of a GRP agonist and one or more agents that enhance the epithelial supply, provide high therapeutic levels of the GRP agonist in the subject. The improved delivery methods and compositions of the present invention provide therapeutically effective delivery of a GRP for prevention or treatment of a variety of conditions and diseases in human subjects. The GRP can be administered via a variety of epithelial routes, for example, by contacting the GRP to a nasal mucosal epithelium, a bronchial or pulmonary mucosal epithelium, the oral buccal surface, the oral mucosal surface and the small intestine, or an epidermal surface. In exemplary embodiments, the methods and compositions are directed to, or formulated for, intranasal delivery (eg, nasal mucosal delivery or intranasal mucosal delivery). The aforementioned GRP formulations and preparative and delivery methods of the invention provide improved epithelial delivery of a GRP to mammals subjects. These compositions and methods may involve combinatorial formulation or coordinated administration of one or more GRP with one or more agents that enhance epithelial delivery. Among the agents that enhance the epithelial supply to be selected to achieve these formulations and method are: (A) solubilization agents; (B) agents that modify the load; (C) pH control agents; (D) degradative enzyme inhibitors; (E) mucosal or mucolytic separation agents; (F) ciliates; (G) agents that enhance the penetration of the membrane (e.g., (i) a surfactant, (ii) a bile salt, (iii) a phospholipid or fatty acid additive, mixed mycelium, liposome or carrier, (iv) a alcohol, (v) an enamine, (iv) a NO-donor compound, (vii) a long-chain antipathetic molecule, (viii) a small hydrophobic penetration enhancer; (ix) derived from salicylic or sodium acid; ) a glycerol ester of acetoacetic acid (xi) a derivative of cyclodextrin or beta-cyclodextrin, (xii) a medium chain fatty acid, (xiii) a chelating agent, (xiv) an amino acid or salt thereof, (xv) an N-acetylamino acid or salt thereof, (xvi) a degrading enzyme to a selected membrane component, (xvii) an inhibitor of fatty acid synthesis, (xviii) a cholesterol synthesis inhibitor, or (xiv) any combination of the agents that intensify the penetration of the membrane of (i) - (xviii)); (H) epithelial binding physiology modulating agents, such as nitric oxide (NO) stimulators; chitosan, and chitosan derivatives; (I) vasodilating agents; (J) agents that intensify selective transport; and (K) stabilization of delivery vehicles, carriers, supports or species forming complexes with which the GRP is / are effectively combined, associated, contained, encapsulated or bound to stabilize the active agent for improved epithelial delivery. In various embodiments of the invention, a GRP is combined with one, two, three, four or more of the agents that enhance the epithelial supply mentioned in (A) - (K), above. These agents that enhance the epithelial supply can be mixed alone, or together, with the GRP, or otherwise combined together with these in a pharmaceutically acceptable formulation or delivery vehicle. The formulation of a GRP with one or more of the agents that enhance epithelial delivery in accordance with the teachings herein (optionally including any combination of two or more agents that enhance epithelial delivery, selected from (A) - (K)) , provide increased bioavailability of binding peptides that regulate glucose after delivery thereof to an epithelial surface of a mammalian subject.

In addition, by adding agents that enhance the epithelial supply to the formulation, the modification of the GRP, such as through the addition of a hydrophobic group, can be used to effect the bioavailability of the peptide. Thus, the present invention is a method for suppressing appetite, promoting weight loss, reducing the absorption of food, or treating obesity and / or diabetes in a mammal, comprising transepithelial administration of a formulation comprised of a GRP. The present invention further provides the use of a GRP for the production of medicament for the transepithelial administration of a GRP to treat hyperglycemia, diabetes mellitus, metabolic syndrome, dyslipidemia, suppression of appetite, promotion of weight loss, reduction of food absorption, or treat obesity in a mammal. A mucosally effective dose of GRP within the pharmaceutical formulations of the present invention comprises, for example, between about 0.001 pmol to about 100 pmol per kg of body weight, between about 0.01 pmol to about 10 pmol per kg of body weight, or between about 0.1 pmol to about 5 pmol per kg of body weight. In exemplary additional embodiments, the GRP dosage is between about 0.5 pmol to about 1.0 pmol per kg of body weight. In a preferred embodiment, an intranasal dose will vary from 0.1-100 g / kg, or approximately 7-7000 g, more preferably 0.5-30 g / kg, or 35-2100 g. More specific doses of the intranasal GRP include 20 yg, 50 yg, 100 yg, 150 yg, 200 yg up to 400 yg, 500 yg, 800 up to 1000 yg and 1200 up to 1800 yg. The pharmaceutical formulations of the present invention can be administered one or more times per day, or 3 times per week or once a week between one week and at least 96 weeks or even for the life of the patient or individual subject. In certain embodiments, the pharmaceutical formulations of the invention are administered one or more times daily, twice daily, four times daily, six times daily, or eight times daily. Agents that enhance the epithelial supply are employed, which intensify the supply of GRP in or through a cell layer, including a nasal mucosal surface. For passively absorbed drugs, the relative contribution of paracellular and transcellular trajectories to drug transport depends on the pKa, division coefficient, molecular radioactivity and drug loading, the pH of the luminal environment in which the drug is delivered, and the area of the absorption surface. The agent that intensifies the epithelial supply of the present invention, can be a pH control agent. The pH of the pharmaceutical formulation of the present invention is a factor that affects the absorption of GRP via paracellular and transcellular pathways to drug transport. In one embodiment, the pharmaceutical formulation of the present invention is adjusted from pH to between about pH 2 to 8. In a further embodiment, the pharmaceutical formulation of the present invention is adjusted from pH to between about pH 3.0 to 6.0. In a further embodiment, the pharmaceutical formulation of the present invention is adjusted to a pH between about 3.5 to 5.5. In general, the pH is 4.5 + 0.5. As noted above, the present invention provides improved methods and compositions for epithelial delivery of GRP to mammalian subjects for the treatment or prevention of a variety of diseases and conditions. Examples of mammalian subjects suitable for treatment and prophylaxis in accordance with the methods of the invention include, but are not limited to, non-human and human primates, livestock species, such as horse, cow, sheep and goat, and research species and domestic animals, which include dogs, cats, mice, rats, guinea pigs and rabbits. To provide better understanding of the present invention, the following definitions are provided: Glucagon-like peptides (GLP) Included within the invention are. analogs, fragments, mimetics and functional derivatives of GLP proteins and peptides. Incretins are hormones derived from the stomach, which stimulate insulin secretion in response to nutrient absorption (in a glucose-dependent manner). Two incretins that originate naturally include glucose-dependent insulinotropic peptide (GIP) and glucagon like peptide-1 (GLP-1). GLP-1 is released from cells in the intestine in response to food. GLP-1 binds to GLP-1 receptors in beta cells of the pancreas, stimulating the release of insulin. GLP-1 [7-36] NH2, also known as proglucagon [78-107] and more commonly as "GLP-1," has an insulinotropic effect, stimulating insulin secretion; GLP-1 also inhibits glucagon secretion [Orskov, et al., Diabetes 42: 658-61, 1993; D'Alessio, et al., J. Clin. Invest. 97: 133-38, 1996]. GLP-1 is reported to inhibit gastric vacuum [Williams B. , et al., J. Clin. Encocrinol Metab. 81 :( 1): 327-32, 1996; Wettergren A., et al., Dig. Dis. Sci. 38: (4): 665-73, 1993], and secretion of gastric acid. [Schjoldager B.T., et al., Dig. Dis. Sci. 34 (5): 703-8, 1989; O'Halloran D.J., et al., J. Endocrinol. 126 (1): 169-73, 1990; Wettergren A., et al., Dig. Dis. Sci. 38: (4): 665-73, 1993]. GLP-1 [7-37], which has an additional glycine residue in its carboxy terminus, it also stimulates the secretion of insulin in humans [Orskov, et al., Diabetes 42: 658-61, 1993]. A receptor coupled to adenylate cyclase of transmembrane G protein is thought to be responsible for the insulinotropic effect of GLP-1, it is reported to have been cloned from a cell line a. beta. [Thorens, Proc. Nati Acad. Sci. USA 89: 8641-45, 1992]. A major limitation of GLP-1 for therapeutics is its rapid degradation by the ubiquitous enzyme dipeptidyl dipeptidase-IV (DDP-IV). DPP-IV inhibitors (LAF237; MK-0431) have been used to improve the duration of endogenous GLP-1 activity. The US Food and Drug Administration (FDA) approved JANUVIA ™ (sitagliptin phosphate), Merck & Co. , Inc., an oral DPP-IV inhibitor available in the United States for the treatment of type 2 diabetes. A method for the treatment of metabolic diseases in a mammal comprises co-administration of a compound capable of binding to a secondary binding site of DPP-IV and DPP-IV-like enzymes and at least one anti-diabetic agent are described in U.S. Patent Application No. 20060234940. Incretin mimics are a class of drugs that mimic the antidiabetic or hormone-lowering actions of glucose of human incretin that originates naturally as GLP-1. The mimetic actions of incretin include, stimulate the body's ability to produce insulin in response to high blood sugar levels, inhibit the release of the hormone glucagon, reduce the absorption of nutrients in the bloodstream, reduce the gastric vacuum ratio, promote satiety and reduce the absorption of food. Incretin mimetics were developed for use in the treatment of type 2 diabetes and include the following: GLP-1 derivatives (Lyraglutide and CJC-1131), and Exenatide. CJC-1131 (ConjuChem, Montreal, Canada) has a reactive linker that allows covalent binding to serum albumin resulting in increased resistance to DPP-IV degradation. The liraglutide (Novo Nordisk, Copenhagen, Denmark) is a derivative of GLP-1 designed to overcome the effects of DPP-VI degradation via acylation with a fatty acid chain. The liragulide structure is shown in WHO Drug Information, Vol. 17, No. 2 (2003). The invention includes modifications of GRP by binding a hydrophobic group, such as fatty acids to the peptide. Additional examples of modified derivatives of GLP-1 with desirable pharmacokinetic properties are described in Knudsen et al., J. Med. Chem. 43: 1664-1669, 2000, and are hereby incorporated by reference. These GLP-1 compounds are derivatized with fatty acids to prolong their action by facilitating the binding to serum albumin. HE describe the following precursor peptides and acyl substitutions: K8R26 '34-GLP-I (7-37) (K8:? -Glu-Cl6); K18R26 '34-GLP-I (7 -37) (K18: Y-G1U-C16); K23R26 '34-GLP-I (7 -37) (K23:? -Glu-Cl 6); R34-GLP-K7-37) (K26:? -Glu-Cl 6); K27R2S '34-GLP-1 (7-37) (K7: y-Glu-C16); R26-GLP-1 (7-37) (K34: Y ~ G1U-C16); K36R26'34-GLP-1 (7-37) (K36: Y-G1U-C16); R26 '34-GLP-I (7-38) (K38:? -Glu-Cl 6); GLP-l (7-37) (K26'34: bis-C16-diacid); GLP-I (7-37) (K26 '34: bis-Y-Glu-Cl 6); GLP-K7-37) (K26'34: bis-Y-Glu-C14; GLP-I (7-37) (K26 '34: bis-C12-diacid); R34-GLP-1 (7-37) ( K26: C16-diacid); R34 GLP-I (7-37) (K26: C14-diacid); R34-GLP-1 (7-37) (K26: Y-G1U-C18); R34-GLP-1 ( 7-37) (K26:? -Glu-Cl4); R3-GLP-1 (7-37) (K26: y-Glu-C12); deamino-H7R34-GLP-l (7-37) (K26: y -Glu-Cl 6); R34-GLP-1 (7-37) (K26: GABA-C16); R34-GLP-1 (7-37) (K26: p-Ala-C16); R3-GLP-1 (7-37) (K26: Iso-Nip-C16); deamino-H7R26-GLP-I (7-37) (K34: Y-G1U-C16); deamino-HR26-GLP-I (7-37) (K34: C8); deamino-H7R26-GLP-1 (7-37) (K34: Y-G1U-C8); K36'34-GLP-1 (7-36) (K36: C20-diacid); K36'34-GLP-1 (7-36) (K36: C16-diacid); K36'34-GLP-1 (7-36) (? 36:? - G1U-C18); R26'34-GLP-1 (7-38) (K38: Cl 6-diacid); R26 '34-GLP-I (7-38) (K38: C12-diacid); R26 '34-GLP-I (7-38) (K38: y-Glu-C18); R26'34-GLP-K7-38) (K38: y-Glu-C14); C8R26 '34-GLP-I (7-38) (K38: y-Glu-C16); E37R6'34-GLP-1 (7-38) (K38: y-Glu-Cl 6); E37C8R26 '34-GLP-I (7-38) (K38: Y-G1U-C16); and E37C8R26 '3-GLP-1 (7-38) (K38: Y ~ G1U-C18). The amino acid sequence of GLP-1 is given i. to. by Schmidt, et al., Diabetologia 28: 704-707, 1985. Human GLP-1 is a peptide of 37 amino acid residues that is originates from the preproglucan which is synthesized, i. a in the L cells in the distal ileum, in the pancreas and in the brain. The processing of preproglucan to GLP-1 (7-36) amide, GLP-1 (7-37) and GLP-2, occurs mainly in the L cells. Although the interesting pharmacological properties of GLP-1 (7-37) and analogs thereof, has attracted much attention in recent years, only little is known about the structure of these molecules. The secondary structure of GLP-1 in mycelia has been described by Thorton, et al., Biochemistry 33: 3532-3539, 1994), but in normal solution, GLP-1 is considered a very flexible molecule. GLP-1 and GLP-1 analogs and fragments thereof are used i. a in the treatment of Type 1 and Type 2 diabetes and obesity. The GLP-1 analogs, GLP-1 fragment and functional derivatives of GLP-1 described in Holst, J., Expert Opin. Emerg. Drugs 9 (1): 155-161, 2004; Rolin, R., et al., Am. J. Physiol. Endocrinol Metab. 283: The 5-E752, 2002; Deacon, C., Diabetes 53: 2181-2187, 2004; Perry, T., et al., Trends Pharmacol. Sci. 24 (7): 377-383, 2003; Holz, G., et al., Curr. Med. Chem. 10 (22): 2471-2481, 2003; Naslund, E., et al., Regul. Pept. 106: 89-95, 2002; patent applications WO 87/06941; O 90/11296; WO 91/11457; EP 0708179-A2 and EP 0699686-A2, are incorporated by reference herein in their entirety; WO 87/06941 describes fragments of GLP-1, which include GLP-1 (7-37), and functional derivatives thereof and its use as an insulinotropic agent. WO 90/11296 discloses GLP-1 fragments, including GLP-1 (7-36), and functional derivatives thereof, which have insulinotropic activity which exceeds the insulinotropic activity of GLP-1 (1-36) or GLP-1 (1-37) and its use as insulinotropic agents. WO 91/11457 discloses analogs of active GLP-1 peptides 7-34, 7-35, 7-36, and 7-37 which can be used as GLP-1 moieties. Document 0708179-A2 (Eli Lilly &Co.) discloses GLP-1 analogs and derivatives that include an N-terminal imidazole group and optionally an unbranched Ce-Cι acyl group attached to the lysine residue at position 34. The document EP 0699686-A2 (Eli Lilly &Co.) describes certain N-terminal truncated fragments of GLP-1, which are reported to be biologically active. The amino acid sequence of GLP-1 (1-37) is: HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 1). The amino acid sequence of GLP-1 (7-37) is: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 2). The amino acid sequence of GLP-1 (7-36) is: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR (SEQ ID NO: 3). The amino acid sequence of GLP-1 (7-34) is: HAEGTFTSDVSSYLEGQAAKEFIAWLVK (SEQ ID NO: 4).

The amino acid sequence of GLP-1 (9-36) is: EGTFTSDVSSYLEGQAAKEFIAWLVKGR (SEQ ID NO: 5), The GLP-1 analogs listed above, have increased resistance to DPP-IV. The amino acid sequence of the GLP-1 analogue GG is: HGEGTFTSDVSSYLEGQAAKEFIAWLVKGR (SEQ ID NO: 6). The amino acid sequence of the GLP-1 analog GGi is: HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRPSS (SEQ ID NO: 7). The amino acid sequence of the GLP-1 analog GG2 is: HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRPSSGAP (SEQ ID NO: 8). The amino acid sequence of the GLP-1 analog GG3 is: HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRPSSGAPPPS (SEQ ID NO: 9). The amino acid sequence of the GLP-1 GLP-1 ET analog is: HAEGTFTSDVSSYLEGQAAKEFIA LVKGGPSSGAPPPS (SEQ ID NO: 10). The amino acid sequence of the synthetic analog of LGP-1 NN2211 is: HAEGTFTSDVSSYLEGQAAK * EFIAWLVRGRG (SEQ ID NO: 11) wherein K * at position 26 of the amino acid chain is modified by acylation to generate a hexadecanoyl side chain (i.e. ,? -? - e- (? -Glu (?? - a-hexadecanoilo) .The amino acid sequence of the synthetic analog GLP-1 CJC-1131 is: HA * EGTFTSDVSSYLEGQAAKEFIAWLVKGRK * (SEQ ID NO: 12) where A * at position 8 of the amino acid chain is a D-alanine substituted by an L-alanine and the K * at position 37 of the amino acid chain has a linker [2- [2- [2-maleimidopropionamido] (ethoxy) ethoxy] acetamide in its amino group e. The amino acid sequence of the synthetic GLP-1 analog LY315902 is: des-HAEG FTSDVSSYLEGQAAREFIAWLVK * GRG (SEQ ID NO: 13), where the histidine residue in the N-terminus (de-H), does not contain an amino group and the K * at position 34, is modified by acylation to generate an octanoyl side chain (i.e., K_ (oxtanoyl)). In accordance with the invention, GLP-1 also includes free bases, acid addition salts or metal salts, such as potassium or sodium salts of the peptides and GLP-1 peptides that have been modified by such processes as amidation , glycosylation, acylation, sulfation, phosphorylation, acetylation, cyclization and other known covalent modification methods.

Exendins and Exendin Agonists Included within the invention are analogs, fragments and functional derivatives of proteins and peptides of exendins. The exendins are peptides that were first isolated from the salivary secretions of Gila monster, a lizard found in Arizona, and the Mexican embroidered lizard. Exendin-3 is present in secretions salivars of Heloderma horrídum, and exendin-4 is present in the salivary secretions of Heloderma suspectum [Eng, J., et al., J. Biol. Chem. 265: 20259-62, 1990; Eng., J., et al., J. Biol. Chem. 267: 7402-05, 1992]. The exendins have some sequence similar to several members of the glucagon-like peptide family, where the highest homology, 53%, is for the incretin hormone GLP-1 [7-36] H .2 [Goke, et al. , J. Biol. Chem. 268: 19650-55, 1993]. The generic name for synthetic exendin-4 is exenatide [WHO Drug Information, Vol. 18, No. 1, 2004]. Exenatide is a synthetic Exendin-4. Exenatide mirrors the effects of GLP-1, but is more potent due to its resistance to degradation of DPP-IV. BYETTA © is the commercially available version of exenatide (Amylin &Lilly). The US FDA approved the injection of BYETTA (Exenatide) as an adjunctive therapy to type 2 diabetes where the treatment of oral metformin and / or sulphonylurea are not adequate to achieve glycemic control. In addition to improving glycemic control, subjects in studies using exenatide also experience weight loss. The present invention is directed to new methods for treating diabetes and conditions that could be benefited by reducing plasma glucose or by slowing and / or reducing gastric vacuum or inhibiting the absorption of food comprising intranasal administration of a exendin, an exendin analogue, an exendin agonist, a modified exendin, an exendin modified analog, or a modified exendin agonist, or any combination thereof, for example: Exendin-3: His Ser Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Wing Val Arg Leu Phe lie Glu Trp Leu Lys Asn Gly Gly Pro Ser Ser Gly Wing Pro Pro Pro Ser (SEQ ID NO: 14) , or, exendin-4 (synthetic exendin-4 (exenatide)): His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe lie Glu Trp Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser where the C-term serine is amidated (SEQ ID NO: 15), or insulinotropic fragments of exendin-4: Exendin-4 (1-31) His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Wing Val Arg Leu Phe lie Glu Trp Leu Lys Asn Gly Gly Pro (SEQ ID NO: 16); y.sup.31 Exendin-4 (1-31) His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Wing Val Arg Leu Phe lie Glu Trp Leu Lys Asn Gly Gly Tyr (SEQ ID NO: 17), or inhibitory fragments of exendin-4: Exendin-4 (9-39) Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe lie Glu Trp Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser (SEQ ID NO: 18), or other preferred exendin agonists: exendin-4 (1-30) His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe lie Glu Trp Leu Lys Asn Gly Gly (SEQ ID NO: 19), exendin-4 (1-30) amide His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe lie Glu Trp Leu Lys Asn Gly Gly-NH.sub.2 (SEQ ID NO : 20), exendin-4 (1-28) amide His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe lie Glu Trp Leu Lys Asn-NH.sub.2 (SEQ ID NO: 21), .sup.14 Leu, .sup.25 Phe exendin-4 amide His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu Wing Val Arg Leu Phe lie Glu Phe Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser-NH.sub.2 (SEQ ID NO: 22), .sup.14 Leu, .sup.25 Phe exendin-4 (1-28) amide His Gly Glu Gly Thr Phe Thr Being Asp Leu Being Lys Gln Leu Glu Glu Glu Wing Val Arg Leu Phe lie Glu Phe Leu Lys Asn-NH.sub.2 (SEQ ID NO: 23), and .sup.14 Leu, .sup.22 Wing, .sup .25 Phe exendin-4 (1-28) amide His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Ala lie Glu Phe Leu Lys Asn-NH.sub.2 (SEQ ID NO : 24). or sequences incorporated by reference that have been described in U.S. Patent No. 5,424,286; U.S. Patent No. 6,506,724; U.S. Patent No. 6,528,486; U.S. Patent No. 6,593,295; U.S. Patent No. 6,872,700; U.S. Patent No. 6,902,744; U.S. Patent No. 6,924,264; and U.S. Patent No. 6,956,026. or other compounds which bind effectively to the receptor in which exendin exerts its actions which are beneficial in the treatment of diabetes and conditions that could be benefited by reducing plasma glucose or slowing and / or decreasing gastric emptying or inhibiting absorption of food. The use of exendin-3 and exendin-4 as insulinotrophic agents for the treatment of diabetes mellitus and the prevention of hyperglycemia is described in U.S. Patent No. 5,424,286. The exendins have also been shown to be used in the mation of triglyceride levels and to treat dyslipidemia. Thus, the invention provides peptides or peptide fragments, synthetically made or purified from natural sources, which include the biological activity of the exendins or fragments thereof, as described by the present specification. In accordance with the present invention, the exendins also include free bases, acid addition salts or metal salts, such as potassium or sodium salts of the peptides, and exendin peptides that have been modified by such processes as amidation, glycosylation, acylation, sulfation, phosphorylation, acetylation , cyclization and other well-known covalent modification methods. Thus, in accordance with the present invention, the peptides described above are incorporated into the formulations suitable for transepithelial delivery, especially intranasal and dermal delivery.

Biological Membranes "Biological membrane" is defined as a membrane material present within a living organism, preferably an animal, more preferably a human, that separates one area of the organism from another. In many cases, the biological membrane separates the organism with its environment or external surroundings. Non-limiting examples of biological membrane include, mucous membranes and skin in the human being.

Agents that intensify the epithelial supply "Agents that intensify the epithelial supply", they are defined as chemicals and other excipients which, when added to a formulation comprising water, salts and / or common buffers and GRP (the control formulation), pre a formulation that pres a significant increase in the transport of GRP through a biological membrane as measured by the maximum concentration of blood, serum or brain spinal fluid (Cmax) or by the area under the AUC curve, in a plot of concentration against time. The epithelial biological membranes may include the nasal, oral, intestinal, buccal, bronchopulmonary, vaginal, rectal and dermal mucosal surfaces. Agents that intensify transepithelial delivery are sometimes called carriers.

Agents that intensify the mucosal supply "Agents that intensify the mucosal supply" are defined as chemicals and other excipients that, when added to a formulation comprising water, salts and / or common buffers and GRP (the control formulation), pre a formulation that pres a significant increase in the transport of GRP through a mucosa, as measured by the maximum concentration of blood, serum or brain spinal fluid (Cmax) or by the area under the curve, AUC, in a trace of concentration against time. A mucosa includes, mucosal surfaces nasal, oral, intestinal, buccal, bronchopulmonary, vaginal and rectal, and in fact, includes all the membranes that secrete mucus that line all the body cavities or passages that communicate with the outside. Agents that intensify the mucosal supply are sometimes called carriers.

Endotoxin-free formulation "Endotoxin-free formulation" means a formulation which contains a GRP and one or more agents that enhance the epithelial supply that is substantially free of endotoxins and / or related pyrogenic substances. Endotoxins include toxins that are confined within a microorganism and are released only when the microorganisms are broken or eliminated. The pyrogenic substances include, thermostable substances, which induce fever (glycoproteins) of the outer membrane of bacteria and other microorganisms. Both substances can cause fever, hypotension and stroke if administered to humans. Producing formulations that are free of endotoxins may require special equipment, skilled artisans, and can be significantly more expensive than making formulations that are not free of endotoxins. Because the intravenous administration of GLP or amylin, simultaneously with infusion of endotoxins in rodents, has been shown to prevent hypotension and even death associated with the administration of endotoxin alone (U.S. Patent No. 4,839,343), producing endotoxin-free formulations of these therapeutic agents, could not be expected to be necessary for non-parenteral (non-injected) administration.

Uninfused administration "Uninfused administration" means any method of delivery that does not involve an injection directly into an artery or vein, a method which forces or triggers (typically a fluid), into something and especially into a part of the body by means of a needle, syringe or other invasive methods. Uninfused administration includes subcutaneous injection, intramuscular injection, intraperitoneal injection and methods without injection of delivery to a biological membrane.

Methods and Compositions of Provision Improved methods and compositions for epithelial administration of GRP to mammalian subjects, optimizes GRP dosage programs. The present invention provides epithelial delivery of GRP formulated with one or more agents that enhance epithelial delivery, wherein the dosage release of GRP is substantially normalized and / or sustained by an effective delivery period of GRP release intervals from approximately 0.1 to 2.0 hours; 0.4 to 1.5 hours; 0.7 up to 1.5 hours; or 0.8 to 1.0 hours; after epithelial administration. The sustained release of GRP achieved can be facilitated by repeated administration of exogenous GRP using methods and compositions of the present invention.

Compositions and Methods of Sustained Release Improved compositions and methods for epithelial administration of GRP to mammalian subjects optimizes GRP dosing schedules. The present invention provides improved (eg, nasal) epithelial delivery of a formulation comprising GRP in combination with one or more agents that enhance epithelial delivery and an agent or agents that enhance sustained release. Agents that enhance the epithelial supply of the present invention, provide an effective increase in delivery, for example, an increase in the maximum plasma concentration (Cmax) to improve the therapeutic activity of epithelially administered GRP. A second factor that affects the therapeutic activity of GRP in the blood plasma and the CNS is the residence time (RT). Agents that intensify sustained release, in combination with agents that intensify intranasal delivery, increase Cmax and increase the residence time (RT) of GRP. The polymeric delivery vehicles and other agents and methods of the present invention that provide formulations that enhance sustained release, for example, are described herein as polyethylene glycol (PEG). The present invention provides an improved method of delivery of GRP and dosage form for the treatment of symptoms related to obesity, diabetes, hyperglycemia, metabolic syndrome, coronary syndrome, colon cancer, exendin cancer, breast cancer, myocardial infarction, promotion of neurogenesis, suppression of appetite, promotion of weight loss, and reduction of food absorption in mammalian subjects . Within the epithelial delivery formulations and methods of the invention, GRP is often combined or coordinately administered with a carrier or vehicle suitable for epithelial delivery. As used herein, the term "carrier" means pharmaceutically acceptable solid or liquid filler, diluent or encapsulant. A liquid carrier containing water may contain pharmaceutically acceptable additives such as acidifying agents, alkalizing agents, antimicrobial preservatives, antioxidants, buffering agents, chelating agents, complexing agents, solubilizing agents, humectants, solvents, agents that they increase the viscosity and / or suspension, tonicity agents, wetting agents or other biocompatible materials. A tabulation of ingredients listed by the above categories can be found in U.S. Pharmacopeia National Formulary, 1857-1859, 1990. Some examples of the materials which can serve as pharmaceutically acceptable carriers are sugars, such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; jelly; talcum powder; excipients such as cocoa butter and wax suppositories; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution, ethyl alcohol and phosphate buffer solutions, as well as other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweeteners, flavoring and perfuming agents, preservatives and antioxidants, may also be present in the compositions, in accordance with the wishes of the formulator. Examples of pharmaceutically acceptable antioxidants include water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite and the like; water-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol and the like; and metal chelating agents such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like. The amount of active ingredient that can be combined with the carrier materials to produce a dosage form will vary depending on the particular mode of administration. Within the epithelial delivery compositions and methods of the invention, various agents that enhance delivery are employed, which improve the delivery of GRP in or through a cell layer. In this sense, the supply of GRP through the epithelium can occur "intracellularly" or "paracellularly". The magnitude in which these trajectories contribute to the total flow and The bioavailability of GRP depends on the environment of the biological membrane, the psycho-chemical properties of the active agent, and the properties of the epithelium. Paracellular transport involves only passive diffusion, while transcellular transport can occur through passive, facilitated or active processes. In general, polar solutes, passively transported, hydrophilic, diffuse through the paracellular route, while more lipophilic solutes use the transcellular route. Absorption and bioavailability (eg, as reflected by a permeability coefficient or physiological assay), for various solutes, passively and actively absorbed, can be easily evaluated in terms of both paracellular and transcellular delivery components, by any GRP selected within of the invention. For passively absorbed drugs, the relative contribution of paracellular and intracellular trajectories to drug transport depends on the partition coefficient, pKa, molecular radioactivity and drug loading, the pH of the luminal environment in which the drug is delivered, and the area of the absorption surface. The paracellular route represents a relatively small fraction of accessible surface area of the nasal mucosal epithelium. In general terms, it has been reported that cell membranes occupy a mucosal surface area that is a thousand times larger than the area occupied by spaces paracellular Thus, the smallest accessible area, and the size and charge-based discrimination against macromolecular permeation could suggest that the paracellular route could be a generally less favorable route than the transcellular delivery for drug transport. Surprisingly, the methods and compositions of the invention provide significantly improved transport of biotherapeutics in and through the mucosal epithelium via the paracellular route. Therefore, the methods and compositions of the invention successfully direct both paracellular and transcellular routes, alternatively or within a single method or composition. As used herein, agents that enhance epithelial delivery include agents which improve release or solubility (eg, from a formulation delivery vehicle), rate of diffusion, capacity and timing of penetration, absorption, residence time, stability , effective half-life, sustained or maximal concentration levels, separation and other desired epithelial delivery characteristics (eg, measurements at the delivery site, or at a selected target site of activity such as bloodstream or central nervous system) of GRP or other biologically active compounds. The intensification of the epithelial supply can thus occur through any of a variety of mechanisms, for example, increasing diffusion, transport, persistence. or stability of GRP, increasing the fluidity of the membrane, modulating the capacity or action of calcium and other ions that regulate intracellular or paracellular permeation, solubilization of membrane components (for example, lipids), alkylation of protein sulfhydryl levels and it does not protein in mucosal tissues, increase the flow of water through the cell layer, modulate the physiology of epithelial attachment, reduce the viscosity of the mucus that overlaps the mucosal epithelium, reduce mucociliary clearance rates and other mechanisms. As used herein, an "effective amount of GRP" contemplates the effective delivery of GRP to a site targeted for drug activity in the subject that may involve a variety of delivery or transfer routes. For example, a given active agent can find its shape through separations between mucosal cells and reach an adjacent vascular wall, while by another route the agent can, either passively or actively, be taken up into mucosal cells to act within the cells or be discharged or transported out of the cells to reach a secondary target site, such as the systemic circulation. The methods and compositions of the invention can promote the translocation of active agents along one or more alternate routes, or they can act directly on the mucosal tissue or proximal vascular tissue to promote the absorption or penetration of the active agents. The promotion of absorption or penetration in this context is not limited to these mechanisms. As used herein, "maximum concentration (Cmax) of GRP in a blood plasma", "concentration of area under the curve (AUC) of GRP in a blood plasma", "time at maximum plasma concentration (tmax) of GRP in a plasma blood ", are pharmacokinetic parameters known to a person skilled in the art. Laursen, et al., Eur. J. Endocrinology 235: 309-315, 1996. The "concentration versus time curve" measures the concentration of GRP in a blood serum of a subject against the time after administration of a GRP dosage to the subject either by routes of intranasal, intramuscular, subcutaneous or other parenteral administration. The "Cmax" is the maximum concentration of GRP in the blood serum of a subject after a single dose of GRP to the subject. "Tmax" is the time to reach the maximum concentration of GRP in a blood serum of a subject after the administration of a single dose of GRP to the subject. As used herein, "concentration of area under the time curve (AUC) of GRP in a blood plasma", is calculated in accordance with the linear trapezoidal rule and with addition of residual areas. A reduction of 23% or an increase of 30% between two dosages could be detected with a probability of 90% (error ß type II = 10%). The "supply rate" or "absorption rate" is estimated by comparing the time (tmax) to reach the maximum concentration (Cmax). Both Cmax and tmax are analyzed using nonparametric methods. Comparisons of the pharmacokinetics of intramuscular, subcutaneous, intravenous and intranasal GRP administrations were performed by analysis of variance (ANOVA). For pair-wise comparisons, a Bonferroni-Holmes sequential procedure is used to evaluate the significance. The dose response relationship between the three nasal doses is estimated by regression analysis. P < 0.05 is considered significant. The results are given as mean values +/- SEM. While the mechanism of absorption promotion may vary with different agents of the invention that enhance the epithelial supply, agents useful in this context will not substantially adversely affect the tissue and will be selected in accordance with the physicochemical characteristics of the particular GRP or other agent of the invention. intensification of supply or asset. In this context, agents that intensify the supply that increase the penetration or permeability of tissues mucosal, will often result in some alteration of the mucosal protective permeability barrier. For such agents that enhance the supply to be of value within the invention, it is generally desired that any significant change in the permeability of the biological membrane be reversible within a time structure appropriate to the desired duration of drug delivery. In addition, there should be no cumulative, substantial toxicity, or any permanent deleterious change induced in the barrier properties of the biological membrane with long term use. Within certain aspects of the invention, the agents that promote absorption for coordinated administration or combinatorial formulation with GRP of the invention, are selected from small hydrophilic molecules, including but not limited to, dimethyl sulfoxide (DMSO); dimethylformamide, ethanol, propylene glycol, and 2-pyrrolidones. Alternatively, long chain amphipathic molecules, for example, deacylmethylsulfoxide, azone, sodium lauryl sulfate, oleic acid and bile salts, can be employed to improve the penetration of the biological membrane of the GRP. In additional aspects, surfactants (e.g., polysorbates) are employed as adjunct compounds, processing agents, or formulation additives to improve the transepithelial delivery of the GRP. Agents such as DMSO, "polyethylene glycol, and ethanol, may if present in sufficiently high concentrations in the delivery environment (for example, by pre-administration or incorporation into a therapeutic formulation), enter the aqueous phase of the mucosa and alter its solubilizing properties, thereby improving the division of the GRP of the vehicle into the biological membrane. Agents that enhance the additional epithelial supply that are employed within the coordinated administration and processing methods and combinatorial formulations of the invention include, but are not limited to, mixed mycelia, enamines; nitric oxide donors (e.g., S-nitroso-N-acetyl-DL-penicillamine, NOR1, NOR4, which are preferably co-administered with a NO scavenger such as carboxy-PITO or diclofenac sodium); sodium salicylate; glycerol esters of acetoacetic acid (for example, glyceryl-1,3-diacetoacetate or 1,2-isopropylidene glycerin-3-acetoacetate); and other agents that promote the release-diffusion or intra- or trans-epithelial penetration that are physiologically compatible for epithelial delivery. Other agents that promote absorption are selected from a variety of carriers, bases and excipients that improve the epithelial supply, stability, activity or trans-epithelial penetration of the GRP. These include, among other things, cyclodextrins and β-cyclodextrin derivatives (eg, 2-hydroxypropyl-p- cyclodextrin and heptakis (2,6-di-O-met il-β-cyclodextrin). These compounds, optionally conjugated with one or more of the active ingredients and in addition, optionally formulated in an oleaginous base, improve the bioavailability in the epithelial formulations of the invention. Further enhancing absorption agents adapted for epithelial delivery include medium chain fatty acids, including mono and diglycerides (e.g., extracts of coconut oil sodium caprate, Capmul), and triglycerides (e.g., amylodextrin, Estaram 299). , Migliol 810). The epithelial and prophylactic therapeutic compositions of the present invention can be supplemented with any agent that promotes penetration, which facilitates the absorption, diffusion or penetration of GRP through the biological membrane barriers. The penetration promoter can be any promoter that is pharmaceutically acceptable. Thus, in more detailed aspects of the compositions of the invention, it is provided that they incorporate one or more agents that promote penetration, selected from sodium salicylate and salicylic acid derivatives (acetyl salicylate, choline salicylate, salicylamide, etc.). ); amino acids and salts thereof (e.g., monoaminocarboxylic acids such as glycine, alanine, phenylalanine, proline, hydroxyproline, etc.), hydroxy amino acids such as serine; acidic such as aspartic acid, glutamic acid, etc .; and basic amino acids such as lysine etc. -including its salts of alkali metals or alkaline earth metals); and N-acetylamino acids (N-acetylalanine, N-acetylphenylalanine, N-acetylserine, N-acetylglycine, N-acetyllysine, N-acetylglutamic acid, N-acetylproline, N-acetylhydroxyproline, etc.) and their salts (alkali metal salts) and alkaline earth metals). Also provided as agents that promote penetration with the methods and compositions of the invention, are substances which are generally used as emulsifiers (e.g., sodium oleyl phosphate, sodium lauryl phosphate, sodium lauryl sulfate, sodium myristyl sulfate, polyoxyethylene alkyl ethers, polyoxyethylene alkyl esters, etc.), caproic acid, lactic acid, malic acid and citric acid and alkali metal salts thereof, pyrrilidinecarboxylic acids, alkylpyrrolidonecarboxylic acid esters, N-alkyl pyrrolidones, acyl proline esters and the like. Within several aspects of the invention, improved nasal mucosal delivery formulations and methods are provided, which allow the delivery of GRP and other therapeutic agents within the invention through biological membrane barriers between administration and selected target sites. Certain formulations are specifically adapted for a cell, tissue or organ selected target, or even a particular disease state. In certain aspects, formulations and methods provided by endo or efficient, selective transcytosis of GRP specifically routed together with a defined intracellular or intercellular path. Typically, GRP is efficiently charged at effective levels of concentration in a carrier or other delivery vehicle, and is delivered and maintained in a stabilized form, for example, in the nasal mucosa and / or during passage through intracellular compartments and membranes to a remote target site of drug action (e.g., the blood stream or a defined tissue, organ or extracellular compartment). GRP can be provided in a delivery vehicle or otherwise modified (e.g., in the form of a prodrug), wherein the release or activation of GRP is activated by a physiological stimulus (e.g., pH change, enzymes lysosomal, etc.). Often, GRP is pharmacologically inactive until it reaches its target site by activity. In many cases, the GRP and other formulation components are non-toxic and non-immunogenic. In this context, carriers and other formulation components are generally selected for their ability to be rapidly degraded and excreted under physiological conditions. At the same time, the formulations are chemically and physically stable in dosage form for effective storage.

Peptides and Protein and Mimetic Analogs Included within the definition of biologically active peptides and proteins for use within the invention, are synthetic, therapeutically or prophylactically active peptides (comprised of two or more covalently linked amino acids), proteins, peptides or fragments of proteins, peptides or analogs of proteins and chemically modified derivatives or salts of peptides or active proteins. A wide variety of useful analogs and mimetics of GRP are contemplated for use within the invention, and can be produced and tested for their biological activity in accordance with known methods. Often, the GRP peptides or proteins or other biologically active peptides or proteins for use within the invention, are muteins that are readily obtainable by substitution, addition or partial deletion of amino acids within a peptide or protein sequence that originates naturally or native (for example, mutant that originates naturally, of native type, or allelic variant). Additionally, biologically active fragments of peptides or proteins are included. Such mutant derivatives and fragments substantially retain the desired biological activity of the native peptide or proteins. If of peptides or proteins having carbohydrate chains, biologically active variants marked by alterations in these carbohydrate species, are also included within the invention. As used herein, "conservative amino acid substitution" refers to the general exchange capacity 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 have aliphatic hydroxyl side chains is serine and threonine; a group of amino acids that have amide-containing side chains, is asparagine and glutamine; a group of amino acids that have aromatic side chains is phenylalanine, tyrosine and tryptophan; a group of amino acids that have basic side chains is lysine, arginine, histidine; and a group of amino acids that have side chains containing 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. In the same way, 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. Additionally, the substitution of a basic waste such as lysine, arginine or histidine by another or the substitution of an acidic residue such as aspartic acid or glutamic acid for another, is also contemplated. Exemplary conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine and asparagine-glutamine. By aligning an analogous peptide or protein optimally with a corresponding native peptide or protein, and using appropriate assays, for example, adhesion protein or receptor binding assay, to determine a selected biological activity, protein and peptide analogues can be readily identified. operable for use within the methods and compositions of the invention. Analogs of operable proteins and peptides are typically specifically immunoreactive with antibodies raised to the corresponding native peptide or protein. A method for stabilizing solid protein formulations of the invention is to increase the physical stability of lyophilized, for example, purified protein. This will inhibit aggregation via hydrophobic interactions, as well as via covalent pathways that can increase as the proteins unfold. Stabilizing formulations in this context often include polymer-based formulations, for example, a biodegradable hydrogel formulation / delivery system. As noted above, the critical role of water in Structure of protein, function and stability is well known. Typically, proteins are relatively stable in the solid state with volume of water removed. However, solid therapeutic protein formulations may become hydrated after storage at high humidity or during delivery of a sustained release composition or device. The stability of proteins in general falls with the increase of hydration. Water can also play a significant role in the aggregation of solid protein, for example, by increasing protein flexibility resulting in increased accessibility of reactive groups, providing a mobile phase for reagents, and serving as a reagent in various deleterious processes such as beta-elimination and hydrolysis. Protein preparations containing between 6% and 28% water are the most unstable. Below this level, the mobility of water links and internal portions of protein are low. Above this level, the mobility of water and portions of protein reach those of complete hydration. Up to a point, the increased susceptibility to solid phase aggregation with increased hydration has been observed in several systems. However, at higher water content, less aggregation is observed due to the dilution effect. In accordance with these principles, a method effective to stabilize peptides and proteins against solid state aggregation for mucosal delivery, is to control the water content in a solid formulation and maintain the water activity in the formulation at optimal levels. This level depends on the nature of the protein, but in general, proteins maintained below their "monolayer" water coverage will exhibit superior solid state stability. A variety of additives, diluents, bases and delivery vehicles are provided within the invention, which effectively control the water content to improve the stability of the protein. These reagents and effective carrier materials as anti-aggregation agents in this regard include, for example, polymers of various functionalities, such as polyethylene glycol, dextran, dimethylaminoethyl dextran and carboxymethylcellulose, which significantly increase stability and reduce solid phase aggregation of peptides and proteins mixed together with them or bound to them. In some cases, the activity or physical stability of the proteins can also be improved by various additives to aqueous solutions of the peptide or protein drugs. For example, additives such as polyols (including sugars), amino acids, proteins such as collagen and gelatin and various salts, can be used.

Certain additives, in particular sugars and other polyols, also impart significant physical stability to lyophilized, for example, dried 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 peptide or protein aggregation during solid phase incubation under various conditions. These additives can also improve the stability of solid proteins embedded with polymer matrices. . Still further additives, eg, sucrose, stabilize proteins against aggregation in the 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 compositions within the invention. For example, polypeptide microparticles can be prepared by simply lyophilizing or by spray drying a solution containing several stabilizing additives described above. The sustained release of non-aggregated peptides and proteins can thereby be obtained over a prolonged period of time. Various additional preparation components and methods, as well as specific formulation additives, are provided herein, which provide formulations for epithelial delivery of peptides and prone aggregation proteins, wherein the peptide or protein is stabilized in a substantially pure, non-aggregated form using a solubilizing agent. A range of components and additives are contemplated for use within these methods and formulations. Exemplary of these solubilizing agents are cyclodextrins (CDs), which selectively bind hydrophobic side chains of polypeptides. These CDs have been found to bind to hydrophobic patches of proteins in a manner that significantly inhibits aggregation. This inhibition is selective with respect to both the CD and the protein involved. Such selective inhibition of protein aggregation provides additional advantages within the intranasal delivery methods and compositions of the invention. Additional agents for use in this context include CD dimers, trimers and tetramers with variant geometries controlled by linkers that specifically block the aggregation of peptides and proteins. Still solubilizing agents and methods for incorporation within the invention involve the use of peptides and peptide mimetics to selectively block protein-protein interactions. In one aspect, the specific binding of hydrophobic side chains reported for CD multimers, extends to proteins via the use of peptides and peptide mimetics that similarly block protein aggregation. A wide range of suitable methods and anti-aggregation agents are available for incorporation into the compositions and processes of the invention.

Load Modification and pH Control Agents and Methods To improve the transport characteristics of biologically active agents (which include GRP, other peptides and active proteins, and small molecular and macromolecular drugs) for improved delivery through biological membrane barriers hydrophobic, the invention also provides techniques and reagents for charge modification of selected biologically active agents or agents that enhance the delivery described herein. In this sense, the relative permeabilities of macromolecules are in general, related to their division coefficients. The degree of ionization of molecules, which is dependent on the pKa of the molecule and the pH at the surface of the biological membrane, also affects the permeability of the molecules. The permeation and division of biologically active agents, including GRP and analogs of the invention, for epithelial delivery, can be facilitated by load alteration or charge dispersion of the active agent or permeabilizing agent, which is achieved, for example, by alteration of charged functional groups, by modifying the pH of the delivery vehicle or solution in which the active agent is delivered, or by coordinated administration of a reagent that alters the pH or charges with the active agent. Consistent with these general teachings, the epithelial delivery of charged macromolecular species, including GRP and other peptides and biologically active proteins, within the methods and compositions of the invention, is substantially enhanced when the active agent is delivered to the epithelial surface in a state of electric charge substantially not ionized or neutral. Certain GRPs and other components of biologically active proteins and peptides of epithelial formulation for use within the invention will be load modi? Ed to provide an increase in the positive charge density of the peptide or protein. These modifications also extend to cationization of peptide and protein conjugates, carriers and other delivery forms described herein. The Cationization offers a convenient means to alter the biodistribution and transport properties of proteins and macromolecules within the invention. Cationization is undertaken in a manner that substantially preserves the biological activity of the active agent and limits potentially adverse side effects, including tissue damage and toxicity. A "buffer" is generally used to maintain the pH of a solution at a value that is close to constant. A buffer maintains the pH of a solution, or even when smaller amounts of strong acid or strong base are added to the solution, preventing or neutralizing load changes at concentrations of hydrogen and hydroxide ions. A buffer in general, consists of a weak acid and its appropriate salt (or a weak base and its appropriate salt). The salt appropriate for a weak acid contains the same negative ions as present in the weak acid (see, Lagowski, Macmillan Encyclopedia of Chemistry, Vol. 1, Simon &Schuster, New York, 1997, pp. 273-4). The Henderson-Hasselbach equation, pH = pKa + loglO [A -] / [HA], is used to describe a buffer, and is based on the standard equation for weak acid dissociation, HA - H + + TO- . Examples of commonly used buffer sources include the following: glutamate, acetate, citrate, glycine, histidine, arginine, lysine, methionine, lactate, formate, glycolate, tartrate and mixtures thereof. The "buffering capacity" means the amount of acid or base that can be added to a buffer solution before a significant pH change occurs. If the pH falls within the range of pK-1 and pK + 1 of the weak acid, the buffering capacity is appreciable, but outside this range, it falls to such a magnitude that it is of little value. Therefore, a given system only has a useful buffer in a range of one pH unit on either side of the pK of the weak acid (or weak base) (see Dawson, Data for Biochemical Research, Third Edition, Oxford Science Publications, 1986, p.419). In general, suitable concentrations are chosen so that the pH of the solution is close to the pKa of the weak acid (or weak base) (see Lide, CRC Handbook of Chemistry and Physics, 86th Edition, Taylor &Francis Group, 2005-2006 , pp. 2-41). In addition, solutions of strong acids and bases are not normally classified as buffer solutions, and do not exhibit buffering capacity between the values of pH 2.4 to 11.6.

Inhibitory Agents of Degradative Enzyme and Methods Another excipient that can be included in a transepithelial preparation is a degrading enzyme inhibitor. Polymer-enzyme inhibitor complexes Exemplary mucoadhesives that are employed within epithelial delivery formulations and methods of the invention include, but are not limited to: Carboxymethylcellulose-pepstatin (with anti-pepsin activity); poly (acrylic acid) -Bigman-Birk inhibitor (anti-chymotrypsin); poly (acrylic acid) -chimiostatin (anti-chymotrypsin); poly (acrylic acid) -elastatinal (anti-elastase); Carboxymethylcellulose-elastatinal (anti-elastase); Polycarbophil-elastatinal (anti-elastase); Chitosan-antipain (anti-trypsin); poly (acrylic acid) -bacitrin (anti-aminopeptidase N); Chitosan-EDTA (anti-aminopeptidase N, anti-carboxypeptidase A); Chitosan-EDTA-antipain (antitrypsin, anti-chymotrypsin, anti-elastase). As described in more detail below, certain embodiments of the invention will optionally incorporate a new chitosan derivative or chemically modified form of chitosan. A new derivative for use within the invention is denoted as polymer ß- [1- > 4] -2-guanidino-2-deoxy-D-glucose (poly-GuD). Any inhibitor that inhibits the activity of an enzyme to protect biologically active agents can be usefully employed in the compositions and methods of the invention. Enzyme inhibitors useful for the protection of biologically active proteins and peptides include, for example, inhibitors of dipeptidyl aminopeptidase (DPP) IV, soybean trypsin inhibitor, exendin trypsin inhibitor, chymotrypsin inhibitor and trypsin inhibitor and chymotrypsin isolated from potato tubers (Solanum tuberosum L). A combination or mixtures of inhibitors can be employed. Additional inhibitors of proteolytic enzymes for use within the invention include, ovomucoid enzyme, gabaxate mesylate, alpha 1-antitrypsin, aprotinin, amastatin, bestatin, puromycin, bacitracin, leupepsin, alpha2-macroglobulin, pepstatin and white egg trypsin inhibitor or soy. These and other inhibitors can be used alone or in combination. The inhibitors can be incorporated in or attached to a carrier, for example, a hydrophilic polymer, coated on the surface of the dosage form which is in contact with the nasal mucosa, or incorporated in the superficial surface phase, in combination with the biologically active agent in a separately administered (e.g., pre-administered) formulation (e.g., oral lozenges). The amount of the inhibitor, for example, of proteolytic enzyme inhibitor that is optionally incorporated in the compositions of the invention, will vary depending on (a) the properties of the specific inhibitor, (b) the number of functional groups present in the molecule (which it can be reacted to introduce ethylenic unsaturation necessary for copolymerization with hydrogel-forming monomers), and (c) the number of groups lectin, such as glycosides, which are present in the inhibitory molecule. They may also depend on the specific therapeutic agent that is proposed to be administered. Generally speaking, a useful amount of an enzyme inhibitor is from about 0.1 mg / ml to about 50 mg / ml, often, from about 0.2 mg / ml to about 25 mg / ml, and more commonly, from about 0.5 mg / ml up to 5 mg / ml of the formulation (ie, a separate protease inhibitor formulation or formulation combined with the inhibitor and biologically active agent). In the case of trypsin inhibition, suitable inhibitors can be selected from for example, aprotinin, BBI, soybean trypsin inhibitor, chicken ovomucoid, chicken ovoinhibitor, human exendin trypsin inhibitor, camostat mesylate, flavonoid inhibitors, antipain, leupeptin , p-aminobenzamidine, AEBSF, TLCK (tosylisin chloromethyl ketone), APMSF, DFP, PMSF and poly (acrylate) derivatives. In the case of inhibition of chymotrypsin, suitable inhibitors can be selected from for example, aprotinin, BBI, soybean trypsin inhibitor, chemostatin, benzyloxycarbonyl-Pro-Phe-CHO, FK-448, chicken ovoinhibitor, biphenylboronic acid complexes of sugar, DFP, PMSF, β-phenylpropionate, and poly (acrylate) derivatives. In the case of inhibition of elastase, suitable inhibitors can be selected from, for example, elastin, methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone (MPCMK), BBI, soybean trypsin inhibitor, chicken ovoinhibitor, DFP and PMSF. Additional enzymatic inhibitors for use within the invention are selected from a wide range of non-protein inhibitors that vary in their degree of potency and toxicity. As described in more detail below, the immobilization of these adjunct agents to matrices or other delivery vehicles, or development of chemically modified analogs, can be easily implemented to reduce or even eliminate the toxic effects, when they are encountered. Among this broad group of candidate enzyme inhibitors for use within the invention, are organophosphorus inhibitors, such as diisopropyl fluorophosphate (DFP) and phenylmethylsulfonyl fluoride (PMSF), which are potent, irreversible serine protease inhibitors (eg, trypsin and chymotrypsin). The additional inhibition of acetylcholinesterase by these compounds, makes them highly toxic in uncontrolled supply settings. Another candidate inhibitor, 4- (2-aminoethyl) -benzenesul fonyl fluoride (AEBSF), has comparable inhibitory activity with DFP and PMSF, but is markedly less toxic. The (4-aminophenyl) -methanesulfonyl fluoride hydrochloride (APMSF) is another potent trypsin inhibitor, but it is toxic in uncontrolled settings. Contrary to these inhibitors, 4- (4-isopropylpiperadinocarbonyl) phenyl (1-4-isopropylpiperadinocarbonyl) phenyl methanesulfonate 1,2,3-tetrahydro-l-naphtanoate (FK-448) is a low toxic substance, which represents a specific and potent inhibitor of chymotrypsin. Additional representatives of this non-protein group of inhibitory candidates, and which also exhibit low toxic risks, are camostat mesylate (N, N'-dimethyl carbamoylmethyl-p- (p'-guanidino-benzoyloxy) phenylacetate methanesulfonate). Still another type of enzyme inhibiting agent for use within the methods and compositions of the invention are amino acids and modified amino acids that interfere with the enzymatic degradation of specific therapeutic compounds. For use in this context, amino acids and modified amino acids are substantially non-toxic and can be produced at low cost. However, due to their low molecular size and good solubility, they are easily diluted and absorbed in mucosal environments. However, under appropriate conditions, amino acids can act as competitive, reversible inhibitors of protease enzymes. Certain modified amino acids may exhibit a much stronger inhibitory activity. A modified amino acid desired in this context is known as a "transition state" inhibitor. The inhibitory activity The strength of these compounds is based on their structural similarity to a substrate in their transition state geometry, while they are generally selected for having a much higher affinity for the active site of an enzyme than the substrate itself. The transition state inhibitors are competitive, reversible inhibitors. Examples of this type of inhibitor are derivatives of amineboronic acid, such as boro-leucine, boron-valine and boron-alanine. The boron atom in these derivatives can form a tetrahedral boronate ion that resembles the transition state of peptides during their hydrolysis by aminopeptidases. These amino acid derivatives are reversible and potent inhibitors of aminopeptidases and it is reported that boro-leucine is more than 100 times more effective in enzyme inhibition than bestatin and more than 1000 times more effective than puromycin. Another modified amino acid for which a strong protease inhibitory activity has been reported is N-acetylcysteine, which inhibits the aminopeptidase N enzymatic activity. This adjunct agent exhibits mucolytic properties that can be employed within the methods and compositions of the invention , to reduce the effects of the mucus diffusion barrier. Still other enzymatic inhibitors useful for use within the methods of coordinated administration and combinatorial formulations of the invention, can be selected peptides and modified peptide enzyme inhibitors. An important representative of this class of inhibitors is the cyclic dodecapeptide, bacitracin, obtained from Bacillus licheniformis. In addition to these types of peptides, certain peptides and tripeptides exhibit weak, non-specific inhibitory activity towards some proteases. By analogy with amino acids, their inhibitory activity can be improved by chemical modifications. For example, dipeptide analogues of phosphyric acid are also "transition-state" inhibitors, with strong inhibitory activity towards aminopeptidases. They have reportedly been used to stabilize leucine enkephalin nasally administered. Another example of a transition-state analogue is the modified pentapeptide pepstatin, which is a very potent inhibitor of pepsin. Structural analysis of pepstatin, testing the inhibitory activity of several synthetic analogues, demonstrates the Main function-structure molecule responsible for the inhibitory activity. Another special type of modified peptide includes inhibitors with an aldehyde function terminally localized in its structure. For example, the benzyloxycarbonyl-Pro-Phe-CHO sequence, which completely covers the known primary and secondary specificity requirements of chymotrypsin, has been found to be an inhibitor. powerful reversible of this proteinase target. Chemical structures of additional inhibitors with a terminally localized aldehyde function, eg, antipain, leupeptin, chemostatin and elastatinal, are also known in the art, as are the structures of other known reversible, modified peptide inhibitors, such as phosphoramidon, betamine , puromycin and amastatin. Due to their comparably high molecular mass, polypeptide protease inhibitors are more receptive than smaller compounds to concentrated delivery in drug carrying matrices. Additional agents for protease inhibition within the formulations and methods of the invention involve the use of complexing agents. These agents mediate enzymatic inhibition by avoiding the intranasal environment (or preparative or therapeutic composition) of divalent cations, which are co-factors for many proteases. For example, agents that form EDTA and DTPA complexes, as adjunct agents combinatorially formulated or coordinately administered, in adequate concentration, will be sufficient to inhibit the proteases selected to thereby improve the intranasal delivery of biologically active agents according to the invention. Additional representatives of this class of inhibitory agents are EGTA, 1, 10-phenanthroline and hydroxyquinoline. In addition, because of their propensity to chelate divalent cations, these and other complexing agents are employed within the invention as direct, absorption-promoting agents. As noted in detail elsewhere herein, it is also contemplated to use various polymers, particularly mucoadhesive polymers, as agents that inhibit the enzyme within the coordinated administration, methods and multi-processing and / or combinatorial formulation compositions of the invention. For example, poly (acrylate) derivatives, such as poly (acrylic acid) and polycarbophil, can affect the activity of several proteases, including trypsin, chymotrypsin. The inhibitory effect of these polymers can also be based on the formation of divalent cation complexes such as Ca2 + and Zn2 +. It is further contemplated that these polymers can serve as conjugated standards or associates for additional enzymatic inhibitory agents, as described above. For example, a chitosan-EDTA conjugate has been developed and is employed within the invention, which exhibits a strong inhibitory effect towards the enzymatic activity of zinc-dependent proteases. The mucoadhesive properties of polymers after the covalent attachment of other enzyme inhibitors in this context are not expected to be substantially compromised, nor is the general utility of such polymers as a delivery vehicle for biologically active agents within the invention, is expected to be diminished. In contrast, the reduced distance between the delivery vehicle and the mucosal surface provided by the mucoaldehyde mechanism will minimize the presystemic metabolism of the active agent, while the covalently bound enzyme inhibitors will remain concentrated at the drug delivery site, minimizing the effects of undesired dilution of inhibitors, as well as toxic and other collateral effects caused thereby. In this way, the effective amount of a coordinately administered enzyme inhibitor can be reduced to the exclusion of dilution effects. Mucoadhesive polymer-enzyme inhibitor complexes that are employed within the mucosal formulations and methods of the invention include, but are not limited to: Carboxymethylcellulose-pepstatin (with anti-pepsin activity); poly (acrylic acid) -Bigman-Birk inhibitor (anti-chymotrypsin); poly (acrylic acid) -chimiostat ina (anti-chymotrypsin); poly (acrylic acid) -elastatinal (anti-elastase); Carboxymethylcellulose-elastatinal (anti-elastase); Polycarbophil-elastatinal (anti-elastase); Chitosan-antipain (anti-trypsin); poly (acrylic acid) -bacitrin (anti-aminopeptidase N); Chitosan-EDTA (anti-aminopeptidase N, anti-carboxypeptidase A); Chitosan-EDTA-antipain (anti- trypsin, anti-chymotrypsin, anti-elastase).

Mucus and Mucolytic Separating Agents and Methods Effective delivery of biotherapeutic agents via intranasal administration should take into account the reduced drug transport rate through the protective mucous lining of the nasal mucosa, in addition to the loss of drug due to binding to the mucosa. glycoproteins of the mucous layer. Normal mucus is a viscoelastic, gel-like substance that consists of water, electrolytes, mucins, macromolecules and displaced epithelial cells. It serves mainly as a cytoprotective cover and lubricant for the underlying mucosal tissues. The mucus is secreted by randomly distributed secretory cells located in the nasal epithelium and other mucosal epithelia. The structural unit of mucus is mucin. This glycoprotein is mainly responsible for the viscoelastic nature of the mucus, although other macromolecules may also contribute to this property. In mucous membranes of the respiratory tract, such macromolecules can locally produce secretory IgA, IgM, IgE, lysozyme and broncotransferrin, which also play an important role in the host defense mechanism. The coordinated administration methods of the present invention, optionally incorporate agents of separation of mucus or mucolytics, which serve to degrade; thin or transparent mucus of the intranasal mucosal surfaces to facilitate the absorption of biotherapeutic agents intranasally administered. Within these methods, a mucus or mucolytic separation agent is coordinately administered as an adjunct to improve the intranasal delivery of the biologically active agent. Alternatively, an effective amount of mucus or mucolytic removal agent is incorporated as a processing agent within a multiple processing method of the invention, or as an additive within a combinatorial formulation of the invention, to provide an improved formulation which improves the intranasal supply of biotherapeutic compounds by reducing the effects of intranasal mucus barrier. A variety of mucus or mucolytic separation agents are available for incorporation into the methods and compositions of the invention. Based on their mechanisms of action, mucus and mucolytic separation agents can often be classified into the following groups: proteases (e.g., pronase, papain), which unfold the nucleus of the mucin glycoprotein protein; sulfhydryl compounds that divide the disulfide bonds of the mucoprotein; and detergents (for example, Triton X-100, Tween 20), which break non-covalent bonds inside the mucus. Additional compounds in this context include, but are not limited to, bile salts and surfactants, for example, sodium deoxycholate, sodium taurodeoxycholate, sodium glycolate and lysophosphatidylcholine. The effectiveness of bile salts that cause structural breakage of the mucus, is of the order of deoxycholate > taurocholate > glycolate. Other effective agents that reduce the viscosity of mucus or adhesion to improve intranasal delivery according to the methods of the invention include, for example, short chain fatty acids and mucolytic agents that work by chelation, such as N-acyl collagen peptides, acids biliary and saponins (the latter functions in part, chelating Ca2 + and / or Mg2 +, which play an important role in maintaining the structure of the mucous layer). Additional mucolytic agents for use within the methods and compositions of the invention include, N-acetyl-L-cysteine (ACS), a potent mucolytic agent that reduces both the viscosity and adhesion of bronchopulmonary mucus and is reported to modestly increase nasal bioavailability of human growth hormone in anesthetized rats (from 7.5 to 12.2%). These and other agents that separate the mucus or mucolytics are contacted with the nasal mucosa, typically in a range of concentration from about 0.2 to 20 mM, in coordination with the administration of the biologically active agent, to reduce the polar viscosity and / or elasticity of the intranasal mucus. Still other mucus or mucolytic separation agents can be selected from a range of glycosidase enzymes, which are capable of unfolding glycosidic linkages within the mucus glycoprotein. The α-amylase and β-amylases are representative of this class of enzymes, although their mucolytic effect may be limited. On the contrary, the bacterial glycosidases which allow these microorganisms to permeate the mucous layers of their hosts. For combinatorial use with the majority of biologically active agents within the invention, including peptides and therapeutic proteins, non-ionogenic detergents are in general also employed as mucus or mucolytic separation agents. These agents will typically not modify or substantially impair the activity of therapeutic polypeptides.

Agents that Intensify Viscosity Suspension agents or agents that increase viscosity can affect the release rate of a drug from the formulation and absorption of dosage. As a result, viscosity enhancers can be used to modify the permeation of some glucose-regulating peptides. Some examples of materials which can serve as pharmaceutically acceptable viscosity enhancing agents are methylcellulose (MC); hydroxypropylmethylcellulose (HPMC); carboxymethylcellulose (CMC); cellulose; jelly; starch; beta starch; poloxamers; pluronic; Sodium CMC; sorbitol; acacia; povidone; carbopol; polycarbophil; chitosan; chitosan microspheres; alginate microspheres; Chitosan glutamate; amberlite resin; hyaluronan; ethyl cellulose; maltodextriña DE; drum-shaped corn starch (DDWM); degradable starch microspheres (DSM); deoxyglycolate (GDC); hydroxyethylcellulose (HEC); hydroxypropylcellulose (HPC); microcrystalline cellulose (MCC); polymethacrylic acid and polyethylene glycol; sulfobutyl ether B cyclodextrin; biospheres of cross-linked eldexomer starch; sodium taurodihydrofusidate (STDHF); N-trimethyl chitosan chloride (TMC); degraded starch microspheres; amberlite resin; nanoparticles of chitosan; crospovidone dried by dew; Dextran microspheres dried by spray; microcrystalline cellulose spray dried; and cross-linked eldexomer starch microspheres.

Cilostatic Agents and Methods Due to the self-cleaning ability of certain mucosal tissues (eg nasal mucosal tissue), mucociliary clearance is necessary as a protective function (for example, to remove dust, allergens and bacteria), which has been considered in general, that this function should not be substantially impaired by mucosal medications. Mucociliary transport in the respiratory tract is a particularly important defense mechanism against infections. To achieve this anointing, the ciliary blow in the passages of the respiratory and nasal passages moves a layer of mucus next to the mucosa to remove the inhaled particles and microorganisms. Cryostatic agents find use within the methods and compositions of the invention, to increase the residence time of mucosally administered (eg, intranasally) GRPs, analogues and mimetics and other biologically active agents described herein. In particular, the delivery of these agents with the methods and compositions of the invention, is significantly improved in certain aspects by coordinated administration or combinatorial formulation of one or more ciliatatic agents that function to reversibly inhibit the activity of mucosal cells, to provide a reversible, temporary increase in the residence time of the mucosally administered active agents. For use within these aspects of the invention, the ciliatic factors mentioned above, either specific or indirect in their activity, are all candidates for successful use as ciliates in appropriate quantities (depending on the concentration, duration and mode of supply), in such a way that they provide a temporary reduction ( that is, reversible) or cessation of mucociliary clearance to a mucosal site! of administration to improve the supply of GRP, analogs and mimetics, and other biologically active agents described herein, without adverse unacceptable side effects. Several bacterial ciliostatic factors isolated and characterized in the literature can be used within the embodiments of the invention. The ciliostatic factors of the bacterium Pseudomonas aeuroginosa include a phenazine derivative, a pyo compound (2-alkyl-4-hydroxyquinolines), and a rhamnolipid (also known as a hemolysin). The pyo compound produces ciliostasis at concentrations of 50 g / ml and without obvious ultrastructural lesions. The phenazine derivative also inhibits ciliary mobility but causes some membrane disruption albeit at concentrations substantially greater than 400 g / ml. Limited exposure of tracheal explants to the rhamnolipid results in ciliostasis, which is associated with membranes altered ciliary More extensive exposure to rhamnolipids is associated with the removal of dynein branches of axonemes.

Surface Active Agents and Methods Within more detailed aspects of the invention, one or more membrane penetration agents may be employed within an epithelial delivery method or formulation of the invention, to improve the epithelial supply of GRP, analogues and mimetics and other biologically active agents described herein. The agents that enhance penetration of the biological membrane in this context can be selected from: (i) a surfactant, (ii) a bile salt, (iii) an additive of phospholipid, mixed mycelia, liposomes, or carrier, (iv) ) an alcohol, (v) an enamine, (vi) a donor compound NO, (vii) a long chain antipathetic molecule, (viii) a small hydrophobic penetration enhancer; (ix) derivative of salicylic or sodium acid; (x) a glycerol ester of acetoacetic acid (xi) a cyclodextrin or beta-cyclodextrin derivative, (xii) a medium chain fatty acid, (xiii) a chelating agent, (xiv) an amino acid or salt thereof, (xi) xv) an N-acetylamino acid or salt thereof, (xvi) a degrading enzyme to a selected membrane component, (xvii) a fatty acid synthesis inhibitor, (xviii) a cholesterol synthesis inhibitor; or (xiv) any combination of the agents that intensify the penetration of the membrane mentioned in (i) - (xviii)). Certain active surface agents (surfactants), are easily incorporated within the epithelial delivery formulations and methods of the invention as agents that enhance epithelial absorption. These agents, which can be coordinately administered or combinatorially formulated with GRP, analogs and mimetics, and other biologically active agents described herein, can be selected from a broad assembly of known surfactants. The surfactants, which in general fall into three classes: (1) polyoxyethylene nonionic ethers; (2) bile salts such as sodium glycolate (SGC) and deoxycholate (DOC); and (3) fusidic acid derivatives such as sodium taurodihydrofusidate (STDHF). The mechanisms of action of these various classes of surface active agents typically include solubilization of the biologically active agent. For proteins and peptides which often form aggregates, the active properties of the surface of these absorption promoters may allow interactions with proteins, such that smaller units such as surfactant-coated monomers may be more easily maintained in solution. . Examples of other surface active agents are didecanoyl L-a-phosphatidylcholine (DDPC), polysorbate 80 and polysorbate 20. These monomers are presumably more transportable units than aggregates. A second potential mechanism is the protection of peptide or protein from proteolytic degradation by proteases in the mucosal environment. Both the bile salts and some derivatives of fusidic acid reportedly inhibit protein proteolytic degradation by nasal homogenates at concentrations less than or equivalent to those required to improve protein absorption. This inhibition of protease may be especially important for peptides with short biological half-lives.

Degradation of Enzymes and Inhibitors of Fatty Acid and Cholesterol Synthesis In related aspects of the invention, GRP, analogs and mimetics, and other biologically active agents for biological membrane administration, are formulated or coordinated with an agent that intensifies penetration, selected of a degradation enzyme, or a metabolic stimulating agent or inhibitor of the synthesis of selected fatty acids, sterols or other components of the epithelial barrier, US Pat. No. 6,190,894. For example, degradative enzymes such as phospholipase, hyaluronidase, neuraminidase, and chondroquitinase, can be employed to improve mucosal penetration of GRP, analogs and mimetics, and other biologically active agents without causing irreversible damage to the mucosal barrier. In one embodiment, chondrokinase is employed within a method or composition as provided herein, to alter the glycoprotein or glycolipid constituents of the mucosal barrier permeability, thereby, enhancing the mucosal absorption of GRP, analogs and mimetics, and other biologically active agents described herein. With regard to inhibitors of synthesis of mucosal barrier constituents, it is noted that free fatty acids account for 20-25% of epithelial lipids by weight. Two enzymes that limit the proportion in the biosynthesis of free fatty acids are acetyl CoA carboxylase and fatty acid synthetase. Through a series of stages, free fatty acids are metabolized into phospholipids. Thus, inhibitors of free fatty acid synthesis and metabolism for use with the methods and compositions of the invention include, but are not limited to, acetyl-CoA carboxylase inhibitors such as 5-tetradecyloxy-2-furancarboxylic acid (TOFA ); fatty acid synthetase inhibitors; Phospholipase A inhibitors such as gomisin A, 2- (p-amylancinamyl) amino-4-chlorobenzoic acid, bromophenacyl bromide, monoalide, 7,7-dimethyl-5,8-eicosadienoic acid, nicergoline, cepharanthin, nicardipine, quercetin, dibutyryl-cyclic AMP, R-24571, N-oleoylethanolamine, N- (7-nitro-2, 1, 3-benzoxadiazol-4-yl) phosphostidyl serine, cyclosporin A, topical anesthetics, including dibucaine, prenylamine, retinoids , such trans and 13-cis-retinoic acids, -7, trifluoperazine, R-24571 (calmidazolium), l-hexadocyl-3-trifluoroethyl glycero-sn-2-phosphomenthol (MJ33); calcium channel blockers including nicardipine, verapamil, diltiazem, nifedipine, and nimodipine; antimalarials that include quinacrine, mepacrine, chloroquine and hydroxychloroquine; beta blockers that include propanalol and labetalol; calmodulin antagonists; EGTA; timersol; glucocorticosteroids including dexamethasone and prednisolone; and non-steroidal anti-inflammatory agents including indomethacin and naproxen. Esteróles free, mainly cholesterol, account for 20-25% of epithelial lipids by weight. The enzyme that limits the rate in cholesterol biosynthesis is reductase of 3-hydroxy-3-methylglutaryl (HMG) CoA. Cholesterol synthesis inhibitors for use within the methods and compositions of the invention include, but are not limited to, competitive CoA reductase inhibitors (HMG), such as simvastatin, lovastatin, fluindostatin (fluvastatin), pravastatin, mevastatin, as well as also other CoA HMG reductase inhibitors, such as cholesterol oleate, cholesterol and phosphate sulfate, and oxygenated sterols, such as cholesterol 25-OH-- and 26-OH--; squalene synthetase inhibitors; squalene epoxidase inhibitors; DELTA7 or DELTA24 reductase inhibitors, such as 22,25-diazacolesterol, 20, 25-diazacolestenol, AY9944, and triparanol. Each of the inhibitors of fatty acid synthesis or inhibitors of sterol synthesis, can be coordinately administered or combinatorially formulated with one or more GRP, analogs and mimetics, and other biologically active agents described herein, to achieve improved epithelial penetration of the active agents. An effective concentration range for the sterol inhibitor in a therapeutic formulation or adjunct for mucosal delivery is generally from about 0.0001% to about 20% by weight of the total, more typically from about 0.01% to about 5%.

Nitric Oxide Donor Agents and Methods Within other related aspects of the invention, a nitric oxide (NO) donor is selected as an agent that enhances the penetration of the biological membrane to improve the epithelial supply of one or more GRP, analogs and mimetics and other biologically active agents described herein. Several NO donors are known in the technique and are employed in effective concentrations within the methods and formulations of the invention. Non-exemplary donors include, but are not limited to, nitroglycerin, nitroprusside, N0C5 [3- (2-hydroxy-1- (methyl-ethyl) -2-nitrosohydrazino) -1-propanamine], N0C12 [W-ethyl-2- (1-ethyl-hydroxy-2-nitrosohydrazino) -etanamine], SNAP [S-nitroso-N-acetyl-DL-penicillamine], N0R1 and N0R4. Within the methods and compositions of the invention, an effective amount of a NO selected donor is coordinately administered or combinatorially formulated with one or more GRP, analogs and mimetics, and / or other biologically active agents described herein, in or through the epithelium.

Agents for Modulating the Epithelial Binding Structure and / or Physiology The present invention provides pharmaceutical compositions containing one or more GRP, analogs or mimetics, and / or other biologically active agents in combination with epithelial enhancing agents described herein formulated in a pharmaceutical preparation for the epithelial supply. The permeabilizing agent reversibly enhances paracellular mucosal epithelial transport, typically by modulating the epithelial junction structure and / or physiology on a mucosal epithelial surface in the subject.

This effect typically involves inhibition by the perothelializing agent of homotypic or heterotypic binding between the epithelial membrane adhesive proteins of 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. Examples of these are antibodies, antibody fragments or single chain antibodies that bind to the extracellular domains of these proteins. In still further detailed embodiments, the invention provides permeabilizing peptides and peptide analogs and mimetics to enhance paracellular mucosal epithelial transport. Subject peptides and peptide analogs and mimetics typically function within the compositions and methods of the invention by modulating the epithelial binding structure and / or physiology in an animal subject. In certain embodiments, the peptides and peptide analogs and mimetics effectively inhibit homotypic and / or heterotypic binding of an epithelial membrane adhesive protein, selected from a binding adhesion molecule (JAM), occludin, or claudin. One such agent that has been extensively studied is the bacterial toxin of Vibrio cholerae known as the "zonula occludens toxin" (ZOT). This toxin mediates increased intestinal mucosal permeability and causes symptoms of disease that include diarrhea in infected subjects. Fasano, et al, Proc. Nat. Acad. Sci., U.S. A. 8: 5242-5246, 1991. When tested on rabbit ileal mucosa, ZOT increases intestinal permeability by modulating the structure of intercellular tight junctions. More recently, it has been found that ZOT is capable of reversibly opening tight junctions in the intestinal mucosa. It has also been reported that the ZOT is able to reversibly open tight junctions in the nasal mucosa. U.S. Patent No. 5,908,825. Within the methods and compositions of the invention, ZOT, as well as various analogs and mimetics of ZOT that function as agonists and antagonists of ZOT activity, are employed to enhance the nasal delivery of biologically active agents - increasing paracellular absorption in and through of the nasal mucosa. In this context, ZOT typically acts by causing a structural rearrangement of tight junctions marked by altered location of the ZO1 binding protein. Within these aspects of the invention, ZOT is coordinately administered or combinatorially formulated with the biologically active agent in an amount effective to provide significantly improved absorption of the active agent, reversibly increasing the permeability of the nasal mucosa without substantial adverse side effects.

Vasodilating Agents and Methods Still another class of agents that promote absorption that show beneficial utility within the coordinated administration and combinatorial formulation methods and compositions of the invention are vasoactive compounds, more specifically vasodilators. These compounds function within the invention to modulate the structure and physiology of the submucosal vasculature, increasing the rate of transport of GRP, analogs and mimetics and other biologically active agents or through the epithelium and / or specific target tissues or compartments (e.g. the central nervous or systemic circulatory system). Vasodilating agents for use within the invention, typically cause relaxation of the submucosal blood spleens by either a decrease in cytoplasmic calcium, an increase in nitric oxide (NO) or by inhibiting the long chain kinase of myosin. In general, they are divided into 9 classes; calcium antagonists, potassium channel openers, ACE inhibitors, angiotensin-II receptor antagonists, α-adrenergic receptor antagonists, and imidazole; ß? -adrenergic agonists, phosphodiesterase inhibitors, eicosanoids and NO donors. Due to chemical differences, the pharmacokinetic properties of calcium antagonists are similar. The Absorption in the systemic circulation is high, and these agents therefore undergo considerable first-pass metabolism by the liver, resulting in individual variation in pharmacokinetics. Except for the newer drugs of the dihydropyridine type (amlodipine, felodipine, isradipine, nilvadipine, nisoldipine and nitrendipine), the half-life of calcium antagonists is short. Therefore, maintaining an effective drug concentration for many of these may require multiple dose delivery, or controlled release formulations, as described elsewhere herein. Treatment with the potassium channel opener minoxidil may also be limited in manner and level of administration due to potential adverse side effects. ACE inhibitors prevent the conversion of angiotensin-I to angiotensin-II, and are more effective when renin production is increased. Since ACE is identical to kinase-II, which inactivates the potent endogenous vasodilator, bradykinin, inhibition of ACE causes a reduction in bradykinin degradation. ACE inhibitors provide the added benefit of cardioprotective and cardioreparative effects, preventing and reversing cardiac fibrosis and ventricular hypertrophy in animal models. The predominant elimination pathway of most ACE inhibitors is via renal excretion. By therefore, renal deterioration is associated with reduced elimination and a dosage reduction of 25 to 50% is recommended in patients with moderate to severe renal impairment. With respect to NO donors, these compounds are particularly useful within the invention for their additional effects on mucosal permeability. In addition to the donors NOT noted above, NO complexes with nucleophiles called NO / nucleophiles, or NONOates, spontaneously and non-enzymatically release NO when dissolved in aqueous solution at physiological pH. In contrast, nitro vasodilators such as nitroglycerin, require specific enzymatic activity for NO release. NONOates release NO with a defined stoichiometry and at predictable rates varying from < 3 minutes for diethylamine / NO to approximately 20 hours for diethylenetriamine / NO (DETANE). With certain methods and compositions of the invention, a selected vasodilating agent is coordinately administered (eg, systemically or intranasally, simultaneously or in combinatorially effective temporal association) or combinatorially formulated with one or more GRP, analogs and mimetics and other biologically active agents in an effective amount to intensify the absorption of the mucosa of the active agents to achieve a target tissue or compartment in the subject (e.g. liver, hepatic portal vein, tissue or CNS fluid, or blood plasma).

Selective Agents that Intensify Transport and Methods The compositions and delivery methods of the invention, optionally incorporate an agent that enhances selective transport, which facilitates the transport of one or more biologically active agents. These agents that intensify transport can be employed in a combinatorial formulation or administration protocol coordinated with one or more of the GRPs, analogs and mimetics described herein, to coordinately provide one or more additional biologically active agents through the transport barriers of the biological membrane, to improve the epithelial supply of the active agents to reach a target tissue or compartment in the subject (e.g., the mucosal epithelium, liver, tissue or CNS fluid or blood plasma). Alternatively, agents that enhance transport may be employed in a coordinated administration protocol or combinatorial formulation to directly enhance the epithelial delivery of one or more of the GRPs, analogs and mimetics, with or without enhanced delivery of an additional biologically active agent.

Selective intensifying transport agents for use within this aspect of the invention include, but are not limited to, glycosides, sugar-containing molecules, and binding agents such as lectin binding agents, which are known to interact specifically with the transport components of the epithelial barrier. For example, specific bioadhesive ligands, including several plant and bacterial lectins, which bind to the cell surface sugar portions by receptor-mediated interactions, can be employed as conjugated transport mediators or carriers to improve nasal delivery , for example, mucosal of biologically active agents within the invention Certain bioadhesive ligands for use within the invention, will mediate the transmission of biological signals to target epithelial cells that activate the selective absorption of the adhesive ligand by specialized cellular transport processes (endocytosis Transcytosis.) These transport mediators can therefore be employed as a "carrier system" to stimulate or direct the selective uptake of one or more GRP, analogs and mimetics and other biologically active agents in and / or through the mucosal epithelium. These and other agents that intensify the trans selective bearing, significantly intensify the epithelial supply of biopharmaceuticals macromolecules (particularly peptides, proteins, oligonucleotides and polynucleotide vectors) with the invention. Lectins are plant proteins that bind to specific sugars found on the surface of glycoproteins and glycolipids of eukaryotic cells. Concentrated lecithin solutions have a "muco-active" effect and several studies have demonstrated rapid receptor-mediated endocytosis (SMR) of lectins and lectin conjugates (eg, concavalin A conjugated to colloidal gold particles) through mucosal surfaces . Additional studies have reported that the absorption mechanisms for lectins can be used to direct the intestinal drug in vivo. In some of these studies, polystyrene nanoparticles (500 nm) were covalently coupled to tomato lectin and reported yields improve systemic absorption after oral administration to rats. In addition to plant lectins, the invasion and microbial adhesion factors provide a rich source of candidates for use as adhesive / selective transport carriers within the mucosal delivery methods and compositions of the invention. Two components are necessary for the processes of bacterial adhesion, a bacterial "adhesin" (adhesion or colonization factor) and a receptor on the surface of the host cell. The Bacteria that cause mucosal infections need to penetrate the mucosal layer before attaching them to the epithelial surface. This binding is usually mediated by bacterial fibrins or pilus structures, although other components of the cell surface can also take part in the process. Adherent bacteria colonize the mucosal epithelium by multiplication and initiation of a series of biochemical reactions within the target cell through the mechanisms of signal transduction (with or without the help of toxins). Associated with these invasive mechanisms, a wide variety of bioadhesive proteins (eg, invasive, internalin), originally produced by various bacteria and viruses, are known. These allow the extracellular binding of such microorganisms with an impressive selectivity for host species and even specific targets. The signals transmitted by such receptor-ligand interactions activate the transport of living microorganisms, intact in, and eventually through the epithelial cells by endo and transcytotic processes. Such naturally occurring phenomena can be hardened (for example, by forming complexes of biologically active agents such as GRP with an adhesin), in accordance with the teachings herein for improved delivery of biologically active compounds in or through a biological membrane and / or to other sites target designated drug action. Various plant and bacterial toxins that bind to epithelial surfaces in a manner similar to lectin, specifically, are also employed within the methods and compositions of the invention. For example, diphtheria toxin (DT) enters host cells rapidly by EMR. Similarly, subunit B of the heat-labile toxin of E. coli binds to the boundary of the intestinal epithelial cell dome in a highly specific lectin-like manner. The absorption of this toxin and transcytosis to the basolateral side of the enterocytes, has been reported in vivo or in vitro. Other investigators have expressed the domain of the diphtheria toxin transmembrane in E. coli as a maltose binding fusion protein and chemically coupled to high molecular weight poly-L-lysine. The resulting complex is successfully used to mediate the internalization of an in vitro reporter gene. In addition to these examples, Staphylococcus aureus produces a series of proteins (eg, staphylococcal enterotoxin A (SEA), SEB, toxic apoplexy syndrome toxin 1 (TSST-1), which acts both as superantigens and toxins. These proteins have been reported to have facilitated transcytosis dependent on the dose of SEB and TSST-1 in Caco-2 cells.The viral hemagglutinin comprises another type of transport agent to facilitate the mucosal delivery of biologically active agents within the methods and compositions of the invention. The initial stage in many viral infections is the binding of surface proteins (hemagglutinins) to mucosal cells. These binding proteins have been identified by many viruses, including rotavirus, varicella zoster virus, Semliki forest virus, adenovirus, potato leaf roll virus, and reovirus. These and other exemplary hemagglutinins can be used in a combinatorial formulation (eg, a mixture or conjugate formulation) or administration protocol coordinated with one or more of the GRPs, analogs and mimetics described herein, to coordinate intensively the mucosal delivery of one or more additional biologically active agents. Alternatively, the viral hemagglutinins may be employed in a combinatorial formulation or coordinated delivery protocol to directly enhance the mucosal delivery of one or more of the GRPs, analogs and mimetics, with or without enhanced delivery of an additional biologically active agent. A variety of selective transport mediating factors are also available for use within the invention. Mammalian cells have developed a classification of mechanisms to facilitate the internalization of specific substrates and directed these to compartments defined. Collectively, this process of membrane deformation is called "endocytosis" and includes phagocytosis, pinocytosis, receptor-mediated endocytosis (RME mediated by clathrin), and potocitosis (RME mediated by clathrin). The RME is a highly specific cellular biological process by which, as the name implies, several ligands bind to cell surface receptors and are subsequently internalized and trafficked into the cell. In many cells, so endocytosis processes are active that the surface of the entire membrane is internalized and replaced in less than half an hour. Two classes of receptors are proposed based on their orientation in the cell membrane; the amino terminus of Type I receptors is located on the extracellular side of the membrane, while Type II receptors have this same extreme protein in the intracellular environment. Still other embodiments of the invention utilize transferrin as an EMR carrier or stimulant of epithelially delivered biologically active agents. Transferrin, an 80 kDa ion transport glycoprotein, is efficiently taken up in cells by RME. The transferrin receptors are found on the surface of most proliferating cells, in high numbers in erythroblasts and in many kinds of tumors. Transcytosis of transferrin (Tf) and transferrin conjugate, as reports, are they intensified in the presence of Brefeldin? (BFA), a fungal metabolite. In other studies, BFA treatment has been reported to rapidly increase apical endocytosis of both ricin and HRP in MDCK cells. Thus, BFA and other agents that stimulate receptor-mediated transport can be employed with the methods of the invention as combinatorially formulated (e.g., conjugates) and / or coordinately administered agents to improve transport mediated by the receptor. biologically active agents, including, GRP, analogs and mimetics.

Polymeric Delivery Vehicles and Methods Within certain aspects of the invention, GRP, analogs and mimetics, other biologically active agents described herein, and agents that enhance delivery as described above, are, individually or combinatorially, incorporated within an epithelially-shaped formulation. administered (eg, nasally), which includes a biocompatible polymer that functions as a carrier or base. Such polymeric carriers include polymeric powders, matrices or microparticulate delivery vehicles, among other forms of polymers. The polymer can be plant, animal or synthetic origin. Often, the polymer is crosslinked. Additionally, in these The GRP, analog or mimetic delivery systems can be functionalized in a manner where they can be covalently bound to the polymer and provided inseparably from the polymer in a simple manner. In other embodiments, the polymer is chemically modified with an inhibitor of enzymes or other agents which can degrade or inactivate biologically active agents and / or agents that enhance delivery. In certain formulations, the polymer is partially or completely insoluble in water but the polymer expandable in water, for example, a hydrogel. The polymers employed in this aspect of the invention are desirably interactive with water and / or hydrophilic in nature to absorb significant amounts of water, and often form hydrogels when placed in contact with water or aqueous medium for a sufficient period of time to achieve balance with water. In more detailed embodiments, the polymer is a hydrogel which, when placed in contact with excess water, absorbs at least twice its weight of water in equilibrium when exposed to water at room temperature. U.S. Patent No. 6,0004,583. Supply systems based on biodegradable polymers are preferred in many biomedical applications because such systems are broken either by hydrolysis or by enzymatic reaction in non-toxic molecules. The degradation rate is controlled by manipulating the composition of the biodegradable polymer matrix. These types of systems can therefore be employed in certain settings for long-term release of biologically active agents. Biodegradable polymers, such as poly (glycolic acid) (PGA), poly- (lactic acid) (PLA), and poly (D, L-lactic-co-glycolic acid) (PLGA), have received considerable attention as carriers of Possible drug supply, since the degradation products of these polymers have been found to have low toxicity. During the normal metabolic function of the body, these polymers are degraded into carbon dioxide and water. These polymers have also exhibited excellent biocompatibility. To prolong the biological activity of GRP, analogs and mimetics, and other biologically active agents described herein, as well as optional supply enhancing agents, these agents can be incorporated into polymer matrices, for example, polyorthoesters, polyanhydrides, or polyesters. These yields sustain the activity and release of the active agents, for example, as determined by the degradation of the polymer matrix. Although the encapsulation of biotherapeutic molecules within synthetic polymers can stabilize them during storage and delivery, the biggest obstacles of polymer-based release technology are the loss of activity of the molecules therapeutic during formulation processes that often involve heat, sonication or organic solvents. Absorption promoting polymers contemplated for use within the invention may include, chemically or physically modified derivatives and versions of the aforementioned types of polymers, in addition to other synthetic or naturally occurring polymers, gums, resins and other agents, as well as also mixtures of these materials with each other or with other polymers, as soon as the alterations, modifications or mixing do not adversely affect the desired properties, such as water absorption, hydrogel formation and / or chemical stability for useful application. In more detailed aspects of the invention, polymers such as nylon, acrylon and other synthetic polymers normally hydrophobic, can be sufficiently modified by reaction to become expandable in water and / or form gels stable in aqueous media. The polymers that promote the absorption of the invention can include polymers from the group of homo and copolymers based on various combinations of the following vinyl monomers: acrylic and methacrylic acids, acrylamide, methacrylamide, hydroxyethyl acrylate or methacrylate, vinylpyrrolidones, as well as polyvinyl alcohol. and its co and terpolymers, polyvinylacetate, its co and terpolymers with the monomers listed above and 2-acrylamido-2-methyl-propanesulfonic acid (A PS ©). Very useful are copolymers of the monomers listed above with copolymerizable functional monomers such as acrylate acrylate or methacrylamide or methacrylate esters wherein the ester groups are straight or branched chain alkyl, aryl derivatives having up to four aromatic rings, which they may contain alkyl substituents of 1 to 6 carbons; spheroidal, sulphate, phosphate or cationic monomers, such as N, -dimethylaminoalkyl (meth) acrylamide, dimethylaminoalkyl (meth) acrylate, (meth) acryloxyalkyltrimethylammonium chloride (meth) acryloxyalkyldimethylbenzylammonium. Polymers that promote additional absorption for use within the invention, are those classified as dextrans, dextrins and from the class of materials classified as natural gums and resins, or from the class of natural polymers such as processed collagen, chitin, chitosa, pulalan , zooglan, alginates and modified alginates such as "Kelcoloide" (a modified alginate of polypropylene glycol), gellan gums such as "Kelocogel", xanthan gums such as "Keltrol", estastins, alpha hydroxybutyrate and their copolymers, hyaluronic acid and their derivatives, polylactic and glycolic acids. A very useful class of polymers applicable withinof the present invention are olefinically unsaturated carboxylic acids containing at least one activated carbon to carbon olefinic double bond, and at least one carboxyl group; that is, an acid or functional group easily converted to an acid containing a double olefinic bond which readily functions in the polymerization due to its presence in the monomeric molecule, either in the alpha-beta position with respect to a carboxyl group, or as part of a terminal methylene group. Olefinically unsaturated acids of this class include such materials as acrylic acids typified by acrylic acid itself, alpha-cyano acrylic acid, beta methacrylic acid (crotonic acid), alpha-phenyl acrylic acid, beta-acryloxy propionic acid, cinnamic acid, acid p-chloro cinnamic, 1-carboxy-4-phenyl butadiene-1,3, itaconic acid, citraconic acid, mesaconic acid, glutaconic acid, aconitic acid, maleic acid, fumaric acid, and tricarboxy ethylene. As used herein, the term "carboxylic acid" includes the carboxylic acids and those acid anhydrides, such as maleic anhydride, wherein the anhydride group is formed by the removal of the water molecule from two carboxyl groups located in the same carboxylic acid molecule. Representative acrylates useful as agents that promote absorption with the invention include methyl acrylate, ethyl acrylate, propyl acrylate, isopropyl acrylate, butyl acrylate, isobutyl acrylate, methyl methacrylate, methyl ethacrylate, ethyl methacrylate, octyl acrylate, heptyl acrylate, octyl methacrylate, isopropyl methacrylate, 2-ethylhexyl methacrylate, nonyl acrylate, hexyl acrylate, n -hexyl methacrylate, and the like. Higher alkyl acrylic esters are decyl acrylate, isodecyl methacrylate, lauryl acrylate, stearyl acrylate, behenyl acrylate and melissyl acrylate and methacrylate versions thereof. Mixtures of two or three or more long chain acrylic esters can be successfully polymerized with one of the carboxylic monomers. Other monomers include olefins including, alpha olefins, vinyl ethers, vinyl esters and mixtures thereof. Other vinylidene monomers, including acrylic nitriles, can also be used as agents that promote absorption with the methods and compositions of the invention, to improve the delivery and absorption of one or more GRP, analogs and mimetics and other biologically active agents. , which include improving the delivery of active agents to a target tissue or compartment in the subject (e.g., liver, hepatic portal vein, tissue or CNS fluid or blood plasma). Useful alpha, beta-olefinically unsaturated nitriles are preferably monoolefinically unsaturated nitriles, having 3 to 10 carbon atoms, such as acrylonitrile, methacrylonitrile and the like. More preferred are acrylonitriles and methacrylonitriles. Acrylic amides containing from 3 to 35 carbon atoms including monoolefinically unsaturated amides can also be used. Representative amides include acrylamide, methacrylamide, Nt-butylacrylamide, N-cyclohexylacrylamide, higher alkylamides, wherein the alkyl group in the nitrogen contains from 8 to 32 carbon atoms, acrylic amides including N-alkylol amides of carboxylic acids alpha, beta- olefinically unsaturated including those having from 4 to 10 carbon atoms, such as N-methylol acrylamide, N-propanol acrylamide, N-methylol methacrylamide, N-methylol maleimide, N-methylol maleamic acid esters, N-methylol-p -vinyl benzamide and the like. Still further useful promoting materials are alpha-olefins containing from 2 to 18 carbon atoms, more preferably from 2 to 8 carbon atoms; dienes containing from 4 to 10 carbon atoms; vinyl esters and allylesters such as vinylacetate; vinylaromatics such as styrene, methylstyrene and chloro-styrene; vinyl and allylethers and ketones such as vinyl methyl ether and methyl vinyl ketone; chloroacrylates; cyanoalkylacrylates such as alpha-cyanomethylacrylate and the alpha, beta and gamma cyanopropylacrylates; Alcoxyacrylates such as methoxyethylacrylate; haloacrylates such as chloroethylacrylate; vinylharules and vinyl chloride, vinylidene chloride and the like; divinyl, diacrylates and other polyfunctional monomers such as divinyl ether, diethylene glycol diacrylate, ethylene glycol dimethacrylate, methylene-bis-acrylamide, allyl pentaerythritol, and the like; and bis (beta-haloalkyl) alkenyl phosphonates such as bis (beta-chloroethyl) vinylphosphonate and the like as known to those skilled in the art. The copolymers wherein the carboxy-containing monomer is a minor constituent, and the other vinylidene monomers present as the main components are readily prepared in accordance with the methods described herein. When hydrogels are used as agents that promote absorption with the invention, these may be composed of synthetic copolymers of the group of acrylic and methacrylic acids, acrylamide, methacrylamide, hydroxyethylacrylate (HEA) or methacrylate (HE A), and vinylpyrrolidones which are Interactive and expandable with water. Specific illustrative examples of useful polymers, especially for the delivery of peptides or proteins, are the following types of polymers: (meth) acrylamide and 0.1 to 99% by weight (meth) acrylic acid; (meth) acrylamides and 0.1-75% by weight of (meth) acryloxyethyl trimethylammonium chloride; (meth) acrylamide and 0.1-75% by weight of (meth) acrylamide; acrylic acid and 0.1-75% by weight of alkyl (meth) acrylates; (meth) acrylamide and 0.1-75% by weight of AMPS.RTM (trademark of Lubrizol Corp.); (meth) acrylamide and 0 to 30% by weight of alkyl (meth) acrylamides and 0.1-75% by weight of AMPS.RTM; (meth) acrylamide and 0.1-99% by weight of HEMA; (meth) acrylamide and 0.1 to 75% by weight of HEMA and 0.1 to 99% of (meth) acrylic acid; (meth) acrylic acid and 0.1-99% by weight of HEMA; 50% mole of vinyl ether and 50% mole of maleic anhydride; (meth) acrylamide and 0.1 to 75% by weight (meth) acryloxyalkyldimethylbenzylammonium chloride; (meth) acrylamide and 0.1 to 99% by weight of vinylpyrrolidone; (meth) acrylamide and 50% by weight of vinylpyrrolidone and 0.1-99.9% by weight of (meth) acrylic acid; (meth) acrylic acid and 0.1 to 75% by weight of AMPS.RTM and 0.1-75% by weight of alkyl (meth) acrylamide. In the above examples, alkyl means Ci to C30, preferably Ci to C22 linear and branched and C4 to Ci6 cyclic; where (met) is used, it means that the monomers with and without the methyl group are included. Other very useful hydrogel polymers are expandable, but insoluble versions of poly (vinylpyrrolidone) starch, carboxymethylcellulose and polyvinyl alcohol. Additional polymeric hydrogel materials useful with the invention include complexes (poly) hydroxyalkyl (meth) acrylate: anionic and cationic hydrogels: poly (electrolyte); poly (vinylalcohols) that have a low residual acetate: an expandable mixture of cross-linked agar and crosslinked carboxymethylcellulose: an expandable composition comprising methylcellulose mixed with poorly cross-linked agar; a copolymer expandable in water produced by a finely divided copolymer dispersion of maleic anhydride with styrene, ethylene, propylene or isobutylene; a water expandable polymer of N-vinyl lactams; expandable sodium salts of carboxymethylcellulose; and similar. Other retention polymers and gellable fluid absorbers employed to form the hydrophilic hydrogel for mucosal delivery of biologically active agents within the invention include pectin; polysaccharides such as agar, acacia, karaya, tragacanth, some and guar and their reticulated versions; acrylic acid polymers, copolymers and salt derivatives, polyacrylamide; polymers of maleic indole anhydride expandable in water; starch graft copolymers; polymers and copolymers of acrylate type with water absorption capacity of approximately 2 to 400 times their original weight; polyglucan diesters; a mixture of cross-linked poly (vinylalcohol) and poly (N-vinyl-2-pyrrolidone); polybutylene-polyethylene block copolymer gels; carob gum; polyester gels; polyurea gels; polyether gels; polyamide gels; polyimide gels; gels polypeptide; polyamino acid gels; polyeleculose gels; crosslinked maleic anhydride acrylic anhydride polymers; and polysaccharides. Synthetic hydrogel polymers for use within the invention can be made by an infinite combination of several monomers in various proportions. The hydrogel can be crosslinked and in general, has the ability to retain and absorb fluid and expand or swell to a state of extended equilibrium. Hydrogels typically swell or expand upon delivery to the nasal mucosal surface, absorbing approximately 2-5, 5-10, 10-50 up to 50-100 or more times twice their water weight. The optimum degree of expandability for a given hydrogel will be determined by different biologically active agents, depending on such factors as molecular weight, size, solubility and diffusion characteristics of the active agent ported or trapped or encapsulated within the polymer, and the specific spacing and movement of cooperative chain associated with each individual polymer. The hydrophilic polymers used with the invention are soluble in water but expandable in water. Such water expandable polymers are as typically referred to as hydrogels or gels. Such gels can be conveniently produced from water soluble polymer by the process of crosslinking the polymers by a suitable crosslinking agent. However, stable hydrogels can also be formed from specific polymers under defined conditions of pH, temperature and / or ionic concentration, in accordance with methods known in the art. Typically, the polymers are crosslinked, that is, crosslinked in the extent that the polymers possess good hydrophilic properties, have improved physical integrity (compared to uncrosslinked polymers of the same or similar type) and exhibit improved ability to retain within the network of gel both the biologically active agent of interest and the additional compounds for co-administration together with these, such as cytokine or enzyme inhibitor, while retaining the ability to release the active agents, at the appropriate time and location. In general, hydrogel polymers for use with the invention are crosslinked with a difunctional crosslinking in the amount from 0.01 to 25% by weight, based on the weight of the monomers forming the copolymer, and more preferably, from 0.1 to 20 percent by weight. percent by weight and more often from 0.1 to 15 weight percent of the crosslinking agent. Another useful amount of a crosslinking agent is 0.1 to 10 percent by weight. Tri, tetra or higher multifunctional crosslinking agents can also be used. When such reagents are used, amounts lower may be required to achieve the equivalent crosslink density, i.e., the degree of crosslinking or network properties that are sufficient to effectively contain the biologically active agents. The crosslinking agents can be covalent, ionic or hydrogen bonds with the polymer having the ability to expand in the presence of fluids containing water. Such crosslinkers and crosslinking reactions are known to those skilled in the art and in many cases, are dependent on the polymer system. In this way, a crosslinked network can be formed by free radical copolymerization of unsaturated monomers. Polymeric hydrogels can also be formed by crosslinking preformed polymers by reacting functional groups found in polymers, such as alcohols, acids, amines with such groups as glyoxal, formaldehyde or glutaraldehyde, bis anhydrides and the like. The polymers can also be crosslinked with any polyene, for example, decadiene or trivinylcyclohexane; acrylamides such as N, N-methylene-bis (acrylamide); polyfunctional acrylates such as trimethylolpropanetriacrylate; or polyfunctional vinylidene monomer containing at least 2 terminal groups CH2 < , which include for example, copolymers of divinylbenzene, divinylnaphthylene, allyl and the like. In certain embodiments, the crosslinking monomers for use in the preparation of the copolymers are polyalkenyl polyethers having more than one alkenyl ether group per molecule, which may optionally possess alkenyl groups in which a double olefinic bond is present attached to a group of terminal methylene (for example, made by the etherification of a polyhydric alcohol containing at least 2 carbon atoms and at least 2 hydroxyl groups). Compounds of this class can be produced by reacting an alkenyl halide, such as allyl chloride or allyl bromide, with a strongly alkaline aqueous solution of one or more polyhydric alcohols. The product can be a complex mixture of polyethers with varying numbers of ether groups. The efficiency of the polyether crosslinking agent increases with the number of potentially polymerizable groups in the molecule. Typically, polyethers containing an average of two or more alkenyl ether groupings per molecule are used. Other crosslinking monomers include, for example, diallyl ester, dimethyl ether, allyl or methallyl acrylates and acrylamides, tetravinylsilane, polyalkenylmethanes, diacrylates and dimethacrylates, divinyl compounds, such as divinylbenzene, polyallylphosphate, diallyloxy and phosphite esters and the like. Typical agents are allyl pentaerythritol, allylsucrose, teimethylolpropane triacrylate, 1,6-hexanediol diacrylate, trimethylolpropanediallyl ether, pentaerythritol triacrylate, tetramethylene dimethacrylate, ethylene diacrylate, ethylene dimethacrylate, triethylene glycol dimethacrylate and the like. Allylpentaerythritol, trimethylolpropane diallyl ether and allyl sucrose provide suitable polymers. When the crosslinking agent is present, the polymer blends usually contain between about 0.01 to 20 weight percent, for example, 1%, 5%, or 10% or more by weight of the crosslinking monomer based on the total monomer of carboxylic acid, plus other monomers. In more detailed aspects of the invention, the epithelial supply of GRP, analogs and mimetics and other biologically active agents described herein, is enhanced by retaining the active agents in enzymatically or physiologically protective slow release vehicle or carrier, for example, a hydrogel that covers the active agent of the action of the degradative enzymes. In certain embodiments, the active agent is chemically linked to the carrier or vehicle, which can also be mixed or bound to additional agents such as enzyme inhibitors, cytokines, etc. The active agent can alternatively be immobilized through sufficient physical traps within the carrier or vehicle, for example, a polymeric matrix.

Polymers such as hydrogels used with the invention can incorporate functional linked agents such as glycosides chemically incorporated into the polymer to enhance the intranasal bioavailability of active agents formulated together with these. Examples of such glycosides are glycosides, fructosides, galactosides, arabinosides, mannosides and their substituted alkyl derivatives and natural glycosides such as arbutin, florizin, amygdalin, digitonin, saponin and indicano. There are several ways in which a typical glycoside can bind to a polymer. For example, the hydrogen of the hydroxyl groups of a glycoside or other similar carbohydrate can be replaced by the alkyl group from a hydrogel polymer to form an ether. Also, the hydroxyl groups of the glycosides can be reacted to esterify the carboxyl groups of a polymeric hydrogel to form polymeric esters in situ. Another method is to employ acetobromoglucose condensation with colest-5-en-3-beta-ol in a maleic acid copolymer. Substituted N-polyacrylamides can be synthesized by the reaction of polymers activated with omega-aminoalkyl glycosides; (1) (carbohydrate spacer) (n) -polyacrylamide, "pseudopolysaccharides"; (2) (carbohydrate spacer) (n) -phosphatidylethanolamine (m) -polyacrylamide, neoglycolipids, phosphatidylethanolamine derivatives; (3) (carbohydrate spacer) (n) - biotin (m) -polyacrylamide. These biotinylated derivatives can bind to lectins on the mucosal surface to facilitate the absorption of biologically active agents, for example, a GRP encapsulated in polymer. Within more detailed aspects of the invention, one or more GRPs, analogs and mimetics, and / or other biologically active agents, described herein, optionally include secondary active agents such as, protease inhibitors, cytokines, additional modulators of intercellular binding physiology. , etc., are modified and linked to a polymer matrix or carrier. For example, this can be done by chemically linking a peptide or active protein agent and modifying optional agents with a crosslinked polymer network. It is also possible to chemically modify the polymer separately with an interactive agent such as a glycosidal containing molecule. In certain aspects, the biologically active agents and optional active secondary agents can be functionalized, ie, wherein an appropriate reactive group is identified or is chemically added to the active agents. More often, an ethylenic polymerizable group is added, and the functionalized active agent is then copolymerized with monomers and a crosslinking agent using a standard polymerization method such as polymerization solution (usually in water), emulsion, suspension or dispersion polymerization. Often, the functionalizing agent is provided with a sufficiently high concentration of functional or polymerizable groups to ensure that several sites in the active agents are functionalized. For example, in a polypeptide comprising 16 amine sites, it is generally desired to functionalize at least 2, 4, 5, 6 and up to 8 or more of the sites. After functionalization, the functionalized active agents are / are mixed with monomers and a crosslinking agent comprising the reagents from which the polymer of interest is formed. The polymerization is then induced in this medium to create a polymer containing the bound active agents. The polymer is then combined with water or other suitable solvents and otherwise purified to remove unreacted trace impurities and, if necessary, crushed or broken by physical means such as by agitation, forcing through a mesh, ultrasonication or other suitable means, at a desired particle size. The solvent, usually water, is then removed in such a way as not to denature or otherwise degrade the active agents. A desired method is lyophilization (freeze drying), but other methods are available and can be used (eg, vacuum drying, air drying, spray drying, etc . ). To introduce polymerizable groups into peptides, proteins and other active agents with the invention, it is possible to react reactive amino, hydroxyl, thiol groups and others available with unsaturated groups containing electrophiles. For example, N-hydroxy succinimidyl groups containing unsaturated monomers, active carbonates, such as p-nitrophenyl carbonate, trichlorophenyl carbonates, tresylate, oxycarbonylimidazoles, epoxide, isocyanates and aldehydes, and unsaturated carboxymethylazides and unsaturated ortho-pyridyl disulfide, belong to this category of reagents Illustrative examples of unsaturated reagents are allyl glycidyl ether, allyl chloride, allyl bromide, allyl iodide, acryloyl chloride, allyl isocyanate, allylsulfonyl chloride, maleic anhydride, copolymers of maleic anhydride and allyl ether and the like. All active lysine derivatives, except aldehyde, can generally react with other amino acids such as imidazole groups of histidine and hydroxyl groups of tyrosine and thiol groups of cystine if the local environment enhances the nucleophilicity of these groups. The functionalizing reagents containing aldehyde are specific to lysine. These types of reactions with available groups of lysines, cysteines, tyrosines, have been extensively documented in the literature and are known to those experts in the art. In the case of biologically active agents containing amine groups, it is convenient to react such groups with an acyloyl chloride, such as acryloyl chloride, to introduce the polymerizable acrylic group into the reacted agent. Then during the preparation of the polymer, such as during the crosslinking of the copolymer of acrylamide and acrylic acid, the functionalized active agent, through the acrylic groups, is bound to the polymer and becomes bound thereto. In further aspects of the invention, biologically active agents that include peptides, proteins, nucleosides and other molecules which are bioactive in vivo, are of stabilized conjugation by covalently linking one or more active agents to a polymer that incorporates as an integral part thereof, both a hydrophilic portion, for example, a linear polyalkylene glycol, a lipophilic portion (see for example, U.S. Patent No. 5,681,811). In one aspect, a biologically active agent is covalently coupled with a polymer comprising (i) a linear polyalkylene glycol moiety, and (ii) a lipophilic moiety, wherein the active agent, linear polyalkylene glycol moiety, and the lipophilic moiety, are conformationally arranged relative to each other, such that the active therapeutic agent has a resistance in vivo intensified to enzymatic degradation (i.e., relative to its stability under similar conditions in an unconjugated form devoid of the polymer coupled to it). In another aspect, the stabilized conjugation formulation has a three-dimensional conformation comprising the biologically active agent coupled with a polysorbate complex comprising (i) a linear polyalkylene glycol moiety, and (ii) a lipophilic moiety, wherein the agent active, the linear polyalkylene glycol portion and the lipophilic portion, are conformationally arranged relative to each other, such that (a) the lipophilic portion is externally available in the three-dimensional conformation, and (b) the active agent in the composition has a intensified in vivo resistance for enzymatic degradation. In a related further aspect, a multiple ligand conjugate complex is provided, which comprises a biologically active agent covalently coupled to a portion of the triglyceride structure through a polyalkylene glycol spacer group attached to a carbon atom of the structure portion. of triglyceride, and at least a portion of fatty acid covalently bound either directly to a carbon atom of the triglyceride structure portion or covalently bound through a polyalkylene glycol spacer portion (see example, U.S. Patent No. 5,681,811). In such multi-ligand conjugate therapeutic complexes, the alpha and beta carbon atoms of the bioactive portion of triglycerides may have fatty acid moieties attached by covalently attaching either directly to it, or indirectly by covalently linking it through portions thereof. polyalkylene glycol spaders Alternatively, a fatty acid portion can be covalently linked either directly or through a polyalkylene glycol spacer portion to the alpha and alpha 'carbons of the triglyceride structure portion, with the bioactive therapeutic agent being covalently coupled with the gamma- carbon of the triglyceride structure portion, either directly covalently bound to it or indirectly bound to it through a polyalkylene shell portion. It will be recognized that a wide variety of structural, compositional and conformational forms are possible for the multiple ligand conjugated therapeutic agent complex comprising the triglyceride structure portion, within the scope of the invention. Furthermore, it is noted that in such a multiple ligand therapeutic agent complex, the biologically active agents can be advantageously covalently coupled with the modified structure portion of triglyceride via alkyl spacer groups, or alternatively, other acceptable spacer groups within the scope of the invention. As used in such a context, acceptability of the spacer group refers to specific acceptability characteristics of end use, spherical and compositional application. In still further aspects of the invention, a stabilized conjugation complex is provided, which comprises a polysorbate complex comprising a polysorbate portion that includes a triglyceride structure that is covalently coupled to the alpha, alpha 'and beta carbon atoms. thereof, functionalizing groups including, (i) a fatty acid group; and (ii) a polyethylene glycol group having a biologically active agent or portion covalently attached thereto, for example, attached to an appropriate functionality of the polyethylene glycol group. Such a covalent bond can be either direct, for example, to a terminal hydroxy functionality of the polyethylene glycol group, or alternatively, the covalent bond can be direct, for example, by reactive blocking the hydroxy terminus of the polyethylene glycol group with a carboxy functional group spacer. terminal, so that the resulting capped polyethylene glycol group has a carboxy terminal functionality in which the biologically active agent or portion can be covalently linked.

In still further aspects of the invention, there is provided a stabilized, water-soluble complex of stabilized conjugation, which comprises one or more GRP, analogs and mimetics, and / or other biologically active agents described herein covalently coupled to a modified glycolipid moiety. polyethylene glycol (PEG) physiologically compatible. In such a complex, the biologically active agents can be covalently coupled to the modified glycolipid portion with physiologically compatible PEG by a labile covalent bond in a free amino acid group of the active agent, wherein the labile covalent bond is cleavable in vivo by biochemical hydrolysis and / or proteolysis. The glycolipid portion modified with physiologically compatible PEG may advantageously comprise a polysorbate polymer, for example, a fatty acid ester groups comprising polysorbate polymer, selected from the group consisting of monopalmitate, dipalmitate, monolaurate, dilaurate, trilaurate, monoleate , dioleate, trioleate, monostearate, distearate and tristearate. In such a complex, the glycolipid portion modified with physiologically compatible PEG, can suitably comprise a polymer selected from the group consisting of polyethylene glycol ethers of fatty acids and polyethylene glycol esters of fatty acids, wherein the fatty acids, for example, comprise a fatty acid selected from the group consisting of lauric, palmitic, oleic and stearic acids.

Material Storage In certain aspects of the invention, the combinatorial formulations and / or administration methods coordinated herein, incorporate an effective amount of peptides and proteins which can adhere to charged glass, thereby, reducing the effective concentration in the container. Silanized containers, for example, silanized glass containers, are used to store the finished product to reduce the absorption of the polypeptide or protein into a glass container. In still further aspects of the invention, a kit for the treatment of a mammalian subject comprises a stable pharmaceutical composition of one or more GRP compounds formulated for mucosal delivery to the mammalian subject, wherein the composition is effective in alleviating one or more symptoms of diabetes , obesity, cancer, hyperglycemia, dyslipidemia, metabolic syndrome, coronary syndrome, myocardial infarction, or neurological disorder in said subject without unacceptable adverse side effects. The kit further comprises a pharmaceutical reagent vial for containing one or more GRP compounds. The pharmaceutical reagent vial is composed of pharmaceutical grade polymer, glass or other adequate material. The pharmaceutical reagent vial is, for example, a vial of silanized glass. The kit further comprises an opening for delivery of the composition to a nasal mucosal surface of the subject. The supply opening is composed of a pharmaceutical grade polymer, glass or other suitable material. The supply opening is, for example, a silanized glass. A silanization technique combines a special cleaning technique for the surfaces to be silanized with a silanization process at low pressure. The silane is in the gas phase and at an intensified temperature of the surfaces to be silanized. The method provides reproducible surfaces with stable, homogeneous and functional silane layers that have characteristics of a monolayer. Silanized surfaces prevent binding to the glass of polypeptides or agents that enhance the mucosal delivery of the present invention. The method is useful for preparing silanized pharmaceutical reactive vials to retain GRP compositions of the present invention. The glass trays are cleaned by rinsing with double distilled water (ddH20) before use. The silane tray is then rinsed with 95% EtOH, and the acetone tray is rinsed with acetone. The pharmaceutical reagent vials are sonicated in acetone for 10 minutes. After the sonication of acetone, the reagent vials are rinsed in a tray of ddH20 at least twice. The reactive vials are sonicated in 0.1M NaOH for 10 minutes. While the reactive vials are sonicated in NaOH, the silane solution is made under a hood. (Silane solution: 800 ml of 95% ethanol, 96 L of glacial acetic acid, 25 ml of glycidoxypropyltrimethoxy silane). After NaOH sonication, the reagent vials are rinsed in a ddH20 tray at least twice. The reagent vials are sonicated in silane solution for 3 to 5 minutes. The reagent vials are rinsed in 100% EtOH tray. The reagent vials are dried with prepurified N2 gas and stored in a 100 ° C oven for at least 2 hours before use.

Bioadhesive Delivery Vehicles and Methods In certain aspects of the invention, the combinatorial formulations and / or methods of coordinated administration herein, incorporate an effective amount of a non-toxic adhesive as a compound or adjunct carrier, to improve the epithelial delivery of one or more biologically active agents. The bioadhesive agents in this context exhibit general or specific adhesion to one or more components or surfaces of the target biological membrane. The bioadhesive maintains a gradient of desired concentration of the biologically active agent in or through the mucosa to ensure the penetration of even large molecules (e.g., peptide and proteins) into or through the epithelium. Typically, the use of a bioadhesive within the methods and compositions of the invention, provides an increase of two to five times, often five to ten times in permeability for peptides and proteins in or through the epithelium. This improvement of epithelial permeation often allows effective transmucosal delivery of large macromolecules, for example, to the basal portion of the nasal epithelium or in adjacent extracellular compartments or a blood plasma or CNS or fluid tissue. This improved delivery provides greatly improved effectiveness for the delivery of peptides, bioactive proteins and other macromolecular therapeutic species. These results will depend, in part, on the hydrophilicity of the compound, thereby achieving greater penetration with hydrophilic species compared with water-insoluble compounds. In addition to these effects, the use of bioadhesives to improve the persistence of the drug to the surface of the biological membrane can cause a reservoir mechanism for the prolonged drug supply, with this, the compounds not only penetrate through the biological membrane, but also spread again towards the surface once the material on the surface is exhausted. A variety of bioadhesives suitable in the art for oral administration are described, U.S. Patent Nos. 3,972,995; 4,259,314; 4,680,323; 4,740,365; 4,573,996; 4,292,299; 4,715,369; 4,876,092; 4,855,142; 4,250,163; 4,226,848; 4,948,580; U.S. Patent No. 33,093, which finds use within the new methods and compositions of the invention. The potential of several bioadhesive polymers as a nasal delivery platform, for example mucosal, with the methods and compositions of the invention, can be easily assessed by determining their ability to retain and release GRP, as well as by their ability to interact with surfaces mucosal after the incorporation of the active agent there. In addition, well-known methods will be applied to determine the biocompatibility of selected polymers with the tissue at the site of mucosal administration. When the target mucosa is covered by mucus (ie, in the absence of mucolytic treatment or mucus separation), it can serve as a connecting link to the underlying mucosal epithelium. Therefore, the term "bioadhesive" as used herein also covers mucoadhesive compounds useful for improving the mucosal delivery of biologically active agents within the invention. Without However, adhesive contact with mucosal tissue mediated through adhesion to a mucosal gel layer may be limited by incomplete or temporary binding between the mucus layer and the underlying tissue, particularly on nasal surfaces where rapid detachment of the mucosa occurs. mucus. In this sense, the mucin glycoproteins are continuously secreted and, immediately after, they are released from cells or glands, forming a viscoelastic gel. The luminal surface of the adherent gel layer, however, is continuously eroded by mechanical, enzymatic and / or ciliary action. Where such activities are more prominent or where prolonged adhesion times are desired, the coordinated administration methods and combinatorial formulation methods of the invention may also incorporate mucolytic and / or ciliostatic methods or agents as described hereinbefore. Typically, the mucoadhesive polymers for use within the invention are natural or synthetic macromolecules which adhere to the surfaces of wet mucosal tissue by complex, but not specific, mechanisms. In addition to these mucoadhesive polymers, the invention also provides methods and compositions that incorporate bioadhesives that adhere directly to a cell surface, rather than mucus, by means of specific interactions that include, mediated by the receptor. A example of bioadhesives that work in this specific way, is the group of compounds known as lectins. These are glycoproteins with an ability to specifically recognize and bind to sugar molecules, for example, glycoproteins or glycolipids, which are part of the membranes of intranasal epithelial cells and can be considered as "lectin receptors". In certain aspects of the invention, bioadhesive materials for enhancing the intranasal delivery of biologically active agents comprise a matrix of a hydrophilic polymer, for example, water-soluble or expandable, or a mixture of polymers that can adhere to a moist mucosal surface. These adhesives can be formulated as ointments, hydrogels (see above), gastric films, and other forms of application. Often, these adhesives have the biologically active agent mixed together with it to effect slow release or local delivery of the active agent. Some are formulated with additional ingredients to facilitate the penetration of the active agent through the nasal mucosa, for example, into the circulatory system of the individual. Several polymers, both natural and synthetic, show significant binding to mucus and / or mucosal epithelial surfaces under physiological conditions. The strength of this interaction can be easily measured by mechanical detachment or cutting tests. When applied to a wet mucosal surface, many dry materials will spontaneously adhere, at least slightly. After such initial contact, some hydrophilic materials begin to attract water by absorption, expansion or capillary forces and if this water is absorbed from the underlying substrate or from the polymer-gone interface, adhesion may be sufficient to achieve the goal of improving the absorption of mucosa by biologically active agents. Such "adhesion by hydration" can be almost strong, but the formulations adapted to use this mechanism, should consider expansion, which continues as the dosage is transformed into a hydrated muscilage. This is projected for many hydrocolloids useful with the invention, especially some cellulose derivatives, which are generally non-adhesive when applied in the pre-hydrated state. However, bioadhesive drug delivery systems for epithelial administration are effective within the invention, when such materials are applied in the form of a polymeric dry powder, microsphere, or film-like delivery form. Other polymers adhere to the epithelial surfaces not only when applied dry, but also in a fully hydrated state, and in the presence of excess amounts of water. The selection of a mucoadhesive in this way, requires consideration due to physiological as well as physicochemical conditions, under which contact with the tissue will be formed and maintained. In particular, the amount of water or moisture, usually present at the proposed adhesion site, and the prevalent pH, are known to greatly affect the mucoadhesive bond intensity of different polymers. Several bioadhesive polymeric drug delivery systems have been manufactured and studied in the past 20 years, not always successfully. A variety of such carriers are, however, currently used in clinical applications involving dental, orthopedic, ophthalmological and surgical uses. For example, acrylic-based hydrogels have been used extensively by bioadhesive devices. Acrylic based hydrogels are well studied for bioadhesion due to their flexibility and non-abrasive characteristics in the particularly expanded state, which reduces the abrasion that causes damage to the tissues in contact. In addition, its high permeability in the swollen state allows the unreacted monomer, uncrosslinked polymer chains and the initiator to be rinsed from the matrix after polymerization, which is an important feature for selection of bioadhesive materials for use with the invention. Acrylic-based polymeric devices exhibit very strong bond strength. For controlled epithelial delivery of peptide and protein drugs, the methods and compositions of the invention, optionally include the use of carriers, eg, polymer delivery vehicles, which function in part, to protect the biologically active agent from proteolytic cleavage, while at the same time, they provide enhanced penetration of the peptide or protein into or through the nasal mucosa. In this context, bioadhesive polymers have shown considerable potential to intensify oral drug delivery. As an example, the bioavailability of 9-desglicinamide, 8-arginine vasopressin (DGAVP), intraduodenally administered to rats, together with 1% (w / w) of saline dispersion of the polycarbophil mucoadhesive poly (acrylic acid) derivative, is 3- 5 times increased compared to an aqueous solution of the peptide drug without this polymer. Mucoadhesive polymers of the poly (acrylic acid) type are potent inhibitors of some intestinal proteases. The mechanism of enzyme inhibition is explained by the strong affinity of this class of polymers for divalent cations, such as calcium or zinc, which are essential cofactors of metalloproteinases, such as trypsin and chymotrypsin. The deprivation of proteases from their cofactors by poly (acrylic acid), is reported by induce irreversible structural changes of enzymatic proteins, which are accompanied by a loss of enzymatic activity. At the same time, other mucoadhesive polymers (for example, some cleulose and chitosan derivatives) may not inhibit proteolytic enzymes under certain conditions. Contrary to other enzymatic inhibitors contemplated for use within the invention (for example, aprotinin, bestatin), which are relatively small molecules, trans-nasal absorption of inhibitory polymers is probably minimal in view of the size of these molecules, and thereby , eliminate the possible adverse side effects. Thus, mucoadhesive polymers, particularly of the poly (acrylic acid) type, can serve as both an adhesion promoting adhesive and an enzyme protective agent to enhance the controlled delivery of peptide and protein drugs, especially when they are considered to be of security. In addition to protection against enzymatic degradation, bioadhesives and other agents that promote non-polymeric and polymeric absorption for use with the invention, can directly increase epithelial permeability to biologically active agents. To facilitate the transport of hydrophilic and large molecules, such as peptides and proteins, through the nasal epithelial barrier, mucoadhesive polymers and other agents, they have been postulated to provide enhanced permeation effects beyond what is considered by the prolonged premucosal residence time of the supply system. The time course of drug plasma concentrations reportedly suggests that bioadhesive microspheres cause an acute, but temporary, increase in insulin permeability through the nasal mucosa. Other mucoadhesive polymers for use with the invention, for example, chitosan, reportedly enhance the permeability of certain mucosal epithelia even when applied as an aqueous solution or gel. Another mucoadhesive polymer reported to directly affect epithelial permeability is hyaluronic acid and ester derivatives thereof. A particularly useful bioadhesive agent with coordinated administration and / or combinatorial formulation methods and compositions of the invention is chitosan, as well as its analogues and derivatives. Chitosan is a non-toxic, biocompatible and biodegradable polymer that is widely used for medical and pharmaceutical applications due to its favorable properties of low toxicity and good compatibility. It is a natural polyaminessaccharide prepared from chitin by N-deacylation with alkali. As used within the methods and compositions of the invention, chitosan increases the retention of GRP, analogs and mimetics and other biologically active agents described herein, in a mucosal site of application. This mode of administration can also improve patient compliance and acceptance. As further provided herein, the methods and compositions of the invention will optionally include a novel chitosan derivative or chemically modified form of chitosan. One such novel derivative for use with the invention is denoted as a β- [1- »4] -2-guanidino-2-deoxy-D-glucose (poly-GuD) polymer. Chitosan is the N-deacylated product of chitin, a naturally occurring polymer that has been used extensively to prepare microspheres for oral and intranasal formulations. The chitosan polymer has also been proposed as a soluble protein for parenteral drug delivery. Within one aspect of the invention, o-methylisourea is used to convert an amine chitosan to its guanidino moiety. The guanidinium compound is prepared, for example, by the reaction between quasi-normal solutions of chitosan and o-methylisourea at pH above 8.0. Additional compounds classified as bioadhesive agents for use with the present invention, act by mediating specific interactions, typically classified as "ligand-receptor interactions," between the complementary structures of the bioadhesive compound and a mucosal epithelial surface component. Many natural examples illustrate this form of bioadhesion of specific link, as exemplified by lect-sugar interactions. The lectins are (glyco) proteins of non-immune origin, which bind to polysaccharides or glycocon ugados. Several plant lectins have been investigated as possible agents that promote pharmaceutical absorption. A plant lectin, hemagglutinin from Phaseolus vulgaris (PHA), exhibits high oral bioavailability of more than 10% after feeding to rats. The tomato lectin Lycopersicon esculentum) (TL), seems safe by several modes of administration. In summary, the aforementioned bioadhesive agents are employed in the combinatorial formulations and methods of coordinated administration of the present invention, which optionally incorporate an effective amount and form a bioadhesive agent to prolong persistence or otherwise increase epithelial absorption. one or more GRP, analogs and mimetics, and other biologically active agents. The bioadhesive agents can be coordinately administered as adjuncts or as additives within the combinatorial formulations of the invention. In certain embodiments, the bioactive agent acts as a "pharmaceutical glue", while in other embodiments, the adjunct supply or combinatorial formulation of the bioadhesive agent it serves to intensify the contact of the biologically active agent with the nasal mucosa, in some cases promoting the specific ligand-receptor interactions with "receptors" of epithelial cells and in others increasing the epithelial permeability to significantly increase the drug concentration gradient measured in a Target delivery site (eg, liver, blood plasma, or CNS tissue or fluid). Still additional bioadhesive agents for use with the invention, act as enzyme inhibitors (eg protease), to improve the stability of mucosally administered biotherapeutic agents, supplied coordinately or in combinatorial formulation with the bioadhesive agent.

Liposomes and Micellar Delivery Vehicles The coordinated delivery methods and combinatorial formulations of the present invention optionally incorporate carriers of effective fatty acids or lipids, processing agents, or delivery vehicles to provide improved formulations for epithelial GRP delivery , analogs and mimetics and other biologically active agents. For example, a variety of formulations and methods are provided for mucosal delivery, which comprises one or more of these active agents, such as peptide or protein, mixed or encapsulated by, or coordinately administered with a liposome, mixed micellar carrier or emulsion, to improve chemical and physical stability and increase the half-life of biologically active agents (e.g., reducing susceptibility to proteolysis, chemical modification and / or denaturation) after the mucosal supply. Within certain aspects of the invention, specialized delivery systems for biologically active agents, comprise small lipid vesicles known as liposomes. These are typically made of neutral, biodegradable, non-toxic and non-immunogenic lipid molecules, and can efficiently trap or bind drug molecules, including peptides and proteins, in or on their membranes. The attractive capacity of liposomes as a peptide and protein delivery system within the invention is enhanced by the fact that the encapsulated proteins can remain in their preferred aqueous environment within the vesicles, while the liposomal membrane protects them against proteolysis and other destabilizing factors. Although not all known liposome preparation methods are feasible in the encapsulation of peptides and proteins due to their unique physical and chemical properties, several methods allow the encapsulation of these macromolecules without substantial deactivation. A variety of methods are available for preparing liposomes for use with the invention, U.S. Patent Nos. 4,235,871, 4,501,728, and 4,837,028. For use with the liposome delivery, the biologically active agent is typically entrapped within the liposome, lipid vesicle or is attached to the exterior of the vesicle. Like liposomes, unsaturated long chain fatty acids, which also have enhanced activity for mucosal absorption, can form vesicles with bilayer-like structures (so-called "ufasomes"). These can be formed, for example, by using oleic acid to trap biologically active peptides and proteins for intranasal delivery, for example, mucosal with the invention. Other delivery systems for use with the invention, combine the use of polymers and liposomes to align the advantageous properties of both vehicles such as encapsulation, within the natural polymer fibrin. In addition, the release of biotherapeutic compounds from this delivery system is controllable through the use of covalent crosslinking and the addition of antifibrinolytic agents to the fibrin polymer. The most simplified delivery systems for use within the invention include the use of lipids cationics as carriers or carriers of supply, which can be effectively employed to provide an electrostatic interaction between the lipid carrier and such charged biologically active agents as polyanionic nucleic acids and proteins. This allows efficient packaging of the drugs in a form suitable for mucosal administration and / or subsequent supply to systemic compartments. Additional supply vehicles for use with the invention include medium and long chain fatty acids, as well as mixed micelles of surfactants with fatty acids. Most lipids that originate naturally in the form of esters have important implications with respect to their own transport through mucosal surfaces. The free fatty acids and their monoglycerides which have bound polar groups, have been demonstrated in the form of mixed micelles to act on the intestinal barrier, as penetration enhancers. This discovery of barrier modifying function of free fatty acids (carboxylic acids with a chain length ranging from 12 to 20 carbon atoms) and its polar derivatives, has extensive research stimulated on the application of these agents as enhancers of mucosal absorption . For use with the methods of the invention, the long chain fatty acids, especially fusogenic lipids (unsaturated fatty acids and monoglycerides such as oleic acid, linoleic acid, linoleic acid, monoolein, etc.), provide useful carriers to intensify the mucosal supply of GRP, analogs and mimics and other agents biologically assets described here. The medium chain fatty acids (C6 to C12) and monoglycerides, have also been shown to have enhancing activity in the absorption of intestinal drug, and can be adapted for use with the mucosal delivery formulations and methods of the invention. In addition, the sodium salts of long and medium chain fatty acids are effective delivery vehicles and agents that enhance absorption for epithelial delivery of biologically active agents with the invention. In this way, fatty acids can be used in soluble forms of sodium salts or by the addition of non-toxic surfactants, for example, polyoxyethylated hydrogenated castor oil, sodium taurocholate, etc. Other mixed micellar preparations and fatty acid which are employed with the invention include, but are not limited to, Na (C8) caprylate, Na caprate (CIO), Na (C12) laurate, or Na (C18) oleate, optionally combined with bile salts, such as glycocholate and taurocholate.

Pegylation Additional methods and compositions provided with the invention involve chemical modification of biologically active peptides and proteins by covalent attachment of polymeric materials, for example, dextrans, polyvinylpyrrolidones, glycopeptides, polyethylene glycol and polyamino acids. The resulting peptides and conjugated proteins retain their biological activities and solubility for epithelial administration. In alternate embodiments, GRP, analogs and mimetics and other biologically active peptides and proteins are conjugated to polyalkyleneoxide polymers, particularly polyethylene glycols (PEG). U.S. Patent No. 4,179,337. The amino-reactive PEG polymers for use with the invention include, SC-PEG with molecular masses of 2,000, 5000, 10,000, 12,000, and 20,000; U-PEG-10000; NHS-PEG-3 00-biotin; T-PEG-5000; T-PEG-12000; and TPC-PEG-5000. PEGylation of biologically active peptides and proteins can be achieved by modification of carboxyl sites (eg, aspartic acid or glutamic acid groups in addition to the carboxyl terminus). The utility of PEG-hydrazide in selective modification of carboxyl groups of carbodiimide activated protein under acidic conditions has been described. Alternatively, the bifunctional PEG modification of biologically active peptides and proteins can be employed. In some procedures, charged amino acid residues, which include lysine, aspartic acid and glutamic acid, have a marked tendency to be accessible to the solvent on protein surfaces.

Other Stabilizing Modifications of Active Agents In addition to PEGylation, biologically active agents such as peptides and proteins for use with the invention, can be modified to improve the half-life of circulation by protecting the active agent via conjugation to other known stabilizing or protective compounds, by example, by the creation of fusion proteins with a peptide, active, protein, analog or mimetic, linked to one or more carrier proteins, such as one or more immunoglobulin chains.

Formulation, Preparation, Manufacturing and Administration The epithelial delivery formulations of the present invention comprise GRP, analogs and mimetics, typically combined together with one or more pharmaceutically acceptable carriers and, optionally, other therapeutic ingredients. The carriers must be "pharmaceutically acceptable", in the sense of being compatible with the other ingredients of the formulation and not causing an unacceptable deleterious effect on the subject. Such carriers are described hereinbefore or are otherwise well known to those skilled in the pharmacology art. Desirably, the formulation should not include substances such as enzymes or oxidizing agents with which the biologically active agent to be administered is known to be incompatible. The formulations can be prepared by any of the methods well known in the pharmacy art. With the compositions and methods of the invention, the GRP, analogs and mimetics and other biologically active agents described herein, can be administered to subjects by a variety of mucosal modes of administration, including oral, rectal, vaginal, intranasal, intrapulmonary delivery , or transdermal, or by topical delivery to the eyes, ears, skin or other mucosal surfaces. The compositions and methods of the invention also include dermal administration modes. Optionally, GRP, analogs and mimetics and other biologically active agents described herein, can be coordinately and adjutatively administered by non-mucosal routes, which include dermal patches, topical preparations applied to the skin, intramuscular, subcutaneous, intravenous, intra-atrial routes, intra-articular, intraperitoneal or parenteral. In other alternative embodiments, the biologically active agents can be administered ex vivo by direct exposure to cells, tissues or organs originating from a mammalian subject, for example, as a component of an ex vivo organ or tissue treatment formulation, containing the biologically active agent in a suitable liquid or solid carrier. The compositions according to the present invention are often administered in an aqueous solution as a pulmonary or nasal atomizer and can be dispensed in the form of a spray by a variety of methods known to those skilled in the art. Preferred systems for dispensing liquids such as a nasal spray are described in U.S. Patent No. 4,511,069. The formulations can be presented in multiple dose containers, for example, in the sealed dispensing system described in US Pat. No. 4,511,069. Additional aerosol delivery forms may include, for example, compressed air, jet, ultrasonic and piezoelectric nebulizers, which supply the biologically active agent dissolved or suspended in a pharmaceutical solvent, for example, water, ethanol or a mixture of the same. The pulmonary and nasal spray solutions of the present invention typically comprise the drug or drug to be delivered, optionally formulated with a surface active agent, such as a non-surfactant. ionic (for example, polysorbate 80), and one or more buffers. 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 2.0 and 8, preferably 4.5 + 0.5. Shock absorbers suitable for use with these compositions are as described above or otherwise known in the art. Other components can be added to improve or maintain chemical stability, which include preservatives, surfactants, dispersants or gases. Suitable preservatives include, but are not limited to, phenol, methylparaben, paraben, m-cresol, thiomersal, chlorobutanol, benzalkonium chloride, sodium benzoate, ethanol, phenylethyl ether, benzyl alcohol, and the like. Suitable surfactants include, but are not limited to, oleic acid, sorbitan triolate, polysorbates, lecithin, phosphatidyl cholines and various diglycerides and long chain 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. With alternative modalities, the mucosal formulations are administered as dry powder formulations comprising the biologically active agent in a dry, usually lyophilized form of an appropriate particle size, or with an appropriate particle size range. The minimum particle sizes suitable for deposition with the pulmonary or nasal passages are often approximately 0.5 μm in average mass equivalent to aerodynamic diameter (MMEAD), commonly approximately 1 μMMEAD, and more typically approximately 2 μMEMAD. The maximum particle size suitable for deposition with the nasal passages is often about 10 μMMEAD, commonly about 8 μMMEAD, and more typically about 4 μMMEAD. Intranasally breathable powders with these particle sizes 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 powders of appropriate MMEAD can be administered to a patient via a conventional dry powder inhaler (DPI); which depends on the respiration of the patient, lung inhalation or nasa, to disperse the powder in an aerosolized amount. Alternatively, the dry powder can be administered via air assisted devices that use an external energy source to disperse the powder in an aerosolized amount, for example, a piston pump.

Dry powder devices typically require a powder mass in the range of about 1 mg to 20 mg to produce a single aerosolized dose ("cloud"). If the desired or required dose of the biologically active agent is lower than this amount, the powdered active agent will typically be combined with a powder in dry pharmaceutical volume to provide the total powder mass required. Preferred bulk dry powders include sucrose, lactose, dextrose, mannitol, glycine, trehalose, human serum albumin (HSA) and starch. Other suitable bulky dry powders include, cellobiose, dextrans, maltotriose, pectin, sodium citrate, sodium ascorbate and the like. To formulate compositions for epithelial delivery with the present invention, the biologically active agent can be combined with various pharmaceutically acceptable additives, as well as as a base or carrier for dispersion of the active agents. Desired additives include, but are not limited to, pH control agents, such as arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid, acetic acid, etc. In addition, local anesthetics (eg, benzyl alcohol), isotonic acid agents (eg, sodium chloride, mannitol, sorbitol), absorption inhibitors (eg, Tween 80), solubility enhancing agents (eg, cyclodextrins and derivatives thereof), stabilizers (e.g., serum albumin) and reducing agents (e.g., glutathione), may be included. When the composition for epithelial supply is a liquid, the tonicity of the formulation, measured with reference to the tonicity of 0.9% (w / v) of the physiological saline taken as a unit, is typically adjusted to a value at which it is not will induce substantial irreversible tissue damage in the nasal mucosa at the site of administration. In general, the tonicity of the solution is adjusted to a value of about 1/3 to 3, more typically 1/2 to 2, and more often 3/4 to 1.7. The biologically active agent can be dispersed in a base or vehicle, which can comprise a hydrophilic compound having a capacity to disperse the active agent and any of the desired additives. The base can be selected from a wide range of suitable carriers, including but not limited to copolymers of polycarboxylic acids or salts thereof, carboxylic anhydrides (e.g., maleic anhydride) with other monomers (e.g., methyl (meth) acrylate, acrylic acid, etc.), hydrophobic vinyl polymers such as polyvinylacetate, polyvinyl alcohol, polyvinylpyrrolidone, cellulose derivatives such as hydroxymethylcellulose, hydroxypropylcellulose, etc., and natural polymers such as chitosan, collagen, sodium sodium, gelatin, haluronic acid and non-toxic metal salts thereof. Often, a biodegradable polymer is selected as a base or carrier, for ple, polylactic acid, poly (lactic acid-glycolic acid) copolymer, polyhydroxybutyric acid, poly (hydroxybutyric acid-glycolic acid) copolymers, and mixtures thereof. Alternatively or additionally, synthetic fatty acid esters such as polyglycerol fatty acid esters, sucrose fatty acid ethers, etc., may be employed as carriers. The hydrophilic polymers and other carriers can be used alone or in combination, and the improved structural integrity can be imparted to the carrier by partial crystallization, ionic bonding, crosslinking and the like. The carrier can be provided in a variety of forms, including viscous or fluid solutions, gels, pastes, powders, microspheres and films for direct application to the nasal mucosa. The use of a carrier selected in this context may result in promotion of absorption of the biologically active agent. The biologically active agent can be combined with the base or carrier in accordance with a variety of methods, and the release of the active agent can be by diffusion, carrier 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, for example, isobutyl 2-cyanoacrylate and dispersed in a biocompatible dispersion medium applied to the nasal mucosa, which provides sustained supply and biological activity over a long time. To further improve the epithelial delivery of pharmaceutical agents with the invention, formulations comprising the active agent may also contain a hydrophilic low molecular weight compound as a base or excipient. Such hydrophilic low molecular weight compounds provide a middle passage through which a water soluble active agent, such as a physiologically active peptide or protein, can diffuse through the base to 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 active peptide. The molecular weight of the hydrophilic low molecular weight compound is generally not more than 10000 and preferably not more than 3000. The exemplary low molecular weight hydrophilic compound includes polyol compounds, such as oligo, di and monosaccharides such as sucrose, mannitol, sorbitol, lactose, L-arabinose, D- erythrose, D-ribose, D-xylose, D-mannose, trehalose, D-galactose, lactulose, cellobiose, gentibiose, glycerin and polyethylene glycol. Other examples of low molecular weight hydrophilic compounds used as carriers within the invention include, N-methylpyrrolidone, and alcohols (eg, oligonivilalcohol, ethanol, ethylene glycol, propylene glycol, etc.) - These low molecular weight hydrophilic compounds can 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 pharmaceutically acceptable carriers, substances required at approximate physiological conditions, such as pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, lactate sodium, 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, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate and Similar. The therapeutic compositions for administering the Biologically active agent can also be formulated as a solution, microemulsion or other ordered structure suitable for high concentration of active ingredients. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol and the like), and suitable mixtures thereof. The proper fluidity for solutions can be maintained for example, by the use of a coating such as lecithin, by the maintenance of a desired particle size in the case of dispersible formulations, and by the use of surfactants. In many cases, it will be desirable to include isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol or sodium chloride in the composition. Prolonged absorption of the biologically active agent can be brought about by including in the composition an agent, which retards absorption, for example, salts of monostearate and gelatin. In certain embodiments of the invention, the biologically active agent is administered is a release formulation over time, for example, in a composition which includes a slow release polymer. The active agent can be prepared with carriers that will protect against rapid release, for example, a controlled release vehicle such as polymer, microencapsulated supply or bioadhesive gel. The prolonged supply of the active agent, in various compositions of the invention, can be carried around by including in the composition, agents that retard the absorption, for example, aluminum monostearate hydrogels and gelatin. When controlled release formulations of the biologically active agent are desired, controlled release binders suitable for use in accordance with the invention include, any biocompatible controlled release material which is inserted into the active agent and which is capable of incorporating the biologically active agent. Numerous such materials are known in the art. Useful controlled release binders are materials that are metabolized slowly under physiological conditions after their intranasal delivery (eg, at the nasal mucosal surface, or in the presence of body fluids after transmucosal delivery). Suitable binders include but are not limited to biocompatible polymers and copolymers previously used in the art in sustained release formulations. Such biocompatible compounds are non-toxic and inert to surrounding tissues, and do not activate significant adverse side effects such as nasal irritation, immune response, inflammation or the like. They are metabolized into metabolic products that are also biocompatible and easily eliminated from the body. Exemplary polymeric materials for use in this context include, but are not limited to, polymer matrices derived from copolymeric and homopolymeric polyesters having hydrolysable ester linkages. A number of these are known in the art to be biodegradable and lead to degradation products that have no toxicity or low. Exemplary polymers include polyglycolic acids (PGA) and polylactic acids (PLA), poly (DL-lactic acid-co-glycolic acid) (DLPLA), poly (D-lactic acid-coglycolic acid) (D PLGA) and poly (L-) lactic acid-co-glycolic acid) (L PLGA). Other useful biodegradable or bioerodible polymers include, but are not limited to, such polymers as poly (epsilon-caprolactone), poly (epsilon-aprolactone-CO-lactic acid), poly (e-aprolactone-CO-glycolic acid), poly (beta) -hydroxy butyric acid), poly (alkyl-2-cyanoacrylate), hydrogels such as poly (hydroxyethyl methacrylate), polyamides, poly (amino acids) (ie, L-leucine, glutamic acid, L-aspartic acid and the like), poly (urea ester), poly (2-hydroxyethyl DL-aspartamide), polyacetal polymers, polyorthoesters, polycarbonate, polymaleamides, polysaccharides and copolymers thereof. Many methods for preparing such formulations are generally known to those skilled in the art. Other useful formulations include release compositions controlled, for example, microcapsules, Patents US Nos. 4,652,441 and 4,917,893, copolymers of lactic acid-glycolic acid used in the manufacture of microcapsules and other formulations, US Patent Nos. 4,677,191 and 4,728,721, and sustained release compositions for water soluble peptides, US Patent No. 4,675,189. The nasal spray product manufacturing process in general includes the preparation of a GRP nasal spray diluent, which includes ~ 85% water plus the components of the nasal spray formulation without GRP. The pH of the diluent is then measured and adjusted to the pH of the desired formulation with sodium hydroxide or hydrochloric acid if necessary. The water is used to achieve the final objective volume of the diluent. The GRP nasal spray is prepared by the non-aseptic transience of ~ 85% of the final target volume of the diluent to a screw capped bottle. An appropriate amount of GRP is added and mixed until it is completely dissolved. The pH is measured and adjusted to the desired formulation pH with sodium hydroxide or hydrochloric acid, if necessary. A sufficient amount of diluent is added to reach the final target volume. The bottles covered with thread are filled and fixed with lids. The above description of the manufacturing process represents a method used to prepare the Initial clinical batches of the drug product. This method can be modified during the development process to optimize the manufacturing process. The GRP currently marketed requires sterile manufacturing conditions for compliance with FDA regulations. Parenteral administration that includes GRP for injection or infusion requires a sterile (aseptic) manufacturing process. Current Good Manufacturing Practices (GMP) for sterile drug manufacturing include standards for construction and design characteristics (21 C.F.R. § 211.42 (April 1, 2005)); standards for assessing and approving or rejecting components, drug product containers and closures (§ 211.84); standards for the control of microbiological contamination (§ 211.113); and other special test requirements (§ 211.167). Non-parenteral (non-aseptic) products, such as the intranasal product of the invention, do not require these specialized sterile manufacturing conditions. As can be easily appreciated, the requirements for a sterile manufacturing process are substantially higher and correspondingly more expensive than those required for a non-sterile product manufacturing process. These costs include much higher capitalization costs for facilities, as well as costly manufacturing costs: additional facilities for sterile manufacturing include, additional rooms and ventilation; Additional costs associated with sterile manufacturing include major human resources, extensive quality control and quality assurance, and administrative support. As a result, the manufacturing costs of a GRP product, such as that of the invention, are sufficiently lower than those of a parenterally administered GRP product. The present invention satisfies the need for a non-sterile manufacturing process for GRP. The invention includes a preservative-free GRP drug product. Such a formulation does not contain a condom. In the absence of an antimicrobial excipient, the formulation could be filled under sterile conditions in a condom-free nasal spray device, or incorporated in a dermal patch preparation. The device could be capable of delivering an effective dose without allowing contamination of the formulation within the delivery system. Such a GRP drug product could allow multiple dosing of the same container, thereby, greatly reducing the cost of items relating to a single-use drug product. Advantages of a multi-use condom free GRP formulation are improved stability, alternative means for microbial prevention, and reduced cost of items that allow the product to be more viable for commercialization. Sterile solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients listed above, as required, followed by filtered sterilization. In generating, the dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the other required ingredients of those enumerated above. In the case of sterile powders, the methods of preparation include vacuum drying and freeze drying, which provides a powder of the active ingredient plus any additional desired ingredient of a previously sterile filtered solution thereof. The prevention of the action of microorganisms can be carried out by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. The administration to the skin and mucosal, according to the invention, allows selective self-administration for treatment by patients, provided sufficient safety is placed to control and monitor the dosages and side effects. The administration to the skin and mucosal also overcomes certain disadvantages of other forms of administration, such as injections, which are painful and expose the patient to possible infections and may present problems of drug bioavailability. For pulmonary and nasal delivery, systems for controlled dispensing of therapeutic liquids aerosol as an atomizer are well known. In one embodiment, metered doses of active agent are supplied by means of a specially constructed mechanical pump valve, US Pat. No. 4,511,069. In another embodiment, the inactive agent is supplied by dermal patch technology.

Dosage For treatment and prophylactic purposes, the biologically active agents described herein, can be administered to subjects in a single bolus delivery, via continuous delivery (eg, transdermal, mucosal or continuous intravenous delivery), over a prolonged period of time or in a repeated administration protocol (for example, by an administration protocol repeated every hour, daily or weekly). In this context, a therapeutically effective dosage of GRP may include repeated doses within a treatment regimen or prolonged prophylaxis that will provide clinically significant to alleviate one or more symptoms or detectable conditions associated with a disease or objective condition, as set forth above. The determination of effective dosages in this context is typically based on studies of animal models followed by human clinical trials and is guided by determining the effective dosages and administration protocols that significantly reduce the incidence or severity of symptoms or disease conditions in the target subject. Suitable models in this regard include, for example, subjects of murine, rat, porcine, feline, non-human primate and other accepted models known in the art. Alternatively, effective dosages can be determined using in vitro models (eg, histopathological and immunological assays). Using such models, only ordinary calculations and adjustments are typically required to determine an appropriate concentration and dose to deliver a therapeutically effective amount of the biologically active agents (eg, amounts that are intranasally effective, transdermally effective, intravenously effective or intramuscularly effective to cause a desired answer). In an alternative embodiment, the invention provides compositions and methods for delivery Intranasal GRP, wherein the GRP compounds are / are administered repeatedly through an effective intranasal dosing regimen involving multiple administrations of the GRP to the subject during a daily or weekly schedule to maintain a therapeutically effective elevated and pulsatile reduced level of GRP during a prolonged dosing period. The compositions and methods provide GRP compounds that are self-administered by the subject in a nasal formulation between one and six times daily to maintain a high and pulsatile reduced therapeutically effective level of GRP during an extended dosing period of 8 hours to 24 hours.

Kits The present invention includes kits, packages and multiple container units containing the pharmaceutical compositions described above, active ingredients, and / or means for administering the msimos, for use in the prevention and treatment of diseases and other conditions in mammalian subjects. Briefly, these kits include a container or formulation containing one or more GRP, analogs or mimetics, and / or other biologically active agents in combination with agents that enhance the epithelial delivery described herein, formulated in a pharmaceutical preparation for epithelial delivery. The intranasal formulations of the present invention can be administered using any bottle or syringe or by instillation. An example of a nasal spray bottle is the "Nasal Spray Pump w / Safety Clip, Pfeiffer SAP # 60548, which supplies a dose of 0.1 ml per jet and has a deep tube length of 36.05 cm. It can be purchased from Pfeiffer of America of Princeton, NJ.

Aerosol Nasal Administration of a GRP It has been discovered that GRP can be administered intranasally using a nasal spray or aerosol. This is surprising because many proteins and peptides have been shown to be ported or denatured due to the mechanical forces generated by the actuator in the production of the atomizer or aerosol. In this area, the following definitions are used: 1. Aerosol - A product that is packaged under pressure and contains therapeutically active ingredients that are released after activation of an appropriate valve system. 2. Measured aerosol - A pressurized dosage form comprises metered dose valves, which allow the delivery of a uniform amount of atomizer after each activation. 3. Powdered aerosol - A product that is packaged under pressure and contains therapeutically active ingredients in the form of a powder, in which they are released after the activation of an appropriate valve system. 4. Spray Aerosol - An aerosol product that uses compressed gas as the propellant to provide the necessary force to expel the product as a wet atomization; is generally applicable to solutions of chemical agents in aqueous solvents. 5. Atomizer - A liquid thoroughly divided as by a jet or air vapor. Nasal spray drug products contain therapeutically active ingredients dissolved or suspended in solutions or mixtures of excipients in non-pressurized dispensers. 6. Measured atomizer - One way non-pressurized dosage consisting of valves that allow the dispensing of a specified amount of atomizer after each activation. 7. Suspension atomizer - A liquid preparation containing solid particles dispersed in a liquid vehicle and in the form of droplets in progress or as finely divided solids. The dynamic characterization of aerosol atomizer fluid is emitted by nasal spray pump measured as a drug delivery device ("DDD"). Spray characterization is an integral part of regulatory admissions required by the Food and Drug Administration ("FDA"), for the approval of research development, quality assurance and stability testing procedures for new and existing nasal spray pumps . Through the characterization of the atomizing geometry, it has been found to be the best indicator of the total performance of the nasal spray pumps. In particular, measurements of the atomizer divergence angle (water column geometry), as they exit the device; ellipticity in cross section of the atomizer, uniformity and distribution of particle / got ita (atomizing pattern); and the evolution time of Atomizer development has been found to be the most representative performance quantities in the characterization of nasal spray pumps. During quality assurance and stability testing, jet geometry and atomizer pattern measurements are key identifiers to verify consistency and compliance with the approved data criteria for nasal spray pumps.

Definitions Jet height - the measurement of the tip of the actuator to the point at which the angle of the jet becomes nonlinear due to the breaking of the linear flow. Based on a visual examination of digital images, and establishing an amplitude measurement point that is consistent with a furthest measurement point of atomizing pattern, a height of 30 mm is defined by this study. Main Shaft - the longest rope that can be extracted with the adjusted atomizer pattern that crosses the COMw in base units (mm). Minor axis - the shortest rope that can be extracted with the atomizer pattern that crosses the COMw in base units (mm). Ellipticity ratio - the ratio of the axis main to the minor axis, preferably between 0.1 and 1.5, and more preferably between 1.0 and 1.3 Dio - the diameter of the droplet by which, 10% of the total volume of liquids in the sample consists of droplets of a smaller diameter (pm) . D5o - the diameter of the droplet by which, 50% of the total volume of liquids in the sample consists of droplets of a smaller diameter (pm), also known as the median diameter of mass. D9o - the diameter of the droplet by which 90% of the total volume of liquids in the sample consists of droplets of a smaller diameter (pm). Path - measurement of the amplitude of the distribution. A smaller value, narrower distribution. The path is calculated as (POP - Di o) Dso% RSD - percentage of relative standard deviation, the standard deviation divided by the mean of the series and multiplied by 100, also known as% CV. Volume - the volume of liquid or powder discharged from the supply vehicle with each equation, preferably between 0.01 ml and approximately 2.5 ml and more preferably, between 0.02 ml and 0.25 ml.

EXAMPLES The foregoing description generally describes the present invention, which is further exemplified by the following examples. These examples are described only for purposes of illustration, and are not intended to limit the scope of the invention. Although specific terms and values have been employed in this document, such terms and values will also be understood as examples and not as limiting the scope of the invention.

EXAMPLE 1 Used Materials and Equipment The present example illustrates the reagents, equipment and source of each used in the subsequent Examples of the present application. Table 1 illustrates the sample reagents used in the subsequent Examples.

Table 1 GLP-1 Sample Reagents Table 2 illustrates the source of the MatTek EpiAirway ™ System components that are described in greater detail in Example 2 of the present application.

Table 2 Components of the Epithelial Cell Model System in vivo Table 3 illustrates the source and components of the LDH assay system described in greater detail in Example 2 of the present application.

Table 3 Components of the LDH (Cytotoxicity) and MTT (Cell Viability) Assay.

Table 4 illustrates the instruments and others related laboratory supplies and supplies of each used in this document.

Table 4 Instruments and Other Related Supplies EXAMPLE 2 In vitro Permeation Kinetics of Pharmaceutical Formulations of Peptide-1 (GLP-1) Similar to Glucagon The present example demonstrates the exemplary pharmaceutical formulations of the present invention, which contain the excipients DDPC, EDTA and β-β-DC alone or in combination, enhanced GLP-1 permeation through an epithelial cell monolayer. Table 5 illustrates the formulations selected in the EpiAirway Model System by transepithelial resistance assay (TEER), cell viability assay (TT), lactate dehydrogenase cell death (LHD) assay and tissue permeation assay to determine which formulation achieves the highest degree of GLP-1 tissue permeation and TEER reduction while not resulting in significant cellular toxicity. The triplicate samples of each formulation and controls were evaluated using the membrane inserts of the tracheal / bronchial epithelial cell of the in vitro model of the EpiAirway System by MatTek Corp. (Ashland, MA), catalog # Air-100. The EpiAirway ™ system was developed by MatTek Corp. (Ashland, MA) as a model of the pseudostratified epithelial lining of the respiratory tract. The cells are grown in round cell cultures of porous membrane inserts in an air-liquid interface, which results in differentiation of the cells to a highly polarized morphology. The apical surface is ciliated with a microvellose ultrastructure and mucus from the epithelium (the presence of mucin has been confirmed by immunoblotting). The inserts have a diameter of 0.875 cm, provide a surface area of 0.6 cm2. The cells were plated on the inserts at the factory approximately three weeks before shipment. EpiAirway ™ culture membranes were received the day before the experiments started. They were sent in Medium of Eagle Modified by Dulbecco free of hydrocortisone and free of phenol red (DMEM). Each tissue inserts were placed in a cavity of a 6-well plate containing 0.9 ml of serum-free DMEM. The membranes are then cultured for 24 hours at 37 ° C / CC > 2 to 5% to allow tissues to balance. This DMEM-based medium is free of serum but supplemented with epidermal growth factor and other factors. The medium is always tested for endogenous levels of any cytokine or growth factor which is considered for intranasal delivery, but is free of all factors and cytokines studied to date except insulin. The volume is sufficient to provide contact with the bottoms of the units and their positions, but the apical surface of the epithelium is kept in direct contact with the air. Sterile clamps are used at this stage and all subsequent stages involving the transfer of units to cavities containing liquid to ensure that no trapped air remains between the bottoms of the units and the medium. The EpiAirway ™ model system was used to evaluate the effect of each GLP-1 containing the formulation on TEER, cell viability (MTT assay), cytotoxicity (LDH assay) and permeation. These tests are described below in detail.

Transepithelial Electrical Resistance (TEER) The TEER measurements were read using a Tissue Resistance Measurement Chamber connected to an Epithelial Voltometer with the guide electrode, both from World Precision Instruments. First the antecedent TEER was read for each insert on the day the experiment begins. After the TEER was read, 1 ml of fresh medium was placed in the bottom of each cavity in a 6-well plate. The inserts were drained on absorbent paper and placed in new cavities with fresh medium, while keeping the numbered inserts to correlate with the measurements of antecedent TEER. 100 ul of experimental formulation was added to each insert. The inserts were placed in a shaking incubator at 100 rmp and 37 ° C for 1 hour. The electrodes and a white slice of the tissue culture were equilibrated for at least 20 minutes in fresh medium with the energy before verifying the calibration. The antecedent resistance was measured with 1.5 ml of medium in the Endohm tissue chamber and 300 μ? of medium in a white Millicell-CM insert. The upper electrode was adjusted so that it is immersed in the medium but does not contact the upper surface of the membrane of the insert. The antecedent resistance of the white insert is 5-20 ohms. Prada each TEER determination, 300 μ? from the middle to the insert followed by an incubation for 20 minutes at room temperature before placing it in the Endohm chamber to read the TEER. The resistance was expressed as (measured-white resistance) x 0.6 cm2. All TEER values were reported as a function of the surface area of the tissue. TEER calculated as: TEER = (Rr - Rb) X A where Rx is the resistance of the insert with a membrane, Rb is the resistance of the white insert, and A is the area of the membrane (0.6 cm2). A decrease in the TEER value in relation to the control value (control = approximately 100 ohms-cm2, normalized to 100) indicates a decrease in cell membrane resistance and an increase in permeability of the mucosal epithelial cell. After 1 hour the incubation was complete, the tissue inserts were removed from the incubator. 200 μ? of fresh medium in each cavity of a 24-well plate and tissue inserts were transferred to the 24-well plate. 200 μ? of fresh medium to each tissue insert. TEER was again measured for each insert. After the tissue culture inserts were transferred from the 6-well plate to the 2-well plate, the basal medium was subdivided into three portions and stored in eppendorf tubes. All three subdivisions were placed at -80 ° C until use.

Lactate Dehydrogenase (LDH) Assay The amount of cell death was assayed by measuring the release of LDH from the cells using a CytoTox 96 Cytotoxicity Assay Kit, from Promega Corp. The samples were run in triplicate for each culture insert. tissue in the studio. 50 μ? of the collected medium (stored at 4 ° C) in triplicate in a 96-well plate. Recently, the cell-free medium was used as a target. 50 μ? of substrate solution (12 ml of Assay Buffer added to a fresh bottle of the Substrate Mixture, made in accordance with the kit) to each well and plates were incubated for 30 minutes at room temperature in the dark. After incubation, 50 μ? of stop solution to each well and plates were read on a pQuant optical density plate reader at 490 nm using the KCJr software.

MTT Assay The cell viability of each tissue culture insert was tested by MTT assay. Cell viability was assessed using the MTT assay (Kit MatTek, MTT-100). This kit measures the recovery and transformation of tetrazolium salt or formazan dye. The MTT concentrate was thawed and diluted with the medium to a ratio of 2 ml of MTT: 8 ml of the medium. The diluted MTT concentrate was pipetted (300 μm) into a 24-well plate. The tissue inserts were gently dried, placed in the cavities of the plate, and incubated for three hours in the dark at 37 ° C. After incubation, each insert was removed from the plate, gently stained and placed in a 24-well plate by extraction. The cell culture inserts were then submerged in 2.0 ml of the extractant solution per cavity (to completely cover the sample). The extraction plate was covered and sealed to reduce the evaporation of the extractant. After an overnight incubation at room temperature in the dark, the liquid inside each insert was decanted again into the cavity from which it was taken, and the inserts were discarded. The extractant solution (50 μ?) Of each well was pipetted in triplicate into 96-well microtiter plates, together with the extracted blanks and diluted with the addition of 150 μ? of fresh extractant solution. The optical density of the samples was measured at 550 nm in an optical density plate reader μ?)? 3? using the KCJr software.

Tissue Permeability Test The amount of GLP-1 (7-36) that passes from the apical surface to the basolateral surface of the monolayer of epithelial cell EpiAirway represents the degree of GLP-1 permeation. The amount of GLP-1 protein found on the basolateral surface of the cultured cells is measured by ELISA. The ELISA amide kit (7-36) GLP-1 was purchased from Lineo Research, (St. Charles, MI). The ELISA assay was performed in accordance with the manufacturer's protocol. The collected samples were diluted with assay buffer provided with the kit. Several rounds of in vitro selection were carried out. Permeation percentage was calculated by diluting the measured amount of GLP-1 found on the basolateral side of the cells as measured by ELISA for the total amount of GLP-1 starting material that was added to the apical side of the cells multiplied by 100. First, a study design of the experiment (DOE) was made using different amounts of excipients EDTA,? -β-CD and DDPC. The summary of the results is given in Table 5. All formulations containing one or more excipients show improvement in permeation over the control without excipients (# 3).

Table 5: In vitro Permeation Study of GLP-1 The intranasal pharmaceutical formulations tested in additional in vitro TEER studies, MTT, LDH and% of permeations are shown in Table 6. The results of TEER, MTT, LDH and permeability for these formulations are summarized in Table 7.

Table 6: GLP-1 Formulations Tested in vitro Table 7 Summary of In Vitro GLP-1 Results A measured decrease in TEER relative value for control indicates a decrease in cell membrane resistance or in other words the passage of ionic species from the apical to the basolateral side of the epithelial monolayer. The data presented in Table 7 indicate that all enhancer formulations significantly reduce TEER compared to control formulations. The MTT assay measures cell viability while the LDH assay measures cytotoxicity. The tests are used in combination to determine the effect of pharmaceutical formulations in "healthy" cell. The MTT and LDH results are both expressed as percentages. The MTT percentage was calculated by dividing the MTT value measured for each formulation by the MTT value of the control formulation multiplied by 100. Thus, the MTT positive control is 100% and serves as the baseline comparison for all other formulations. An MTT value below 80% represents a negative effect on cell viability. Similarly, the LDH percentage was calculated by dividing the LDH value for each formulation by the LDH value of the control formulation multiplied by 100. Thus, the LDH positive control is 100% and serves as the baseline comparison for all other formulations. The MTT test results indicate that all but one formulation (# 12 in Table 7) does not reduce cell viability below 80%. These data are also reported by the cytotoxicity assay LDH shows that a majority of the formulations do not show significant levels of cytotoxicity. The permeation of the GLP-1 tissue is expressed as% permeation once it increases over the control formulation (sample # 13). Increased times on control for the excipient containing GLP-1 permeation enhancer formulations of approximately 74 times up to 341 fold over the control. These data indicate that the inclusion of excipients DDPC, EDTA and? -β-CD significantly improves the permeation of GLP-1 through the epithelial cell monolayer. From table 7, formulations # 1, # 2, # 6 and # 12 result in > 200 times improvement of% permeation over the control without excipient (# 13 in Table 17). In summary, the in vitro data indicate that the exemplary pharmaceutical formulation of the present invention comprises 2 mg / ml of GLP, 10 mg / ml of EDTA and 10 mM of Citrate Buffer (sample # 6 in Table 6), exhibiting GLP permeation -1 greater intensified and reduced qualities of TEER while having a minimal negative effect on cell viability. Thus, this formulation represents an ideal candidate for the supply of GLP-1 through a mucosal surface, for example intranasal drug (IN) delivery, in the treatment of human diseases including obesity and diabetes.

EXAMPLE 3 Comparison of In Vitro Permeation Kinetics of Glucagon-Like Peptide-1 (GLP-1) Pharmaceutical The present example demonstrates that the in vitro permeation kinetics of GLP-1 pharmaceutical formulations are sensitive to the EDTA form used in the formulation. Table 8 below illustrates the training selected in the EpiAirway Model System in vitro by transepithelial resistance (TEER assay), cellular bioavailability (TT assay), lactate dehydrogenase (LDH assay, cell death) and tissue permeation. All samples contain 2 mg / ml of GLP-1 except samples # 9 and # 19, the lime serves as controls. Each formulation is made at a total volume of 0.5 ml and is evaluated in triplicate (n = 3). Each sample was evaluated in accordance with the protocols described in detail below in Example 2.

Table 8: Different Forms Containing EDTA Formulations Selected for GLP-1 Permeation Enhancers Abbreviations:? -β-CD = methyl-beta-cyclodextrin, EDTA = disodium edetate, DDPC = L-a-phosphatidylcholine didecanoyl, CB = chlorobutanol, Mg EDTA = magnesium salt of disodium EDTA; Zn EDTA = zinc salt of disodium EDTA; MTT = MTT assay; LDH = LDH assay; TEER = transepithelial resistance.

Results The effect of pharmaceutical formulations comprising agents for improving intranasal delivery, for example, the excipients DDPC, EDTA and -ß-CD in EpiAirway ™ Cell Membrane (mucosal epithelial cell layer) is shown. The results of permeation kinetics are summarized below in Table 9 and represent measurements taken at one hour after the cells were incubated with the formulations shown in Table 8.

Table 9 Results of permeation kinetics for formulations containing different EDTA salts The preceding data indicated that various forms of EDTA salts can be used in pharmaceutical formulations to manipulate the drug's permeation kinetics.

EXAMPLE 4 GLP-1 Stability The present example demonstrates that small molecule excipients, for example β-β-CD, EDTA and DDPC, do not promote the physical stability of GLP-1 in pharmaceutical formulations. In the present example, the stability of GLP-1 was evaluated with the two formulations described below in Table 10. The purpose of the present example is to determine whether the heating of GLP-1 causes protein degradation.

Table 10 GLP-1 Stability Formulations A differential scanning calorimetry (DSC) experiment was performed with a new exploration in the Sample # 1 of Table 10; the sample was subjected to scanning from 5 ° C to 100 ° C at a scanning ratio of 60 ° C / hour. The data was plotted as Cp (cal / ° C) against temperature (° C). A sustained transition peak was observed near 35 ° C in the first heat exploration. The propagation after the transition and the great decrease in heat capacity between 80 and 90 ° C suggests the formation of aggregates. It was observed that the solution was slightly cloudy after the scans, which support the information of a precipitate. The transition peak is narrower than expected for the unfolding of a peptide, this may be the result of aggregation immediately after the unfolding of the peptide. These data indicate that GLP-1 is to denature and / or form aggregates in the formulation at or around 35 ° C. A DSC experiment was performed with a reexploration in sample # 2; the sample was scanned from 5 to 100 ° C at a scanning ratio of 60 ° C / hour. The data was plotted as Cp (cal / ° C) against temperature (° C). A very large peak was observed near 46 ° C in the first heat exploration. The large decrease in heat capacity between 80 and 90 ° C may be due to the formation of aggregates. However, the solution does not look turbid after the scans, suggesting very small aggregates. These data indicated that GLP-1 is for denaturing and / or Form aggregates in the formulation at or around 46 ° C. These data indicated that the addition of excipients as described in Table 10 above for the GLP-1 formulation does not improve their physical stability.

EXAMPLE 5 Evaluation of Kinetics (PK) of Intranasal and Intravenous Administration of Glucagon-Like Peptide-1 (GLP-1) in Selected Pharmaceutical Formulations in Rabbits The present example demonstrates that the bioavailability of GLP-1 by intranasal administration is significantly improved by the inclusion of a dipeptidyl peptidase IV inhibitor (DPP-IV), for example Lys (4-nitro-Z) pyrrolidode, in the pharmaceutical formulation. A pharmacokinetic (PK) study in rabbits was conducted to evaluate the plasma pharmacokinetic properties of GLP-1 with various formulations administered via intranasal (IN) delivery against intravenous (IV) infusion. The complete study design is presented below in Table 11. Formulations # 1 through # 4 represent the IN formulations while formulation # 5 represents the infused IV formulation.

Table 11 Design of the Complete Study for the Pharmacokinetic Evaluation In this study, New Zealand White rabbits (Hra: (NZW) SPF) were used as test objects to evaluate the plasma kinetics of GLP-1 by intranasal administration and intravenous infusion. Rabbits were selected as subject animals 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. Four intranasal formulations and one intravenous formulation of GLP-1 were evaluated in the study. The The composition of the vehicle for each formulation is given in Table 12. The intranasal and intravenous formulations of GLP-1 were manufactured for preparation and final testing. For each of the dosing solutions, the components were provided in two parts, Part A and Part B (see Table 12). The final formulation was created for each of the intranasal groups (Groups 1-4) by mixing equal volumes of Part A (1 ml) and Part B (1 ml). For the intravenous group (Group 5), 1.5 ml of Part A and 3.5 ml of Part B were mixed. Final dosage solutions were used for all groups within 6 hours of preparation.

Table 12 Composition of the Vehicle for Formulations 1 to 5 of GLP-1 The concentration of GLP-1 is constant among the four intranasal formulations. The concentration of Citrate and pH are also considered among the four formulations. The concentration of EDTA is consistent for formulations 1, 3 and 4. Formulation 2 contains PN159, a strong binding modulator previously showed improvement for the delivery of peptides through an epithelial cell layer, but not EDTA. The formulation 5 containing citrate, EDTA and sodium chloride, each at the appropriate concentration for intravenous administration; while the concentration of GLP-1 decreases to provide a total dose of GLP-1 which is 10% of the intranasal dose. Lys (4-nitro-Z) -pyrrolidide is a specific inhibitor of dipeptidyl aminopeptidase (DPP) IV, the primary enzymes responsible for the metabolism of active GLP-1 (amino acid fragment 7-36) for an inactive metabolite (amino acid fragment) 9-36). For evaluation in this study, the concentrations of Lys (4-nitro-Z) -pyrrolidide is varied for each of the intranasal dose groups with the exception of equal concentration between Formulation 1 and Formulation 2. The total dose of Lys ( -nitro-Z) -pyrrolidide is approximately 0.04 mMoles / kg for groups 1 and 2 (intranasal groups), and group 5 (intravenous group).

GLP-1 Assay Method Study samples, quality and standard samples were tested with a Peptide-1 ELISA Kit similar to Glucagon (Active) (Lineo Research Inc. Catalog # EGLP-35K). Each sample was analyzed in duplicate. This assay is based on the capture of active GLP-1 (fragments of amide 7-36 and 7-36) by a monoclonal antibody (specific to the N-terminal region) immobilized in the cavities of a 96-well microtitre plate. and detection by a second antibody labeled with anti-GLP-1 alkaline phosphatase. After washing, methyl umbelliferyl phosphate was added to each cavity, which in the presence of alkaline phosphatase forms the fluorescent product umbelliferone. The amount of fluorescence generated is directly proportional to the concentration of active GLP-1 in an unknown sample, and thus was derived by interpolation from a reference curve using reference standards of known concentration of active GLP-1. Due to similar species between human and rabbit GLP-1, it was anticipated that it can detect endogenous active GLP-1 (ie, rabbit). Endogenous levels of rabbit GLP-1 are measured at time 0. The GLP-1 fragment 9-36 is not detected by the assay, with respect to the source.

Pharmacokinetics Evaluation Pharmacokinetic calculations were performed using inNonlin software (Pharsight Corporation, Version 4.0, Mountain Vie, CA) and a non-compartmental model of extravascular administration. The parameters for evaluation are described in Table 13.

Table 13 Pharmacokinetics Parameters Pharmacokinetic Parameters Analysis The average GLP-1 pharmacokinetic data for both the intranasal and intravenous groups are given in Table 14. The pre-dose (baseline) concentrations of endogenous GLP-1 in serum or plasma are generally below 10 pg / ml. For some samples, the limitations in repeated analysis impede the sample volume to obtain definitive results. A value of <was reported4 pg / ml for these samples. To calculate group mean values, and for pharmacokinetic evaluation, the data was corrected in the baseline when appropriate. The results indicated by < NUMBER conforms to "<NUMBER / 2"; with this procedure, a value of < 4 pg / ml is adjusted to 2 pg / ml. Plasma values of the individual animal, after dosing for GLP-1 exceeds 10 pg / ml baseline value. At the final time point after the intravenous infusion (90 minutes), the plasma concentrations of GLP-1 are at or near baseline values indicating that the infusion and elimination phases are captured by the time structure of sampling used by the study. After intranasal instillation, several animals have plasma concentrations of GLP-1 exceeding baseline levels. The AUCinf was determined to estimate the complete exposure profile for GLP-1.

Table 14 Parameters of Average Pharmacokinetics for GLP-1 in Male Rabbit Plasma after Intranasal Instillation (Groups 1-4) and Intravenous (Group 5) Formulation 1 / Group 1 Concentrations of peak GLP-1 (Tmax) occurred between 5 and 20 minutes (group mean 11 minutes) after dose administration. The mean Cmax of the group is 844.9 pg / ml. The concentrations of plasma remaining below baseline at 90 minutes after dosing, indicating that the elimination was not completed in this time. This is reflected in the mean AUC of 20792.7 min * pg / ml and AUCinf0 of 32.415.9 min * pg / ml. The mean terminal half-life (ti / o) was estimated to be 55.5 minutes.

Formulation 2 / Group 2 The mean Cmax of group 2 was estimated to be 424.0 pg / ml. The Tmax medís was estimated to be 43 minutes; however, there is considerable inter-animal variability for this parameter. The examination of the concentrations against time profile for these animals indicates an absorption phase (increased) and elimination (decrease). The AUCuitim of group 2 is 17,069.8 min * pg / ml. A ti / 2 should not be estimated due to an absence of a clear elimination phase in two animals within the group. However, based on the data collected for the remaining animals in the group, the average ti 2 is 66.8 minutes. The average AUCinf (three animals in which Kel should be determined) is 33.324.4 min * pg / ml.

Formulation 3 / Group 3 The Cmax, Tmax, ti / 2 and mean AUCúitíma of group 3 are 283.9 pg / ml, 25 minutes, 39.3 minutes, and 9331.5 min * pg / ml, respectively. An exact Kel, and thus ti / 2 can not be terminated because an animal in the group has a greater than the expected value in 60 minutes. Thus, this value is not included in the average ti / 2 of the group of 39.3 minutes and mean AUCinf of the group of 13.232.7 min * pg / ml.

Formulation 4 / Group 4 La Cma: < mean for group 4 was estimated to be 154.5 pg / ml. The mean Tmax of the group is 47 minutes, however, two, animals, have their highest concentrations of plasma measures of GLP-1 at 90 minutes. Without a Kel of apparent phase elimination (and ti / 2) and AUCinf should not be determined for these animals. The mean ti 2 and AUCinf for the other three animals are estimated to be 22.4 minutes and 5734.4 min * pg / ml, respectively.

Formulation 5 / Group 5 During the infusion procedure, an animal of group 5 experienced a mechanical failure with the pumping apparatus, which resulted in -100 μ? Additional be supplied between 5 and 10 minutes time points. This is reflected in the concentration value of 10 minutes of > 5000 pg / ml. As the exact conditions for the infusion or should be determined, the data for this animal are not included in the pharmacokinetic evaluation for group 5. The mean Cmax for the 10-minute intravenous infusion is 3183.6 pg / ml. Three animals have a Tmax at 5 minutes and an animal has a Tmax at 10 minutes; although the GLP-1 concentrations are generally similar at the 5 and 10 minute time points for all four animals. A terminal 2 terminal for the group was estimated to be 30.9 minutes. The average AUC of 28,883.2 min * pg / ml captures the majority of the exposure profile, as the AUCinf is only slightly higher, 29,149.5 min * pg / ml. The examination of the log concentration against time profile between 10 minutes (end of infusion) and 20 minutes, suggests a biphasic elimination of GLP-1. The faster phase of a biphasic profile is generally associated with extravascular distribution and elimination, while the β phase is slower, it is considered to represent terminal elimination. The calculation of the elimination ratio during this time structure indicates an initial ti / 2 of 2 minutes or less. This initial ti / 2 should be consistent with achieving an apparent steady state in the last half of the 10-minute infusion period.

Bioavailability The bioavailability of GLP-1 was calculated after intranasal administration using AUCuitima or AUCinf, "These estimates are given in Table 15. In groups 1, 3 and 4 the concentration of inhibitor Lys (4-nitro-Z) -pyrrolidino in the formulation is 0 m, 15 mM or 25 mM respectively. of the inhibitor, the bioavailability of GLP-1 is approximately 2% while the addition of 15 mM of Lys (4-nitro-Z) -pyrrolidide in the formulation (Group 3) increases the bioavailability of GLP-1 to approximately 3% up to 5%. % The bioavailability of GLP-1 is further increased from approximately 7% to 11% in addition to 25 mM Lys (4- nitro-Z) -pyrrolidide in the formulation (Group 1). PN159 polypeptide has been shown to increase the bioavailability of peptides compared to small molecule excipients. When tested, formulation 2 (Group 2) containing PN159 and 25 mM Lys (-nitro-Z) -pyrrolidide has an approximate GLP-1 bioavailability of 6 to 11%, which is equivalent to the bioavailability observed for LPG -1 in the presence of 25 mM of Lys (-nitro-Z) -pyrrolidide without PN159. PN159 has the same effect as 10 mM EDTA in 1N d bioavailability of GLP-1.

Table 15 Bioavailability of GLP-1 in Rabbits Administered with 75 pg / kg via Intranasal Instillation (7.5 and kg / kg intravenous dose for calculation, Group 5) Bioavailability = [Dosage (Group 5) X AUCXXX (Group X) } / [Dosage (Group X) x AUCXXX (Group 5)] x 100 The terminal half-life for GLP-1 is approximately 30 minutes in rabbit and longer than anticipated based on published reports that show a terminal half-life of 10 minutes or less for rat or human (Parkes, D. et al., "Pharmacokinetic Actions of Exendin-4 in the rat: Comparison with Glucagon-like Peptide-1", Drug Development Research 53: 260-267, 2001; Deacon C, Therapeutics Strategies Based on Glucagon-like Peptide-1, Perspectives in Diabetes 53 : 2181-2189, 2004. The ti / 2 for rabbit is consistent between each of the groups and animals, and thus was reported within the study.The terminal / terminal 2 calculation is dependent on the portion of the log concentration against the time curve used for determination of overlap (see Kel's definition in Table 13) For the estimate described above, the modeling program is left to choose the best fit for the Kel estimate The manual selection for the adjustment curve 10 minutes (end of l infusion) up to 90 minutes indicating a terminal half-life in the range of 10 to 12 minutes (data not shown). The separation provides a second means to evaluate the disposition of a drug after administration. The separation can be considered as the intrinsic capacity of the body or its organs to remove a drug from the blood (Basic Ckinical Pharmacokinetics, 2nd ed. , Applied Therapeutics, Inc. Vancouver, Washington). However, in this case, GLP-1 may remain in the blood, but is considered removed for separation assessment purposes. The reason for this is that GLP-1 exists in two different states: an active state consisting of a peptide fragment represented by amino acids 7-36 and an inactive state, a result of metabolism, consisting of a peptide fragment represented by amino acids 9-36. In the conversion to the inactive state, GLP-1 is considered effectively removed from the body. As the assay used in this document to detect GLP-1 is specific to the active form of GLP-1 (7-36), the presence of the active form of GLP-1 in the blood does not interfere with the evaluation of the separation of GLP-1. For intranasal groups, the upper separation value (CL-F) is noted for Group 4 with an average of the group of 15,160.0 ml / min / kg. The mean value for group 3 is 6977.0 ml / min / kg. The separation is similar for Groups 2 and 1, 2716.0 ml / min / kg and 3032.8 ml / min / kg, respectively. As expected, the estimated separation for each group is inversely proportional to the systemic exposure. Separation values for intranasal groups are also adjusted for the bioavailability of GLP-1. The adjusted spacing for Groups 4 and 3 is 7580 ml / min / kg and 1395 ml / min / kg, respectively. The separation values for groups 1 and 2, (-11% bioavailability for each) were determined to be 275 ml / min / kg and 247 ml / min / kg, respectively. The separation adjusted for Groups 1 and 3 are similar to the estimated separation of 259.8 ml / min / kg for the intravenous dose group (Group 5). The total dose of Lys- (4-nitro-Z) -pyrrolidide is approximately 0.004 mMoles / kg per Groups 1, 2 and 5. Blood levels of Lys (4-nitro-Z) -pyrrolidide were not determined in this study, as such the bioavailability after intranasal administration is not known. The presence of Lys (4-nitro-Z) -pyrrolidide in the nasal formulation has the potential to protect active GLP-1, from metabolism within the nasal mucosa, and it is assumed that reasonable bioavailability, within the circulation systemic The protection of the metabolism by the nasal mucosa is consistent with a higher Cmax for GLP-1 when Lys (4-nitro-Z) -pyrrolidide is present in the formulation. However, since both GLP-1 fragments are susceptible to cross-circulation, one assay for total GLP-1 (amino acid fragment 7-36 and 9-36) will be required to confirm a higher percentage is active GLP-1. The protection of the metabolism within the blood is indicated by the similarity in separation values for GLP-1 between the intravenous group (Group 5) and the groups intranasal with the highest inhibitor concentration (Groups 1 and 2).

Summary These data show the surprising and unexpected discovery provided by the intransal pharmaceutical formulations of GLP-1 with the DPP IV inhibitor Lys (4-nitro-Z) -pyrrolidide resulting in increased GLP-1 bioavailability. In addition, the GLP-1 bioavailability is dependent on the concentration of the inhibitor in the formulation. In the absence of the inhibitor, the bioavailability of GLP-1 is approximately 2%. The inclusion of increased Lys (4-nitro-Z) -pyrrolidide at 15 mM, the bioavailability of GLP-1 at approximately 5%, and with 25 mM Lys (4-nitro-Z) -pyrrolidide, the bioavailability for GLP-1 is approximately 11%. The potential for local (nasal tissue) and systemic inhibition of GLP-1 metabolism is indicated by the data obtained. The formulation with PN159, in addition to 25 mM of Lys (4-nitro-Z) -pyrrolidide, is also investigated to evaluate the potential for greater bioavailability with this strong binding modulator. Under the conditions of this study, the effect of PN159 on the bioavailability of GLP-1 is not greater than the effect of EDTA on the formulation.

EXAMPLE 6 Formulations of GLP-1 in Use and Sale Stability "in use" stability is defined as those studies that involve a formulation stored inside a vial fixed with an actuator and atomized in accordance with the appropriate therapeutic regimen (in this case, three times a day (TID)), and placed at specific storage temperatures. Vials with actuators are initially primed, but are not primed between subsequent atomizations. The priming is defined as atomized until a complete atomization is visually apparent, then acting one or more times before dosing. The vials are stored at 30 ° C / 65% relative humidity (RH) every time and are atomized within 10 minutes after removing them from the chamber for each dosage. The vials are operated three times or once a day, depending on the regimen selected for each study. The time between each atomization is at least 1 hour and the visual / physical observations were noted. Studies were conducted in TID / 30 ° C use. The formulations used in this study contain 5 mg / ml of GLP-1, 10 mg / ml of EDTA, 10 mM of Citrate Buffer (pH 3.5), and no condom. The vials were filled, primed and powered by TID, and stored at 30 ° C / 65% RH for 10 days. The recovery and purity in use of GLP-1 after 7 days of spray TID are shown in the Table 16 and 17. Recovery of the peptide in use is greater than 95 ± 2.2% for up to 7 days. The purity of the total peptide in use is 98 ± 2.1% after 10 days TID / 30 ° C / 65% RH.

Table 16 Recovery in use of GLP-1 after TID / 30 ° C / 65% *% tag requirement of T = 0 Estimated values Table 17 Purity in use of GLP-1 After TID / 30 ° C / 65 Stability studies "for sale" are defined as those studies that involve the formulation stored inside a closed (ie, capped) vial, placed in specific storage and accelerated temperature conditions (ie, 5 ° C, 25 ° C, 40 ° C and / or 50 ° C) for specified periods of time. Stability studies were conducted for sale in formulations that have positive results from the in vitro selection rounds. Formulations were prepared and stored at 5 ° C, 25 ° C and 35 ° C. Table 18 shows the formulations that were tested. The results of formulations # 5 and # 6 (containing GLP-1 and EDTA) show that after 56 days of storage the recovery of GLP-1 is 100% at 5 ° C, and > 97% at 25 ° C and > 90% at 35 ° C.

Table 18 Summary of Formulations submitted to Trial for Stability for Sale A stability test was made for sale in the same batch of formulation made for the study of in use (5 mg / ml of GLP-1, 10 mg / ml of EDTA, 10 mM of Citrate Buffer (pH 3.5) and without preservative). The formulation was stored at 5 ° C. The results of stability for sale are shown below in Tables 19 to 20.

Table 19 Stability for sale, Recovery of GLP-1 in Storage at 5 ° C Table 20 Stability for sale, Purity of GLP-1 in Storage at 5 ° C EXAMPLE 7 In Vivo Pharmacodynamics of Intranasal GLP-1 Formulations Three studies compare the pharmacodynamic (PD) actions of GLP-1 (7-36) amide and synthetic exendin-4 (exanatide) in blood glucose after intranasal administration (1N) of GLP-1 in the presence or absence of the DPP-V inhibitor or subcutaneous (SQ) injection of exenatide. The studies were performed in the ZDF rat model of the oral glucose tolerance test (OGTT) used in human studies. The pharmacodynamic actions were evaluated by monitoring the blood glucose and insulin levels in the blood. The concentration of glucose in the blood was determined using a Synchron CX4 analyzer and appropriate Glucose Reagent Kit (Beckman Coulter, Brea, CA USA). The pharmacokinetic parameters were determined using WinNonlin software (Pharsight Corporation, Version 5.01, Mountain View, CA). The concentration of insulin in the blood was determined by ELISA. A concentration of acetaminophen in blood was determined using a Synchron CX4 analyzer and Acetominofen Reagent Kit (Beckman Coulter, Brea, CA USA). Acetaminophen was used as a marker of gastric emptying because acetaminophen generally has negligible absorption of the stomach. Peak concentration time (Tmax) and peak concentrations (Cmax) after dose administration reflect the time at which gastric emptying occurs, and the gastric emptying profile (eg, absorption into the systemic circulation after of the release of the stomach) reflects the duration of the emptying. He AUC reflects the total exposure. Acetaminophen will be administered in Study 2 and Study 3 to monitor gastric emptying.

Study 1 The design of the complete study for evaluation of blood glucose and insulin values in the Study is summarized in Table 21.

Table 21 Study 1: Design of the Pharmacodynamic Study (PD) Group Designations for the Determination of Blood Glucose and Insulin * Male ZDF rats Formulations for rats treated with GLP-1 in Study 1 are Fl = 10 mg / ml EDTA, 10 mM Shock absorber. Citrate (pH 3.5) WITHOUT INHIBITOR; F2 = 10 mg / ml EDTA, 10 mM Citrate Buffer (pH 3.5) and 25 mM H-Lys (4nitro-Z) pyrrolidide (the inhibitor is commercially available from Bachem)); and F3 = 10 mg / ml EDTA, 10 mM Citrate Buffer (pH 3.5) and 25 mM L-proline-boroproline (Pro-boropro was synthesized based on the information in Patent Application 2004/0229829 Al, Bachovchin, Willia, W. et al.). The PD results for study 1 are shown in Table 22. The AUC (0-150) glucose for rats treated with GLP-1 formulations (p / yp / or DPP-IV inhibitor) is -15% lower than exanatide in rats treated with placebo. In study 1, glucose is given at 2 g / kg, while in subsequent studies glucose is given at 1 g / kg. As a result, in Study 1, the glucose reduction observed is relatively lower than the other studies (for example, AUC (0-150) of 0-16%) because the glucose challenge is greater. For study 1, acetaminophen is not dosed to monitor gastric emptying.

Table 22 Study 1:% Calculated Reduction in Glucose AUC GLP-1 compared to Placebo and Exenatide There is no difference in blood glucose between the GLP-1 formulations when administered 20 minutes after the oral glucose dose. There is a difference in the blood glucose profile between the formulations of GLP-1 with inhibitor (F2 and F3) versus without inhibitor (Fl) when administered before the dose, -10 minutes. Both formulations containing a DPP-IV inhibitor (F2 and F3) result in a delay in Cmax (from 45 to about 120 minutes). Significant levels of GLP-1 delay gastric emptying; the rabbit PK study showed a 5% higher BA for the formulation of GLP-1 containing DPP-IV inhibitor than without it. In study 2, it was added acetaminophen to a formulation containing a DPP-IV inhibitor to compare gastric emptying in a GLP-1 formulation without inhibitor. The insulin and glucose measurement data for the Placebo at -10 minutes, Fl for GLP-1 (without inhibitors) at -10 minutes and Fl for GLP-1 (without inhibitors at +20 minutes show a greater insulin increase with Fl of GLP-1 when administered at either -10 and +20 minutes relative to placebo.

Study 2 The PD actions of GLP-1 (amide 7-36) in the presence or absence of the DPP-IV inhibitor in blood glucose after intranasal instillation (GLP-1) in a rat model of the OGTT were evaluated. Study 2. The design of the complete study for blood glucose evaluation and insulin values are summarized in Table 23. OGTT study 2 is conducted as a bolus dose of lg / kg of a glucose solution administered by oral gavage . Acetaminophen was co-administered at a dose of 100 mg / kg into the glucose solution. For purposes of time recording the OGTT was designated 0 minutes in time.

Table 23 Study 2: OGTT Design of Pharmacodynamics and Group Designations for Determination of Blood Glucose and Insulin * Rat ZDF The formulations used in Study 2 include: Fl = 10 mg / ml EDTA, 10 mM Citrate buffer (pH 3.5) WITHOUT INHIBITOR; F3 = 10 mg / ml EDTA, 10 mM Citrate buffer (pH 3.5) and 25 mM L-proline-boroproline. Blood glucose was evaluated at the following time points: preOGTT and 5, 10, 15, 30, 45, 60, 75, 90, 120, 180 and 240 minutes post-OGTT. The blood insulin was calculated in the following periods of time: preOGTT and 15, 30, 60, 120 and 240 minutes post-OGTT. The APAP blood levels were evaluated at the following time points: preOGTT and 5, 10, 15, 30, 45, 60, 75, 90, 120 and 180 minutes post-OGTT.

The results of Study 2 show that there is a reduction in AUC of glucose (correct baseline) compared to the control, especially for formulations without inhibitor (Fl). Both formulations (Fl and F3) with and without inhibitor increase insulin after dosing. The 1 N dose of 100 ug / ml of GLP-1 showed that in the presence of the inhibitor, gastric emptying was delayed. By increasing the dose of GLP-1 to 1N, gastric emptying is also affected without the presence of the inhibitor. When gastric emptying is delayed due to high doses or the presence of the inhibitor, glucose absorption is delayed. Table 24 shows the difference in AUC of glucose for treatment groups compared to Placebo.

Table 24 Summary of Differences in Glucose AUC Study 3 In study 3, the pharmacodynamic (PD) actions of GLP-1 (amide 7-36) without inhibitor and exenatide in blood glucose after intranasal instillation (GLP-1 or saline) or subcutaneous injection of exenatide in a rat model of the OGT. In study 3, a GLP-1 formulation was used that did not have DPP-IV inhibitors (Fl = 10 mg / ml EDTA, 10 mM citrate buffer (pH 3.5) WITHOUT INHIBITOR). The main goal of Study 3 is to compare Fl GLP-1 (IN) to exenatide (SQ). PD was evaluated by monitoring the level of blood glucose and insulin in the blood. Acetaminophen was administered to monitor gastric emptying (from Study 2). In Study 3 the OGTT was conducted using a dose of 1 g / kg bolus of a glucose solution administered by oral gavage. When relevant, acetaminophen, at a dose of 100 mg / kg, was co-administered into the glucose solution. For purposes of time recording, the OGTT was designated 0 minutes of time. The time record for the administration of the treatment dose is relative to the OGTT. The single treatment dose is administered at -10 minutes. Dosage is conducted twice, with the first dose at -10 minutes and the second dose at +35 minutes. Therefore, administration for doses by saline, GLP-1 or exenatide occurs at -10 minutes, +35 minutes or -10 and +35 minutes, in relation to the OGTT. Blood was collected for pharmacodynamic analysis, including AUCO-240 and Cmax calculations. Blood glucose was evaluated at the following time points: preOGTT and post-OGTT (5, 15, 30, 45, 60, 75, 90, 120, 180 and 240 minutes). The preOGTT and post-OGTT blood insulin were evaluated (5, 15, 30, 45, 60, 75, 90, 120 and 180 minutes). The blood levels of acetaminophen preOGTT and post-OGTT were evaluated (15, 30, 45, 60, 75, 90, 120 and 180 minutes). The total design for the evaluation of Study 3 of blood glucose and insulin values are summarized in Table 25. The total design for evaluation of gastric emptying is summarized in Table 26.

Table 25 Study 3: Design of Pharmacodynamic Study and Group Designations for Blood Glucose and Insulin Determinations * Male ZDF rats Table 26 Study 3: Design of the Pharmacodynamic Study and Group Designations for Gastric Emptying Assessments * Male ZDF rats The results of Study 3 show dramatic reduction (-60%) of AUC0-240 of correct blood glucose after intranasal GLP-1 dosing compared to Saline or SQ administration of Exenatide. AUCo-240 and Cmax values are shown in Table 27. The change in glucose concentration corrected for endogenous glucose is shown in Figure 1. These pharmacodynamic results show the surprising and unexpected discovery that intranasal administration of pharmaceutical formulations of GLP- 1 results in a decreased glucose concentration in the blood.

Table 27 Pharmacodynamic Data for Parameters of Blood Glucose Pharmacokinetics After Administration of GLP-1, Exenatide and Saline in Rats Determined in Oral Glucose Tolerance Test The mean insulin data of the group for GLP-1 and placebo treatment groups of Study 3 are shown in Figure 2. Insulin levels in saline-treated rats (placebo) show a slight decrease after OGTT. With a single dose of 100 g / kg of GLP-1 at -10 minutes, there is a marked increase in levels of insulin in the blood in response to an OGTT. The administration of GLP-1 at -10 minutes and +35 minutes results in a sudden increase in insulin immediately after each dose. This pattern indicates that GLP-1 is responsible for the release of insulin in response to elevated blood glucose. Data from acetaminophen group media for the GLP-1 and placebo treatment groups (with standard deviation for the placebo group) are shown in Figure 3. In In study 3, the total exposure determination (AUC) indicated is less than 3% difference in the total amount of acetaminophen absorbed in the systemic circulation among the three treatment groups. The AUC values are 6804 pg * min / kg, 6672 pg * min / kg and 6951 pg * min / kg for the control of saline (placebo), groups 100 pg / kg of GLP-1 and 1000 pg / kg GLP-1 , respectively. However, the absorption profile is different. In rats treated with placebo, the Tmax for acetaminophen is 30 minutes post-dose; with a mean CmaK of the group of 78 pg / ml with a standard deviation of ± 21 pg / ml. A CMA: < slightly lower, 66 ± 21 pg / ml was noted 30 minutes after the dose for the group 100 pg / kg GLP-1; however, acetaminophen concentrations between this group and the control group are similar for more time points evaluated. The Cmax for acetaminophen is lower, 45 ± 30 ug / ml, in the group 1000 pg / ml GLP-1. In addition, the Tmax seems to originate between 30 and 90 minutes after the dose, and blood levels of acetaminophen are markedly higher at the last points of time. The results for the group 1000 pg / kg GLP-1 are consistent with a delay in gastric emptying and a longer profile for gastric emptying after a high dose of GLP-1. The results of Study 3 show that GLP-1 without inhibitor significantly lowers glucose and that the dose can be administered before or after food. The formulation of GLP-1 to 1N decreases glucose AUC while dosing of SQ Exenatide in ZDF rats at either 0.6 or 3 ug / kg does not.

Summary of PD Results These armadodynamic results show the surprising and unexpected discovery that intranasal administration of GLP-1 pharmaceutical formulations results in a decreased glucose concentration in the blood. In addition, the pattern of insulin concentration in the blood indicates that GLP-1 is the factor responsible for the release of insulin in response to elevated blood glucose. The results for the study of acetaminophen are consistent with a delay in gastric emptying and a longer profile for gastric emptying at high doses of GLP-1 even in the absence of the DOO-IV inhibitor. A lower dose of GLP-1, which is also effective in lower blood glucose, does not impact gastric emptying. The decrease in the concentration of glucose in the blood, especially through high insulin levels is an effective treatment for Type II Diabetes. In addition, the delay in gastric emptying can increase satiety and promote weight loss. These data support the efficacy of the intranasal formulation GLP-1 described in these assays for use in the treatment of metabolic disorders such as obesity and diabetes.

EXAMPLE 8 Transmucosal Synthetic Formulations of Exendin-4 with Enhancers A variety of excipients were tested for in vitro optimization of exenatide formulations. Formulations of transmucosal exenatide were generated by combining exenatide and excipients (which include enhancers, solubilizers, surfactants, chelators, stabilizers, buffers, toners and preservatives). Multiple rounds of formulation selection were made and divided into two series, A and B. The A series focuses on changing the concentrations of the excipient of solubilizers (?? - ß-CD), surfactants (DDPC), chelators (EDTA) and stabilizers (gelatin). Shock absorbers such as citrate buffer, tartrate buffer and glutamate (MSG) were also tested. Series B selects alternative excipients for their potential to enhance exenatide permeation. Several concentrations of potential permeation enhancers were tested including cyclodextrins, glycosides, fatty acids, phosphatidylcholines, GRAS compounds, PN159, gelatin and others. In addition to the selection of potential permeation enhancers, it will also be selected by varying concentrations of shock absorber (citrate buffer, tartrate buffer) and toning / stabilizing excipients (mannitol, NaCl). Condoms were tested such as sodium benzoate (NaBz) and benzalkonium chloride (BAK). Table 28 lists the excipients tested in in vitro selection. Out of the unique formulations 372 that were tested, eleven formulations were recommended for use in preclinical rabbit PK studies in vivo, see Table 29.

Table 28 Excipients Tested in Optimization of In Vitro Exempt Formulation Excipients Series B Class DMe- -CD Cyclodextrins 20 - 50 mg / mL ?? - ß-CD Cyclodextrins 20 - 50 mg / mL B-CD Cyclodextrins 10-20 mg / mL n-Decyl-PD-maltopyranoside Glycosides 2.5- 10 mg / mL n-Dodecyl- -D-maltopyranoside Glycosides 2.5 - 10 mg / mL n-Tetradecyl-PD-maltopyranoside Glycosides 2.5- 10 mg / mL n-Octyl-PD-maltopyranoside Glycosides 10-20 mg / mL n-Hexadecyl-D-maltopyranoside Glycosides 2.5-10 mg / mL n-Octyl-PD-galactopyranoside Glycosides 5- 10 mg / mL Octyl-P-glucopyranoside Glycosides 5- 10 mg / mL Octyl-p-glucopyranoside Glycosides 5 - 7.5 mg / mL n-Heptyl-P-D-glucopyranoside Glycosides 2.5-10 mg / mL Dodecanoylsucrose Glycosides 1 - 5 mg / mL Decanoylsucrose Glycosides 1 - 5 mg / mL Sodium Caprate (10) Unsaturated fatty acids 5-50 mg / mL Sodium Caprilate (8) Unsaturated fatty acids 20-100 mg / mL Phosphatidyl choline Phosphatidylcholine 0.177- 1.77 mmol Dimiristoil Glycer Phosphatidylcholine (14: 0) DMPC Phosphatidylcholine 0.177- 1.77 mmol Dilauroyl Glycer Phosphatidylcholine (12: 0) DLPC Phosphatidylcholine 0.177- 1.77 mmol Di Nonanoil Glycero Phosphatidylcholine (9: 0) Di Non-PC Phosphatidylcholine 0.177- 1.77 mmol Dipalmitoyl Glycer Phosphatidylglycerol (16: 0) DPPG Phosphatidylcholines 0.177- 1.77 mmol Dimiristoil Glycero Phosphatidylglycerol (14: 0) DMPG Phosphatidylcholines 0.177- 1.77 mmol Palmitoyl-DL-Carnitine Other 1 - 5 mg / mL Sodium Glycolate Other 1 - 10 mg / mL Nitroso-N-acetyl-penicillamine S Other 0.2 - 1 mg / mL Cremefor EL Other 1 - 5 mg / mL Table 29 Recommended Transmucosal Exempt Formulations Pre-clinical Studies EXAMPLE 9 Formulation of Transcutaneous Exenatide that Induces Opening of Strong Joints in vitro TER, LDH, MTT and in vitro permeation assays were performed for exenatide formulations as described in the protocols in Example 2 above. For all exenatide formulations containing enhancers, TER of approximately 350-700 ohms x cm2 to approximately 5-20 ohms x cm2 after sixty (60) minutes of the incubation period. All exenatide formulations, with the exception of controls, contain EDTA. As a calcium chelator, EDTA is known to open strong bonds by calcium scavenger. In a static environment similar to the in vitro tissue culture system used in this document, the removal of calcium from the solution leads to the opening of significant strong junctions. No reduction or TER was observed in the control of glutamate plus exenatide (SG) containing only exenatide in glutamate buffer with sodium chloride as a tonifier. The control of glutamate plus exenatide indicates that the opening of strong bonds is not an inherent characteristic of exenatide itself. The TER of inserts after sixty (60) minutes exposed to the control of glutamate is similar to those of the inserts exposed to the medium for sixty (60) minutes. The X triton control is the lowest possible TER, which results from the removal of the cellular barrier as expected. To verify that the reduction of TER by the exenatide formulations resulted from modulation of strong bonds by enhancers and not cell death, LDH and MTT assays were performed using the same cell line, MatTek Corp., as used in the TER assays. Exenatide formulations show no increase significant in cytotoxicity as measured by% LDH. The exenatide formulations have less than 20% LDH loss. Similarly, the control medium does not show cytotoxicity. In contrast, the group treated with Triton X control showed significant toxicity, as expected. Cell viability was assessed using the MTT assay (MTT-100, MatTek kit). The exenatide formulations do not show a significant increase in toxicity as measured for% MTT with the exception of three formulations, JW-239-126-1, JW-239-126-19, and J -239-126-24 , which have around 50% MTT. On the other hand, exenatide formulations show greater viability than 80% MTT. Similarly, the control medium does not show cytotoxicity. In contrast, the group treated with Triton X control showed significant toxicity as expected. The results of the permeation study are shown in Table 30.

Table 30 Transmucosal Exempt Formulations from Previous Studies MTT Permeation LDH Times% Perm in Laugh < // o or // or dev. Vol. De al ctrl desv. detour estd Load MSG 1 / or estd 0 // or estd / o AKL-225- 126-2 Full 346 6.0 1 .6 103.2 16.5 2.5 2.0 JW-239-9-21 Full 501 7.2 1 .6 73.3 18.7 7.2 4.1 JW-239- 126-3 Full 365 8.6 1 .7 90.6 20.7 3.4 1 .6 JW-239- 126-7 Full 430 8.2 4.7 84.5 17.9 6.1 2.9 JW-239-126- 14 Full 829 16.7 8.5 52.0 1 1.0 6.3 2.0 JW-239-126-15 Full 361 13.4 14.4 1 12.2 20.9 3.4 4.1 JW-239-126-19 Medium 1426 20.6 7.1 90.1 1 7.9 3.5 2.8 JW-239- 126-24 Medium 2264 32.7 8.2 98.5 15.9 7.3 1.4 JW-239-126-19 Full 492 7.1 1.1 41 .8 6.1 15.4 0.8 JW-239-126-24 Full 469 6.8 1 .1 45.6 6.4 13.9 1 .5 A L-310-27-12 full 306 4.4 1.5 101 .7 1 1.6 10.8 0.6 The permeation results for the formulations shown 300 to 830 times increased in permeation in relation to a control formulation containing only 3 mg / ml of exenatide and monosodium glutamate. The formulations dosed at half the volume shown at 1400 and 2200 times of increase in permeation compared to the control.

EXAMPLE 10 PK Rabbit Results for Transmucosal Exempt Formulations Exendin-4 formulations are shown for in vivo tests in Table 31.

Table 31 Transmucosal Exempt Formulations from Previous Studies A PK study of exendin-4 was conducted in rabbits comparing PK results for exendin-4 administered by IV and IN. IN formulations include IN control (without intensifiers), IN IX enhancer + gelatin, 2X IIN intensifier and IN 2X intensifier + gelatin (formulations shown in Table 31). The results of the PK study are shown in Table 32 and Figure 4.

Table 32 Pharmacokinetics Results for Exendin-4 in Conej The PK results show that IN 2X intesif and IN 2X intensif + gel are the best formulations of exendin-4 developed, tested in the rabbit study. Both IN 2X intensif and IN 2X intensif + gel result in higher PK values than the IV or IN controls.

EXAMPLE 11 Formulations of Transmucosal Exenatide Alternatives Formulations of exendin-4 were developed alternatives for transmucosal administration to test the following: 1) increased storage stability, 2) increased bioavailability of exendin-4 and 3) increased pharmacodynamic effect determined by measuring insulin and glucose levels. Modifications of previously tested exendin-4 formulations were prepared to remove DDPC and gelatin completely but in a formulation. "OEF" was used to refer to formulations containing 80 mg / ml Me-b-CD and 5 mg / ml EDTA (without DDPC or gelatin) in 10 mM acetate buffer. A second change in the formulations previously tested is the addition of arginine to some of the formulations. The OEF formulations are described in Table 33.

Table 33 Transmucosal Exenatide Formulations for In Vitro Permeation Studies Abbreviations: DDPC = didecanoyl L-a-phosphatidylcholine, EDTA = Edetate dihydrate sodium,? -β-DC = methyl ^ -cyclodextriña random.

Permeation Percentage In vitro permeation studies showed that OEF formulations (without DDPC and gelatin) intensifying the permeation of exendin-4 to a greater extent than the previously tested exendin-4 formulations (see formulations previously tested in Table 31) while comparable or better cell viability is provided. The permeation results comparing the formulations are show in Table 34.

Table 34 In vitro Permeation Results for Formulations Exendin-4 after 90 Minutes Pharmacokinetics Study A pharmacokinetic (PK) study in vivo in rabbits demonstrated that the formulations "OEF + arg" and "2x intensif + gel, arg" when administered intranasally produce enhanced bioavailability of exendin-4, comparable to or exceeded that formulations previously tested. The mean peak of the plasma concentration exendin-4 is greater than the formulation OEF + arg. Table 35 shows a comparison of the average PK parameters (Tmax, AUC, ti / 2 and Kel) for the formulations tested. The coefficient of variance for each PK parameter is shown in Table 36, and the absolute bioavailability (% F) for each intranasal formulation is shown in Table 37.

Table 35 Average PK Parameters for Exendin-4 Formulations Table 36 Average Coefficient of Variation Group T c AUClas, AUCinf Formulation # (min) (pg / mL) (min * pg mL) (min * pg / mL) IV 1 0.0 61 .6 46.0 52.0 ÍN Control 2 99.1 164.1 156.5 1 19.0 lx intensif + gel 3 53.5 79.8 80.3 75.8 2x intensif 4 73.8 52.6 51 .0 43.8 2x intensif + gel 5 38.0 36.4 49.7 52.5 2x intensif + gel, arg 6 96.2 72.7 88.8 94.4 OEF + arg 7 46.5 42.7 40.2 48.4 Table 37 Absolute Bioavailability (% F) using AUCuitimo Results for the PK study show that OEF + arg have higher bioavailability% (16.6%). Two other intranasal formulations, 2x intensif and 2x intensif + gel, also have significantly enhanced bioavailability compared to the IN control (12.6% and 11.5%, respectively).

Pharmacodynamic Studies An in vivo study in rabbits provided pharmacodynamic (PD) parameters that show that the formulations "OEF + arg" and "2x intensif + gel, arg" when delivered intranasally produce insulin and glucose responses, comparable to or greater than formulations previously described (see Table 31). Table 38 includes PD parameters for average insulin levels (Tmax, Cmax and AUC) and Table 39 shows the average glucose levels (Tmax and Cmin). Surprisingly, the "2x intensif + gel, arg" formulation elicits the larger PD response even though it does not result in the increased bioavailability increase of exendin-4 in the PK study.

Table 38 PD Parameters for Insulin Levels After Dosage of Formulations in Rabbits T c AUClasl Formulation Group # N (min) (μ ?? / mL) (???? * μ ?? / ???) IV 1 3 15.0 40.2 490.3 IN Control 2 1 60.0 8.0 60.0 l x intensif + gel 3 1 5.0 8.5 1 12.5 2x intensif 4 4 21 .3 34.4 5 1 8.9 2x intensi + gel 5 2 17.5 38.5 247.5 2x intensif + gel, arg 6 2 30.0 58.5 121 1 .3 OEF + arg 7 3 16.7 38.3 513.5 Table 39 Parameters for Glucose Levels After Dosage of Rabbit Formulations Stability Accelerated stability studies show that the "OEF" formulation variants provide enhanced stability for exendin-4 in relation to the "2x intensif + gel" formulation. The stability of the "OEF" formulations are comparable to that of commercially available BYETTA®. After four (4) weeks at 40 ° C, the purity of the "OEF" formulations is 85.6-86.0%, while the purity of "2x intensif + gel" is 83.7%. After four (4) weeks at 50 ° C, the purity of the "OEF" formulations is similar to BYETTA ® (72.2-72.9% versus 72.9) while the "2x intensif + gel" formulation has precipitated. No precipitation was observed in the simple formulations. In summary, accelerated stability studies demonstrated that acetate cushion provides increased physical stability for formulations of exendin-4 relative to the tartrate buffer. Less precipitation was observed in buffered acetate formulations than in tartrate buffered formulations. Generally, mono-ionogenic buffers (acetate, arginine, lactate) provide better stability than poly-ionogenic buffers (tartrate, citrate). The pH range 4.7-5.5 provides better chemical stability for exendin-4 compared to pH = 4.25. Arginine provides slightly improved chemical stability. In addition to methionine and aspartic acid decrease the chemical stability of exendin-4. The optimal osmolality for chemical stability of exendin-4 is 200-250 mOsm / kg H20. Additionally, accelerated stability studies indicate that variants of the "OEF" formulation provide enhanced stability for exendin-4 in relation to the "2x intensif + gel" formulation. The stability of the "OEF" formulations is comparable to that of commercially available BYETTA®. At 40 ° C, after four weeks, the purity of the "OEF" formulations is 85.8-86.0% while the purity of "2x intensif + gel" is 83.7. More dramatically, at 50 ° C, after four weeks, the purity of the "OEF" formulations is comparable to BYETTA® (72.2-72.9% versus 72.9%) while the "2x intensif + gel" formulation has precipitate Although it has not been tested in this study, it is noted that viscosity enhancers can be added to the formulations. Condoms may also be included in the formulations, which may include, but are not limited to, benzalkonium chloride, methyl and propyl parabens and / or chlorobutanol. Examples of other previously tested condoms, which can be included in the formulations to promote antimicrobial effectiveness including the following combinations: 0.033% methylparaben + 0.017% Propylparaben; 0.18% methylparaben + 0.02% Propylparaben; 0.10% up to 0.50% chlorobutanol; 0.10% up to 0.25% chlorobutanol + 0.033% methylparaben + 0.017% Propylparaben; 0.10% up to 0.25% chlorobutanol + 0.18% Methylparaben + 0.02% Propylparaben; 0.5% benzyl alcohol; 0.5% benzyl alcohol + 0.033% methylparaben + 0.017% propylparaben; 0.5% benzyl alcohol + 0.18% methylparaben + 0.02% Propylparaben; 0.5% phenylethanol + 0.1 up to 0.25% chlorobutanol; 0.5% Phenylethanol + 0.033% Methylparaben + 0.017% Propylparaben; 0.5% Phenylethanol + 0.18% Methylparaben + 0.02% Propiparaben; 5 mg / ml EDTA; 0.01-0.1% benzalkonium chloride; 7 mg / ml ethane; 5 mg / ml benzyl alcohol + 2.5 mg / ml phenylethyl alcohol.

Variable concentrations of exenatide can be used to achieve a desired dose, for example, concentrations of 0.5 mg / ml up to 6 mg / ml. The "OEF" and "OEF + arg" formulations can provide stability and improved bioavailability for other GRPs, including, but not limited to, other GLP-1 analogues. A formulation of exendin-4 for transmucosal administration with increased half-life, decreased input costs and increased bioavailability are identified from the OEF improvements to the formulation. The increased half-life means that the commercial product will last longer, allowing less products to expire and reducing its manufacture. Increased bioavailability means efficacy and potentially increased therapeutic utility of the drug product. It can also mean cost savings by reducing the amount of API (exendin-4, GLP-1) required for the efficacy of the drug product. The removal of DDPC potentially allows for greater ease in product approval by the FDA as the DDPC is a new excipient. It also decreases the time required to manufacture the product. In addition, DDPC is a costly excipient and elimination significantly reduces the cost of supplies associated with the drug product. The elimination of gelatin results in a decrease in the time required for the manufacture of the product and also reduces the manufacturing cost with the elimination of an excipient. The invention also includes preservative-free GRP formulations Such formulations do not contain a condom. In the absence of an antimicrobial excipient, the formulation will be filled under sterile conditions in a condom-free delivery device. A preservative-free exenatide formulation may include formulations such as those shown in Tables 40 and 41. Exenatide concentrations may vary from 2-12 mg / ml.

Table 40 Preservative Free Formulation for "2x Gelatine Enhancers" Component Concentration (mg / mL) (mM) Exempt Depends on the required formulation potency Methyl-β-cyclodextrin 80.0 - 29.4 - 30.4" L-Alpha-phosphatidylcholine didecanoyl 2.0 1 .77 Edetate di sodium 5.0 6.72 Tartrate sodium and potassium 7.566 26.81 Tartaric acid 0.479 3.19 Gelatine 2.5 several Purified water or sterile water for CS irrigation Table 41 Preservative-free formulation for "lx intensifiers + Gelatin" Other embodiments may vary levels of permeation enhancing components such as the following: ??? ß-CD (20-80 mg / ml), DDPC (0-2 mg / ml), E DTA (2-10 mg / ml) , tartrate buffer (0 - 30 mM), gelatin, and sodium chloride. Other modalities may contain a different formulation:? E-ß- ?? (80 mg / ml), E DTA (5 mg / ml), arginine (2.8 or 10 mM) and / or acetate buffer (10 mM), and sodium chloride with a pH of 4.9-5.6. The concentrations of these excipients may vary. The The formulations may also contain a viscosity enhancer such as gelatin, hydroxymethylcellulose, carboxymethylcellulose or carbopol. Various concentrations of exenatide can be used to achieve a desired dose. The concentrations can vary from 0.5 mg / ml to 25 mg / ml, for example.

EXAMPLE 12 Pharmacodynamic Study (PD) in Single Dose of Intranasal Administration of Glucose Regulating Proteins (GRPs) in a Rat Model of the Glucose Tolerance Test (OGTT) The pharmacodynamic actions of GLP-1 were tested ( amide 7-36) and exendin-4 e blood glucose after intranasal instillation in a rat model of the oral glucose tolerance test (OGTT). Two formulations of exendin-4 were evaluated and a unique formulation of GLP-1 was used. The GLP-1 (based on EDTA) formulation (# 2) contains GLP-1 (amide 7-36) and the following ingredients: 10 mg / mL disodium EDTA; 10 mM citrate buffer, pH 3.5. The exendin-4 (EDTA-based) formulations (# 2, # 3, # 4, and # 5) contained exendin-4 and the following ingredients: 10 mg / mL disodium EDTA and 10 mM arginine buffer, pH 4.0 . The formulations of exendin-4 (based on PDF) (# 6 and # 7) contain exendin-4 and the following ingredients: 45 mg / ml of ?? - ß-Cd, 1 mg / ml of DDPC, 1 mg / ml of disodium EDTA, 100 mM of sorbitol, 25 mM of lactose, 5 mg / ml of CB, and 10 mM of arginine buffer, pH 4.0 . A summary of the tested formulations is shown in Table 42. The study design and group designations are shown in Table 43.

Table 42 Formulations Used to Evaluate Intranasal Dosing Exendin-4 and GLP-1 Abbreviations: Me- -CD = methyl-beta-cyclodextrin, EDTA edetate disodium, DDPC = L-a-phosphatidylcholine didecanoyl, CB = Chlorobutanol.

Table 43 Study Design and Group Designations Pharmacodynamics were evaluated by monitoring levels of blood glucose and insulin in the blood; Acetaminophen was coadministered with glucose to monitor gastric emptying. The study includes a single dose treatment in ZDF rats of approximately 11 weeks of age (5 rats per treatment group). The OGTT was conducted as a 1 g / kg bolus dose of a glucose solution administered by oral gavage. Acetaminophen was administered at a dose of 100 mg / kg. For purposes of time recording the OGTT was designated as time = 0 minutes. Dosage administration for saline, GLP-1 or exendin-4 is at -10 minutes in relation to the OGTT. Blood glucose was evaluated at the following time points: pre-OGTT (twice) and 5, 15, 30, 45, 60, 75, 90, 120, 180 and 240 minutes post-OGTT. Blood insulin was evaluated at the following time points: pre-OGTT and 15, 30, 45, 60, 75, 90, 120, and 180 minutes post-OGTT. The APAP blood level was evaluated at the following time points: pre-OGTT and 15, 30, 45, 60, 75, 90, 120, and 180 minutes post-OGTT.

Pharmacodynamic results Table 44 shows the results of the armadrodynamics of the glucose level. Peak levels (Cmax) for glucose are 70% control in animals administered with GLP-1; similarly the AUC for glucose for the time structure from 0 to 60 (AUC0-60) minutes post-OGTT is 70% control. Administration of Exendin-4 has minimal effects on Cmax (ranging from 86% to 99% of control) or AUC0-6o (ranging from 91% to 103% of control). There is no clear dose response for exendin-4.

Table 44 Results of Pharmacodynamics in Glucose Level for GRPs: GLP-1 and Exendin-4 Table 45 shows the pharmacodynamic results of the glucose level after correction for endogenous glucose. Peak levels (Cmax) for glucose are 51% control in animals administered with GLP-1; similarly the AUC for glucose for the time structure from 0 to 60 (AUC0-6o) minutes post-OGTT is 46% control, and AUCo-240 (0 to 240 minutes post-OGTT) is 88% control. The administration of exendin-4 in formulations based on EDTA or based on PDF has moderate effects on Cmax (which varies from 73% to 87% of control) or AUC0-6o (which varies from 91% to 103% of control) . No minimum effect was noted for AUCo-24C that varies from 77% to 125% control.

Table 45 Results of Pharmacodynamics of the Glucose Level (Corrected for Endogenous Glucose) for GRPs: GLP-1 Exendin-4.

The results of the insulin response (corrected for endogenous insulin) show that post-OGTT insulin levels in control animals are similar or slightly lower, compared to post-dose values. Nasal administration of GLP-1 at 100 g / kg is associated with up to 2-fold increase in insulin from 5 to 45 minutes post-OGTT. Insulin levels after dosing of exendin-4 to 2, 10 and 20 pg / kg (EDTA-based formulation) demonstrated that a dose-dependent increase in insulin with peak levels is approximately 0.7 times, 1.5 times and 4 times, respectively, below the pre-dose values for each group. The response is limited to approximately 45 minutes post-OGTT. After nasal administration of exendin-4 in the formulation based on PDF at 2 or 10 pg / kg dose, peak insulin levels are 1.5 times or 2.7 times, respectively, below the pre-dose. The response is limited to approximately 45 minutes post-OGTT.

Results of Gastric Emptying Table 46 shows the results of acetaminophen (gastric emptying). In controls, peak levels of acetaminophen (Cmax) of 73 ng / ml occurred at 30 minutes postdose (Tmax). In animals administered GLP-1, a slightly lower Cmax (66 ng / ml) was noted; however, Tmax is greater than suggested and delayed gastric emptying at this dose level. The administration of exendin-4 in the formulation of EDTA at 2 or 10 pg / kg or formulations based on PDR at 2 ug / kg is similar to the control for Tmax (30 to 45 minutes) and Cmax (approximately 70 to 73 ng) / ml). The administration of exedin-4 in EDTA-based training at 20 pg / kg or formulations based on PDF at 10 μg / kg showed a decrease in Cmax (approximately 51 to 54 ng / ml) and at least for 20 g / kg (EDTA-based formulation) a slightly prolonged exposure profile. The results suggest that gastric emptying is impacted at these dose levels.

Table 46 Summary of Results of Acetaminophen GRP (Gastric Emptying) Summary Based on the pharmacological effects of GLP-1 and exendin-4 with respect to glucose-dependent stimulation of released insulin, EDTA-based and PDF-based intanasal formulations effectively deliver the active drug to systemic targets. Stimulation for the insulin released is dose dependent. The modulation of peak levels (Cmax) or exposure (AUC) for blood glucose in animals administered with GLP-1 or exendin-4, compared to controls. The stimulation of insulin release and modulation of gastric emptying, by GLP-1 and exendin-4 are similar pharmacological bases for modulation of the blood glucose profile after an oral glucose load. The inhibition of gastric emptying is a known pharmacological action of GLP-1 and exendin-4. The change in time for floor concentration (Tmax) or peak concentration (Cmax) for acetaminophen is consistent with the pharmacology induced by GLP-1 and exendin-4. These data support the efficacy of the GLP-1 and exendin-4 formulations described in these assays for use in the treatment of metabolic disorders such as obesity and diabetes. Although the foregoing invention has been described in detail by way of example for purposes of clarity of understanding, it will be apparent to the technician that certain changes and modifications are understood by the description and can be practiced without undue experimentation of the scope of the appended claims, which they are presented by means of illustration, without limitation.

Claims (57)

1. CLAIMS Having described the invention as above, property is claimed as contained in the following. 1. A pharmaceutical formulation for epithelial delivery of glucose-regulating peptide (GRP), characterized in that it comprises a GRP and permeability enhancers, wherein the enhancers comprise a solubilizing agent, an active surface agent and an agent that intensifies viscosity. 2. The formulation according to claim 1, characterized in that the enhancers increase the permeability of the GRP by at least 10%. 3. The formulation according to claim 1, characterized in that the enhancers increase the permeability of the GRP by at least 15-fold. 4. The formulation according to claim 1, characterized in that the enhancers increase the bioavailability of GRP by at least about 25-fold. 5. The formulation according to claim 1, characterized in that the enhancers increase the bioavailability of GRP by at least about 50-fold. 6. The formulation in accordance with the claim 1, characterized in that the bioavailability of GRP is at least about 1% relative to a supply by subcutaneous injection. The formulation according to claim 1, characterized in that the bioavailability of GRP is at least about 5% relative to a supply by subcutaneous injection. The formulation according to claim 1, characterized in that the bioavailability of GRP is at least about 10% relative to a supply by subcutaneous injection. 9. The formulation according to claim 1, characterized in that a time for maximum concentration in the animal circulation, Tmax, is less than about 45 minutes. 10. The formulation according to claim 1, characterized in that a time for maximum concentration in the circulation of the animal, Traax, is greater than about 60 minutes. 11. The formulation according to claim 1, characterized in that a time for maximum concentration in the circulation of the animal, Tmax, is less than about 30 minutes. 12. The formulation according to claim 1, characterized in that it also comprises a chelating agent. The formulation according to claim 12, characterized in that the chelating agent is selected from the group consisting of ethylene diamine tetraacetic acid and ethylene glycol tetraacetic acid. 14. The formulation according to claim 1, characterized in that the solubilizing agent is selected from the group consisting of a cyclodextrin, hydroxypropyl-cyclodextrin, sulfobutyl ether-β-cyclodextrin and methyl-p-cyclodextrin. 15. The formulation according to claim 14, characterized in that the solubilizing agent is met? -β-cyclodextrin. 16. The formulation according to claim 1, characterized in that the surface active agent is selected from the group consisting of nonionic polyoxyethylene ether, fusidic acid and its derivatives, sodium taurodihydrofusidate, didecanoyl La-phosphatidylcholine, polysorbate 80, polysorbate 20, polyethylene glycol, cetyl alcohol, polyvinyl pyrrolidone, polyvinyl alcohol, lanolin and sorbitan monooleate. 17. The formulation according to claim 16, characterized in that the surface active agent is didecanoyl L-a-phosphatidylcholine. 18. The formulation in accordance with the claim 1, characterized in that it comprises a GRP, methyl- -cyclodextrin, and didecanoyl L- -phosphatidylcholine. 19. The formulation according to claim 1, characterized in that the GRP is present at a concentration greater than about 50 mg / ml. 20. The formulation according to claim 1, characterized in that the GRP is present at a concentration of about 0.1 to about 50 mg / ml. The formulation according to claim 1, characterized in that the GRP is present at a concentration of about 0.25 to about 10 mg / ml. 22. The formulation according to claim 1, characterized in that the GRP consists of GLP-1, GLP-1 analogs, fragments, or derivatives. 23. The formulation according to claim 1, characterized in that the GRP consists of exendin-4, exendin-4 analogs, fragments, or derivatives. 24. The formulation according to claim 22 or 23, characterized in that the GRP derivative comprises the GRP covalently bound to a hydrophobic portion. 25. The formulation according to claim 24, characterized in that the portion Hydrophobic is an alkali chain. 26. The formulation according to claim 24, characterized in that the hydrophobic portion is a fatty acid chain. 27. The formulation according to claim 1, characterized in that the formulation is a non-sterile formulation. The formulation according to claim 26, characterized in that it also comprises a condom selected from the group consisting of chlorobutanol, methyl paraben, propyl paraben, butyl paraben, benzalkonium chloride, benzethonium chloride, sodium benzoate, sorbic acid, phenol. and ortho-, meta or para-cresol. 29. The formulation according to claim 1, characterized in that the formulation is a sterile formulation. 30. The formulation according to claim 22, characterized in that it also comprises a dipeptidyl aminopeptidase (DPP) IV inhibitor. 31. The formulation according to claim 30, characterized in that the DPP IV inhibitor is lys (4-nitro-Z) -pyrrolidide. 32. The formulation according to claim 31, characterized in that lys (4-nitro-Z) - pyrrolidide is at a concentration of at least about 5 mM. 33. The formulation according to claim 31, characterized in that lys (4-nitro-Z) -pyrrolidide is at a concentration of at least about 30 mM. 34. The formulation according to claim 31, characterized in that lys (-nitro-Z) -pyrrolidide is at a concentration of at least about 50 mM. 35. The formulation according to claim 1, characterized in that the formulation is suitable for intranasal delivery of GLP-1. 36. The formulation according to claim 1, characterized in that the formulation has a pH of about 3 to about 6. 37. The formulation according to claim 1, characterized in that the formulation has a pH of 3.5 ± 0.50. 38. The formulation according to claim 36 or 37, characterized in that it also comprises a citrate buffer. 39. The formulation according to claim 36 or 37, characterized in that it also comprises a monoiontogenic buffer. 40. A use of a GRP in the manufacture of a medicament for treating a metabolic syndrome in a mammal, wherein the medicament comprises a formulation of any one of claims 1-39, and wherein said medicament comprises a pharmaceutically effective amount of the GRP. 41. The use of claim 40, wherein the epithelial administration of the drug to a mammal increases plasma insulin levels. 42. The use of claim 40, wherein epithelial administration of the drug to a mammal reduces blood glucose levels. 43. The use of claim 40, wherein the epithelial administration of the drug to a mammal retards gastric emptying. 44. A method for treating a metabolic syndrome, characterized in that it comprises administering intranasally to a mammal the formulation according to any of claims 1-39. 45. The method according to the claim 44, characterized in that the metabolic syndrome treated is selected from the group consisting of Type 2 diabetes, Type 1 diabetes, impaired glucose tolerance, hyperglycemia, metabolic syndrome (syndrome X and / or insulin resistance syndrome), glucosuria, acidosis metabolic, arthritis, cataracts, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, diabetic cardiomyopathy, obesity, conditions excacerbated by obesity, hypertension, hyperlipidemia, atherosclerosis, osteoporosis, osteopenia, fragility, bone loss, bone fracture, acute coronary syndrome, short stature due to growth hormone deficiency, infertility due to polycystic ovary syndrome, anxiety, depression, insomnia, chronic fatigue, epilepsy, feeding disorders, chronic pain, alcohol addiction, diseases associated with intestinal mobility, ulcers, irritable bowel syndrome, syndrome of inflammatory bowel; short bowel syndrome; and the prevention and progression of disease in diabetes Types 2. 46. The method according to the claim 44, characterized in that the metabolic syndrome treated is type II diabetes. 47. The method according to claim 44, characterized in that the metabolic syndrome treated is obesity. 48. The method according to claim 44, characterized in that the metabolic syndrome treated is hyperlipidemia. 49. The method according to claim 44, characterized in that said administration reduces the glucose levels in the blood. 50. The method according to claim 49, characterized in that said administration reduces blood glucose levels by at least 10%. 51. The method of compliance with the claim 44, characterized in that said administration is post-prandial. 52. The method according to claim 44, characterized in that said administration increases plasma insulin levels. 53. The method according to claim 52, characterized in that said administration increases plasma insulin levels by at least 10%. 54. The method according to claim 44, characterized in that said administration delays gastric emptying. 55. The method according to claim 44, characterized in that said intranasal administration comprises supplying an aerosol comprising about 0.3 to about 30 g of GRP per kg of weight of the mammal. 56. The method according to claim 44, characterized in that said intranasal administration comprises supplying an aerosol comprising 0.5 to 1.0 mg of GRP per 0.1 ml of solution. 57. The method according to claim 44, characterized in that said intranasal administration is given at a dosage frequency of between 0.1 to 24 hours.