MXPA06009331A - Compositions and methods for enhanced mucosal delivery of y2 receptor-binding peptides and methods for treating and preventing obesity - Google Patents

Abstract

Pharmaceutical compositions and methods are described comprising at lease one Y2 receptor-binding peptide, such as peptide YY (PYY), Neuropeptide Y (NPY) or Pancreatic Peptide (PP) and one or more mucosal delivery-enhancing agents for enhanced nasal mucosal delivery of the peptide YY, for treating a variety of diseases and conditions in mammalian subjects, including obesity.

Description

COMPOSITIONS AND METHODS TO IMPROVE THE MUCOSAL SUPPLY OF PEPTIDES THAT JOIN THE RECEIVER Y2 AND METHODS TO TREAT AND PREVENT OBESITY Obesity and its associated disorders are common and very serious public health problems in the United States and throughout the world. Obesity of the upper body is the strongest known risk factor for type-2 diabetes mellitus and is a strong risk factor for cardiovascular diseases. Obesity is a recognized risk factor for hypertension, arteriosclerosis, congestive heart failure, shock, gallbladder disease, ostioarthritis, sleep apnea, reproductive disorders such as polycystic ovary syndrome, breast, prostate and colon cancers, and incidences increased complications of general anesthesia. It reduces the duration of life and carries a serious risk of previous co-morbidities, as well as disorders such as infections, varicose veins, acanthosis nigricans, exema, exercise intolerance, insulin resistance, hypertension, hypercholesterolemia, cholelithiasis, orthopedic damage and thromboembolic disease. Obesity is also a risk factor for the group of conditions called insulin resistance syndrome or "X'X syndrome. It has been shown that when peripherally administered certain peptides that bind to the Y2 receptor in mammals induce weight loss. Peptides that bind to the Y2 receptor are neuropeptides that bind to the Y2 receptor Neuropeptides are small peptides originated from large protein precursors synthesized by peptidergic neurons and endocrine / paracrine cells Frequently the precursors contain biologically multiactive peptides There is a great diversity of neuropeptides In the brain caused by the alternating overlap of the primary genetic transcription and differential processing of the precursor, the neuropeptide receptors serve to discriminate between ligands and to activate the appropriate signals.These peptides that bind to the Y2 receptor belong to the family of peptides including he peptide YY (PYY), neuropeptide Y (NPY) and pancreatic peptide (PP). NPY is a peptide of 36 amino acids and is the most abundant neuropeptide to be identified in the mammalian brain. NPY is an important regulator in both central and peripheral nervous systems and influences a diverse range of physiological parameters, including effects on psychomotor activity, food intake, central endocrine secretion and vasoactivity in the cardiovascular system. High concentrations of NPY are found in sympathetic nerves supplying the coronary, cerebral and renal vasculature and have contributed to vasoconstriction. NPY binding sites have been identified in a variety of tissues, including spleen, intestinal membranes, brain, aortic smooth muscle, kidney, testis and placenta. The neuropeptide Y (NPY) pharmacological receptor is currently defined by the ratio of structural activity within the pancreatic polypeptide family. This family includes NPY, which is synthesized mainly in neurons; PYY, which is synthesized mainly by endocrine cells in the pancreas. These peptides of approximately 36 amino acids have a compact helical structure that involves a "PP-fold" in the middle of the peptide. Specific features include a polyproline helix at residues 1 through 8, a β-turn at residues 9 through 14, an a-helix at residues 15 through 30, a C-terminus projecting outwards into residues 30 a 36 and an amide at the carboxyl terminus, which appears to be critical for biological activity. The peptides have been used to define at least five receptor subtypes known as Yl y2 y3 y4 and Y5. The recognition of the Yl receptor by NPY involves both the N- and C-terminal regions of the peptide; an exchange of Gln34 with Pro34 is reasonably tolerated. The recognition of the Y2 receptor by NPY depends mainly up to four C-terminal residues of the peptide (Arg33 - Gln34 -Arg35 - - - Tyr36 -NH2) preceded by an amphipathic an a-helix; the exchange of Gln34 with Pro34 is not well tolerated. One of the key pharmacological characteristics that distinguishes Yl and Y2 is the fact that the Y2 receptor (and not the Yl receptor) has a high affinity for the carboxyl terminal fragment of the NPY- (13-36) NPY peptide and the fragment PYY, PYY (22-36). It has been shown that when a 36 amino acid peptide called peptide YY (1-36) [PYY (1-36)] [YPIKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY, SEQ ID NO: 1] is administered peripherally by injection to an individual, produces weight loss. and thus it can be used as a drug to treat obesity and related diseases, Morley, J. Neuropsychobiology 21: 22-30 (1989). Subsequently it was found that to produce this effect PYY binds to a Y2 receptor and the binding of a Y2 agonist to a Y2 receptor causes a decrease in the ingestion of carbohydrates, proteins and amount of feed, Leibowitz, S.F. et al. Peptides, 12: 1251-1260 (1991). An alternate molecule of PYY is PYY (3-36) IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY [SEQ ID NO .: 2], Eberlein, Eysselein et al. Peptides 10: 797-803, 1989). This fragment constitutes approximately 40% of the total immunoactivity similar to PYY with in intestinal human and canine extracts and approximately 36% of the total immunoreactivity of PYY with plasma in a fasted state to slightly over 50% after eating. Dipeptidyl peptidase-IV (DPP4) is apparently a cleavage product of PYY. PYY3-36 is, according to reports received, a selective ligand to receptors Y2 and Y5, which appears to be pharmacologically unique in truncated analogs of the N-terminus NPY prefixes (i.e. fragments of the C-terminal). It has also been shown that a fragment of PYY having only residues 22-36 will remain attached to the Y2 receptor. However, if any of the carbonyl terminals of the peptide is split, the lost one loses its ability to bind to the Y2 receptor. Accordingly, a PYY agonist is a peptide, which has a partial sequence of the total length of PYY and can bind to the Y2 receptor in the arcuate nucleus of the hypothalamus. Hereinafter the term PYY refers to the total length of PYY and any fragment of PYY that binds to a receiver Y2. It is known that PYY and PYY3-36 can be administered by intravenous infusion or by injection to treat life-threatening hypotension such as that encountered in shock, especially that caused by endotoxins (US Pat. No. 4,839,343), to inhibit proliferation. of pancreatic tumors in mammals, by administration by perfusion, parenteral, intravenous or subcutaneous and by implantation (US Patent 5,574,010) and to treat obesity (Morley, J. Neuropsychobiology 21: 22- - - (1989) and Patent Application 20020141985). It is also claimed that PYY can be administered by the parenteral, oral, nasal, rectal and typical routes to domestic or human animals in an amount effective to increase the weight gain of said subject by improving the gastrointestinal absorption of a co-transported sodium-dependent nutrient. (U.S. Patent 5,912,227). However, for the treatment of obesity and related diseases, including diabetes, the form of administration should be limited to IV intravenous infusion without formulations optimized in effectiveness for alternative administration of PYY3-36. None of these prior techniques provide formulations containing PYY or PYY (3-36) combined with excipients designed to improve mucosal delivery (ie, nasal, buccal, oral) or teach the value of the peptide formulations that bind to the Y2 receptor. endotoxin-free for administration not infused. In this way, it is necessary to develop formulations and methods to administer PYY3-36. The generation of aerosol formulations can improve the absorption of formulations on mucous surfaces (nasal, buccal, oral, vaginal and rectal) as well as on the surface of the skin. Review: O'Riordan TG. Formulations and Nebulizer performance (Performance of Formulations and Nebulizer). Respir Care 2002 Nov; 47 (11): 1305-12; discussion 1312-3.

However, the physical forces associated with droplet formation frequently destroy or denature proteins and peptides. For example, recombinant human deoxyribonuclease (rhDNase) was substantially denatured during processing as shown by the significantly reduced monomer content. Similarly, albumin was affected by processing and only 50-75% of the monomer was retained compared to 86% of the original material. Bustami RT, Chan HK, Dehghani F, Foster NR. Generation of protein micro-particles by aerosol supply using modified carbon dioxide at high pressure. Pharm Res. 2000 Nov; 17 (11): 1360-6. The physical stability of a formulation of human growth hormone (hGH) peptide hormone to overexposure to air / water interfaces (agitation by mixing) has been investigated. The effect of this stress on the formation of soluble and insoluble aggregates has been studied. Aggregates were characterized and quantified by size exclusion HPLC and UV spectrophotometry. The mixing with stirring of hGH solutions (0.5 mg / mL) in phosphate buffer, pH 7.4, for only 1 min, caused the precipitation of 67% of the drug as insoluble aggregates. These aggregates were non-covalent by nature Katakam M, Bell LN, Banga AK. J Pharm Sci. 1995 Jun; 84 (6): 713-6. SUMMARY OF THE INVENTION The present invention fills the aforementioned needs and satisfies additional objectives and advantages by providing novel methods and compositions, effective for especially nasal mucosal delivery of a peptide that binds to the Y2 receptor such as PYY, Pancreatic peptide (PP) and NPY for Treat obesity, induce satiety in an individual and promote weight loss in an individual and prevent or cure diabetes. In certain aspects of the invention, the peptide that binds to the Y2 receptor is delivered in formulations to the intranasal mucosa so that it can increase the concentration of the peptide that binds to the Y2 receptor by at least 5 pmol, preferably by at least 10 pmol, in the blood plasma of a mammal when a dose of the Y2 receptor agonist formulation is administered intranasally. Moreover, preferred formulations will be able to reach the plasma concentration of the peptide that binds to the Y2 receptor of a mammal by lOpmol, preferably 20 pmol, when the peptide that binds to the Y2 receptor is administered intranasally. When 150 μg is administered intranasally the preferred formulation will be able to reach in the plasma of the mammal the concentration of the Y2 receptor agonist by at least 40 pmol per liter of plasma. When 200 μg of the peptide which binds to the Y2 receptor is intranasally administered, the formulations of the present invention induce at least 80 pmol, per liter of plasma increase in the peptide that binds to the Y2 receptor. In preferred embodiments, high concentrations of the peptide that binds to the Y2 receptor remains elevated in the plasma of the mammal for at least 30 minutes, preferably at least the following 60 minutes of a single intranasal dose of the peptide that binds to the Y2 receptor. Preferably the peptide that binds to the Y2 receptor is a peptide PP, PYY or NPY and the mammal is a human. In a more preferred embodiment the peptide that binds to the Y2 receptor is a PYY peptide, preferably PYY (3-36) and the mammal is human. The present invention also relates to the formulation of a peptide that binds to the Y2 receptor that can reach the concentration of the peptide that binds to the Y2 receptor in blood plasma of a mammal of at least 5 pM when a dose is administered to a mammal containing at least 50 μg of the peptide that binds to the Y2 receptor. In preferred embodiments, high concentrations of the peptide that binds to the Y2 receptor remains elevated in the plasma of the mammal for at least 30 minutes, preferably at least 60 minutes after the single intranasal dose of the peptide that binds to the Y2 receptor.

The present invention also relates to the formulation of a peptide that binds to the Y2 receptor that can reach the concentration of the peptide that binds to the Y2 receptor in the blood plasma of a mammal by at least 20 pM when the mammal is administered a dose containing at least 100 μg of the peptide that binds to the Y2 receptor. In preferred embodiments, elevated concentrations of the peptide that binds to the Y2 receptor remains elevated in the plasma of a mammal for at least 30 minutes, preferably for at least the next 60 minutes at a single intranasal dose of the peptide that binds to the Y2 receptor. . The present invention also relates to the formulation of a peptide that binds to the Y2 receptor administered intranasally to a mammal that can reach the concentration of the peptide that binds to the Y2 receptor in the blood plasma of the mammal by at least 30 pM when administered a dose containing at least 150 μg of the peptide that binds to the Y2 receptor. In preferred embodiments, high concentrations of the peptide that binds to the Y2 receptor remains elevated in the plasma of the mammal for at least 30 minutes, preferably for at least the next 60 minutes at a single intranasal dose of the peptide that binds to the Y2 receptor. Preferably the mammal is a human The present invention also relates to the formulation of a peptide that binds to the Y2 - - receptor administered intranasally to a mammal that can reach the concentration of the peptide that binds to the Y2 receptor of at least 60 pM when The mammal is administered a dose containing at least 200 μg. In preferred embodiments, high concentrations of the peptide that binds to the Y2 receptor remains elevated in the plasma of the mammal for at least 30 minutes, preferably for at least the next 60 minutes at a single intranasal dose of the peptide that binds to the Y2 receptor. Preferably the mammal is a human. The present invention is also directed to an intranasal formulation of a Y2 receptor agonist that is substantially free of proteins or polypeptides that stabilize the formulation. In particular, the preferred formulation is free of proteins such as albumin and collagen-derived proteins such as gelatin. In other aspects of the present invention the transmucosal formulation of the peptide that binds to the Y2 receptor comprises a peptide that binds to the Y2 receptor, water and a solubilizing agent having a pH of 3-6.5. In a preferred embodiment, the solubilizing agent is a cyclodextrin. In another embodiment of the present invention the transmucosal formulation of the peptide that binds to the Y2 receptor comprises a peptide that binds to the Y2 receptor, water, a solubilizing agent, preferably a cyclodextrin and at least one polyol, preferably 2 polyols. In alternative embodiments the formulation may contain one or all of the following: a chelating agent, a surface-acting agent and a buffering agent. In another embodiment of the present invention the formulation comprises a peptide that binds to the Y2 receptor, water, chelating agent and a solubilizing agent. In another embodiment of the present invention the formulation comprises a peptide that binds to the Y2 receptor, water and a chelating agent having a pH of 3-6.5. In another embodiment of the present invention the formulation comprises a peptide that binds to the Y2 receptor, water, chelating agent and at least one polyol, preferably two polyols. Additional embodiments may include one or more of the following: a surface-acting agent, a solubilizing agent and a buffering agent. In another embodiment of the present invention the formulation comprises a peptide that binds to the Y2 receptor, water and at least two polyols, such as lactose and sorbitol. Additional agents that may be added to the formulation include, without limitation, a solubilizing agent, a chelating agent, one or more buffering agents and an action agent on the surface. Improving the intranasal delivery of a peptide agonist that binds to the Y2 receptor according to the methods and compositions of the invention takes into account the effective pharmaceutical use of those agents to treat a variety of diseases and conditions in mammalian subjects. The present invention fills this need provided by a liquid or dehydrated formulation of the peptide that binds to the Y2 receptor wherein the formulation is substantially free of a stabilizer which is a polypeptide or a protein. The PYY liquid formulation comprises water, PYY and at least one of the following additives selected from a group consisting of polyols, surface-acting agents, solubilizing agents and chelating agents. The pH of the formulation is preferably from 3 to about 7.0, preferably from 4.5 to about 6.0, more preferably near 5.0 ± .03. Another embodiment of the present invention is an aqueous Y2 receptor binding formulation of the present invention comprising water, a peptide that binds to the Y2 receptor, a polyol and a surface-acting agent wherein the formulation has a pH of about 3.0 to about 6.5 and the formulation is substantially free of a stabilizer which is a protein or a polypeptide. Another embodiment of the present invention is an aqueous Y2 receptor binding formulation comprising water, a Y2 receptor binding peptide, a polyol and a solubilizing agent wherein the formulation has a pH of from about 3.0 to about 6.5 and the formulation is sub-substantially free of a stabilizer which is a protein or polypeptide. Another embodiment of the present invention is an aqueous peptide formulation that binds to the Y2 receptor comprising water, peptide that binds to the Y2 receptor, a solubilizing agent and a surface-acting agent wherein the formulation has a pH of about 3.0. to about 6.5 and the formulation is substantially free of a stabilizer which is a protein or polypeptide. Another embodiment of the invention is an aqueous peptide formulation that binds to the Y2 receptor comprising water, peptide that binds to the Y2 receptor, a solubilizing agent, a polyol and a surface-acting agent wherein the formulation has a pH of about 3.0 to about 6.5 and the formulation is substantially free of a stabilizer which is a protein or polypeptide. In another aspect of the present invention, the stable aqueous formulation is dehydrated to produce a dehydrated formulation of the peptide that binds to the Y2 receptor comprising peptide that binds to the Y2 receptor and at least one of the following additives selected from a group consisting of polyols, surface-acting agents, solubilizing agents and chelating agents, wherein said dehydrated formulation of the peptide that binds to the Y2 receptor is substantially free of a stabilizer which is a protein or polypeptide such as albumin, collagen or collagen-derived protein such like gelatin. Dehydration can be carried out by various means such as lyophilization, spray dehydration, salt induced precipitation and dehydration, vacuum drying, rotary evaporation or supercritical precipitation of C02. In one embodiment, the peptide that binds to the dehydrated Y2 receptor comprises a peptide that binds to the Y2 receptor, a polyol and a solubilizing agent, wherein the formulation is substantially free of a stabilizer that is a protein. In another embodiment, the dehydrated formulation of the peptide that binds to the Y2 receptor comprises a peptide that binds to the Y2 receptor, a polyol and a surface-acting agent wherein the formulation of the peptide that binds to the Y2 receptor is substantially free of a stabilizer that is a protein or polypeptide. In another embodiment, the dehydrated formulation of the peptide that binds to the Y2 receptor comprises a peptide that binds to the Y2 receptor, a surface-acting agent, and a solubilizing agent wherein the formulation of the peptide that binds to the Y2 receptor is substantially free. of a stabilizer that is a protein or polypeptide.

In another embodiment of the present invention, the dehydrated formulation of the peptide that binds to the Y2 receptor comprises a peptide that binds to the Y2 receptor, a polyol, a surface-acting agent and a solubilizing agent wherein the peptide formulation that is binds the Y2 receptor is substantially free of a stabilizer that is a protein or polypeptide. Any solubilizing agent can be used but one selected from the group consisting of hydroxypropyl-β-cyclodextran, sulfobutylether-β-cyclodextran, methyl-β-cyclodextrin and chitosan is preferred. Generally a polyol is selected from the group consisting of lactose, sorbitol, trehalose, sucrose, mannose and maltose and derivatives and homologs thereof. A satisfactory surface action agent is selected from a group consisting of L-a-phosphatidylcholine didecanoyl (DDPC), polysorbate 20 (Tween 20), polysorbate 80 (Tween 80), polyethylene glycol (PEG), cetyl alcohol, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), lanolin alcohol and sorbitan monooleate. In a preferred formulation, the peptide formulation that binds to the Y2 receptor comprises a chelating agent such as ethylene diamine tetraacetic acid (EDTA) or ethylene glycol tetraacetic acid (EGTA). A preservative such as chlorobutanol or benzalkonium chloride can also be added to the formulation to inhibit microbial growth. The pH is generally regulated using a buffer such as sodium citrate and citric acid and sodium acetate and acetic acid. An alternate buffer may be acetic acid and sodium acetate or succinic acid and sodium hydroxide. The preferred peptide that binds to the Y2 receptor is a peptide PYY, PP or NPY, preferably a PYY peptide (3-36). The present invention also comprises a formulation wherein the concentration of the peptide that binds to the Y2 receptor is 0.1-15.0 mg / ml, preferably 1.0-2 mg / ml and the pH of the aqueous solution is 3.0-6.5 and preferably about 5.0. ± 0.3. The present invention further includes the peptide formulation that binds to the Y2 receptor wherein the concentration of the polyol is between about 0.1% and 10% (w / v) and further where the concentration of the polyol is in the range of about 0.1% to approximately 3% (w / v). The present invention also includes a formulation wherein the concentration of the surface-acting agent is between about 0.00001% and about 5% (w / v) and wherein the concentration of the - Surface action agent is between about 0.0002% and about 0.1% (w / v). The present invention also includes a formulation, wherein the concentration of the solubilizing agent is 1% -10% (w / v) and wherein the concentration of the solubilizing agent is from 2% to 5% (p / v). The final solution can be filtered and freeze-dried, lyophilized, using methods well known to those skilled in the art and following the instructions of the lyophilization equipment manufacturer. This produces a dehydrated formulation of the peptide that binds to the Y2 receptor substantially free of a stabilizer which is a protein. In another embodiment of the present invention, the peptide formulation that binds to the Y2 receptor comprises a peptide that binds to the Y2 receptor and a pharmaceutically acceptable carrier wherein the peptide formulation that binds to the Y2 receptor has at least 1% , preferably 3% and more preferably at least 6% higher permeability in in vitro tissue in a permeability test compared to the control formulation consisting of water, sodium chloride, a buffer and the peptide that binds to the Y2 receptor, as determined by the transepithelial electrical resistance test shown in Examples 2 and 7. In a preferred embodiment, the formulation that binds to the Y2 receptor further comprises at least one excipient selected from a group consisting of a surface active agent., a solubilizing agent, a polyol and a chelating agent. Preferably the peptide that binds to the Y2 receptor is a PYY peptide, an NPY peptide or a PP peptide. In another embodiment of the present invention the formulation of a peptide that binds to the Y2 receptor provides the ability to reach the plasma concentration of a mammal of the peptide that binds to the Y2 receptor by at least 5, preferably 10, 20, 40, 60 , 80 or more pmol per liter of plasma when 100 μl of the formulation is intranasally administered to said mammal. In exemplary embodiments, the improved delivery methods and compositions of the present invention provide effective therapeutic mucosal delivery of the peptide agonist that binds to the Y2 receptor for the prevention or treatment of obesity and eating disorders in mammalian subjects. In one aspect of the invention there are provided pharmaceutical formulations suitable for intranasal administration comprising a therapeutically effective amount of the peptide that binds to the Y2 receptor and as described herein, one or more agents that improve the intranasal delivery, whose formulations are effective by the method of the invention for delivery by nasal mucosa to prevent the beginning or progress of obesity or food disorders in mammalian subjects. The delivery to the nasal mucosa of a therapeutically effective amount of a peptide agonist that binds to the Y2 receptor and one or more agents that improve intranasal delivery produce high therapeutic levels of the peptide agonist that binds to the Y2 receptor in the subject and inhibits food intake in the mammalian subject, reducing obesity symptoms or an eating disorder. The improved delivery methods and compositions of the present invention provide a therapeutically effective mucosal delivery of a Y2 receptor binding peptide for the prevention and treatment of a variety of conditions and conditions in mammalian flocks. The Y2 receptor binding peptide can be administered through a variety of mucosal pathways, for example by contacting the Y2 receptor binding peptide with a nasal mucosal epithelium, a bronchial or oral mucosal epithelium, the oral buccal surface or the surface. oral mucosal and small intestine. In exemplary embodiments, the methods and compositions are directed or formulated for intranasal delivery (e.g., nasal mucosal delivery or intranasal mucosal delivery).

In one aspect of the invention, pharmaceutical formulations suitable for intranasal administration are provided comprising a therapeutically effective amount of a Y2 receptor binding peptide agonist and one or more agents that enhance intranasal delivery as described herein , whose formulations are effective in a nasal mucosal delivery method of the invention to prevent the onset or progression of obesity, diabetes, cancer, or malnutrition or cancer-related exhaustion in a mammalian subject, or to alleviate one or more symptoms clinically very recognized obesity, as well as treating Alzheimer's disease, colon carcinoma, colon adenocarcinoma, pancreatic carcinoma, pancreatic adenocarcinoma, breast carcinoma. In another aspect of the invention, the pharmaceutical formulations and methods are directed to the administration of an agonist of the peptide binding to the Y2 receptor in combination with vitamin E succinate. An agonist of the peptide binding to the Y2 receptor in combination with succinate of Vitamin E can be administered to alleviate the symptoms or prevent the onset or decrease the incidence or severity of the cancer, for example, colon adenocarcinoma, pancreatic adenocarcinoma, or breast cancer. In another aspect of this invention, surprisingly it was found that the use of endotoxin-free Y2 receptor binding peptides, for example PYY (3-36), produces increased mucosal delivery compared to a peptide in which endotoxin is not removed . The use of endotoxin-free Y2 receptor peptides in pharmaceutical formulations is thus allowed for administration by non-infusion routes, including mucosal, nasal, oral, pulmonary, vaginal, rectal supply and the like. The above formulations and preparations and delivery methods of the invention of mucosal Y2 receptor binding peptide provide improved mucosal delivery of a Y2 receptor binding peptide to mammalian subjects. These compositions and methods may involve combinatorial formulation or coordinated administration of one or more Y2 receptor binding peptides with one or more mucosal delivery enhancing agents. Among the mucosal supply enhancing agents to be selected to achieve these formulations and methods are (A) solubilization agents; (B) agents that modify the load; (C) pH control agents; (D) inhibitors of degrading enzymes; (E) mucolytic agents or mucus cleansers; (F) ciliates; (G) membrane penetration enhancing agents (eg, (i) a surfactant, (ii) a bile salt, (iii) a phospholipid or fatty acid additive, mixed micelles, liposome, or vehicle, (iv) a alcohol, (v) an enamine, (iv) a non-donor compound, (vii) - - a long-chain antipathetic molecule (viii) a small hydrophobic penetration enhancer, (ix) sodium or a salicylic acid derivative; x) a glycerol ester of acetoacetic acid (xi) a cyclodextrin or derivative of 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 for a selected membrane component, (xvii) an inhibitor of fatty acid synthesis, (xviii) an inhibitor of cholesterol synthesis; or (xiv) any membrane of (i) - (xviii)); (H) epithelial binding physiology modulating agents, such as nitric oxide (NO) stimulants, chitosan, and chitosan derivatives; (I) vasodilating agents; (J) selective transport improving agents; and (K) stabilizing delivery vehicles, carriers, supports or species that form complexes with which the peptide binding to the Y2 receptor (s) is effectively combined, associated, contained, encapsulated. (n) or joins (n) to stabilize the active agent for improved mucosal delivery. In various embodiments of the invention, the peptide binding to the Y2 receptor is combined with one, two, three, four or more of the mucosal supply enhancing agents mentioned in (A) - (K), above. These mucosal supply enhancing agents can be mixed, alone or together, with the Y2 receptor binding peptide, or otherwise be combined therewith in a pharmaceutically acceptable formulation or delivery vehicle. The formulation of a Y2 receptor binding peptide with one or more of the mucosal delivery enhancing agents according to the teachings herein (optionally including any combination of two or more mucosal delivery enhancing agents selected from (A) - ( K) above) provides increased bioavailability of the Y2 receptor binding peptide after delivery thereof to a mucosal surface of a mammalian subject. Thus, the present invention is a method for suppressing the appetite, which promotes weight loss, decreases the absorption of food, or treats obesity and / or diabetes in a mammal comprising transmucosally administering a formulation comprised of an amino acid binding peptide. receptor Y2, such that when 50 μg of the Y2 receptor is administered transmucosally to the mammal the concentration of the Y2 receptor binding peptide in the plasma of the mammal is increased by at least 5 pmol, preferably at least 10 pmol per liter of plasma. Examples of such formulations are described in the foregoing.

The present invention further provides the use of a Y2 receptor binding peptide for the production of a medicament for transmucosal administration,. of a Y2 receptor binding peptide to suppress the appetite, promote weight loss, decrease food absorption, or treat obesity in a mammal such that when approximately 50 μg of the Y2 receptor is administered transmucosally to the mammal the concentration of the Y2 receptor binding peptide in the plasma of the mammal is increased by at least 5 pmol per liter of plasma. When 100 μg of the Y2 receptor binding peptide is administered intranasally to the mammal, the concentration of the Y2 receptor agonist is increased by at least 20 pmol per liter of plasma in the mammal. When 150 μg is administered intranasally, the concentration of the Y2 receptor binding peptide in plasma in the mammalian blood is increased by at least 30 pM. When 200 μg is administered intranasally, the concentration of the Y2 receptor binding peptide in plasma in the mammalian blood is increased by at least 60 pM. In preferred embodiments, high concentrations of the Y2 receptor binding peptide remains elevated in the plasma of the mammal for at least 30 minutes, preferably at least 60 minutes after a single intranasal dose of the Y2 receptor binding peptide. Preferably the mammal is a human. A mucosally effective dose of the YY peptide 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 further exemplary embodiments, the dose of peptide YY 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 50 μg to 400 μg, preferably 100 μg to 200 μg, more preferably approximately 100 μg to 150 μg. The pharmaceutical formulations of the present invention can be administered one or more times per day (eg, before a food), or 3 times a week or once a week for between one week and at least 96 weeks or even during the life of the product. patient or individual subject. In certain embodiments, the pharmaceutical formulations of the invention are administered one or more times a day, twice a day, four times a day, six times a day, or eight times a day. Agents that improve the intranasal delivery are used to improve the supply of the YY peptide in or through a nasal mucosal surface. To passively absorb drugs, the relative contribution of paracellular and transcellular trajectories for drug transport depends on the pKa, division coefficient, molecular radio and - - charge of the drug, the pH of the luminal environment in which the drug is delivered, and the area of the surface of absoción. The agent that improves the intranasal delivery 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 peptide YY through paracellular and transcellular trajectories for drug transport. In one embodiment, the pharmaceutical formulation of the present invention is adjusted from pH to between about pH 3.0 to 6.5. In a further embodiment, the pharmaceutical formulation of the present invention is adjusted from pH to between about pH 3.0 to 5.0. In a further embodiment, the pharmaceutical formulation of the present invention is adjusted from pH to between about pH 4.0 to 5.0. Generally, the pH is 5.0 + 0.3. The present invention also describes the surprising ability to successfully aerosolize the Y2 receptor binding compound, PYY (3-36), from an aqueous formulation.

Brief Description of the Drawings Figure 1 shows the stability of PYY3-36 at high temperature (40 ° C) at various pHs from 3.0 to 7.4. Figure 2 shows the data for TEER of the permeability enhancers. Figure 3 shows the cellular viabilities of candidate PYY formulations. Figure 4 shows the cytotoxic effects of candidate formulations. In figures 2-4 ENl = PBS pH 5.0 EN2 = L-Arginine (10% w / v) EN3 = Poly-L-Arginine (0.5% w / v) EN4 = Range-Cyclodextrin (1% w / v) EN5 = Alpha-Cyclodextrin (5 % w / v) EN6 = Methyl-Beta-Cyclodextrin (3% w / v) EN7 = n-Capric Sodium Acid (0.075% w / v) EN8 = Chitosan (0.5% w / v) EN9 = L-Alpha-phosphatidylcholine didecanil (3.5% w / v) EN10 = S-Nitroso-N-acetylpenicillamine, (0.02% w / v) EN11 = Pal oto-DL-Carnitine (0.5% w / v) EN12 = Pluronic-127 (0.3% w / v) EN13 = Sodium nitroprusside (0.3% w / v) EN14 = Sodium glycocholate (1% w / v) Figure 5 shows the synergistic contributions of the various components on the permeation of the drug. In figure 5 ENL is DDPC, EN2 is methyl-β-cyclodextrin, and EX1 is EDTA.

- Figure 6 shows PYY3-36 in the plasma of rats, the table represents a dose of 4.1 μg / kg, the triangle represents a dose of 41 μg / kg, and the circle represents a dose of 205 μg / kg. Figure 7 shows the linearity of the dose after intranasal administration of PYY3-36 in rats such as Cmax-Cbas pg / mL v. dose as μg / kg. Figure 8 shows the linearity of the dose after intranasal administration of PYY3-36 in rats such as AUC v. dose as μg / kg. Figure 9 shows the average plasma concentration of PYY v. time in minutes in three human volunteers who were each administered 20 μg of PYY (3-36) intranasally. Figure 10 shows the average plasma concentration of PYY v. time in minutes in three human volunteers who were each administered 50 μg of PYY (3-36) intranasally. Figure 11 shows the average plasma concentration of PYY v. time in minutes in three human volunteers who were each administered 100 μg of PYY (3-36) intranasally. Figure 12 shows the average plasma concentration of PYY v. time in minutes in three human volunteers who were each administered 150 μg of PYY3-36 intranasally. Figure 13 shows the average plasma concentration of PYY v. time in minutes in three human volunteers who were each administered 200 μg of PYY (3-36) intranasally. Figure 14 shows the plasma concentration of PYY as pmol / L v. time for five groups of healthy human volunteers who received PYY (3-36) intranasal.

The doses were 200 μg, 150 μg, 100 μg, 50 μg and 20 μg of PYY3-36. Figure 15 shows the linearity of the Cmax dose of PYY in pg / mL vs. dose of PYY (3-36) administered to human volunteers. Figure 16 shows the linearity of the dose of PYY average AUC in pg / mL vs. dose of PYY (3-36) administered to human volunteers. Figure 17 shows the visual analogue scale (VAS) vs. dose of PYY (3-36) administered to human volunteers. The question was: "How hungry are you?" The lower the score, the less hungry the individual was on a scale of 100 points. Figure 18 shows the visual analog scale (VAS) vs. dose of PYY (3-36) administered to human volunteers. The question was: "How much could you eat?" The lower the score, the lower was the hunger for a - individual on a scale of 100 points. Figure 19 shows the visual analog scale (VAS) vs. dose of PYY (3-36) administered to human volunteers. The question was: "How satisfied are you?" The lower the score, the lower the satisfaction of an individual on a scale of 100 points. Figure 20 shows the percent permeation of PYY (3-36) containing endotoxin vs. PYY (3-36) free of endotoxin). Figure 21A shows a nasal spray pump / impeller which is not engaged. Figure 21B shows the nasal spray pump / impeller which is engaged and expels a spray plume. Figure 22 shows an example of a nasal spray pattern PYY of the present invention.

Detailed Description of the Invention As noted above, the present invention provides improved methods and compositions for the mucosal delivery of the Y2 receptor binding peptide to mammalian subjects for treatment or prevention of a variety of diseases and conditions. Examples of mammalian subjects suitable for the treatment and prophylaxis according to the methods of the invention include, but are not restricted to, human and non-human primates, livestock species, such as horses, cattle, sheep, and goats, and research and domestic species, including dogs, cats, mice, rats, guinea pigs, and rabbits. In order to provide a better understanding of the present invention, the following definitions are provided: LINK PEPTIDES TO RECEIVER Y2 The Y2 receptor binding peptides used in the mucosal formulations of the present invention include the family of pancreatic poppeptides. "as used herein, it is comprised of three naturally occurring bioactive peptide families, PP, NPY, and PYY Examples of the Y2 receptor binding peptides and their uses are described in US Patent No. 5,026,685; U.S. Patent No. 5,574,010; U.S. Patent No. 5,604,203; U.S. Patent No. 5,696,093; U.S. Patent No. 6,046,167; Gehlert et.al., Proc Soc Exp Biol Med 218: 7-22 (1998); Sheikh et al., Am J Physiol, 261: 701-15 (1991), Fournier et al., Mol Pharmacol 45: 93-101 (1994), Kirby et al., J Med Chem 38: 4579-4586 (1995).; Rist et al., Eur J Biochem 247: 1019-1028 (1997), Kirby et al., J Med Chem 36: 3802-3808 (1993), Grundemar et al., Regulatory Peptides 62: 131-136 (1996). U.S. Patent No. 5,696,093 (examples of PYY agonists), U.S. Patent No. 6,046,167 In accordance with the present invention, the Y2 receptor binding peptide includes free bases, acid addition salts or metal salts, such as potassium or sa of sodium or the peptides, the peptides binding to the Y2 receptor that have been modified by such processes as amidation, glycosylation, acylation, sulfation, phosphorylation, acetylation and cyclization, (US Pat. No. 6,093,692; and US Patent. No. 6,225,445 and pegylation. PEPTIDE AGONISTS As used herein, "PYY" refers to PYY (1-36) in native sequence or in variant form, as well as derivatives, fragments, and analogs of PYY from any source, whether natural, synthetic or recombinant. The PYY must be comprised at least of the last 15 amino acid residues or analogs thereof of the sequence PYY, PYY (22-36) (SEQ ID NO: 3). Other PYY peptides, which can be used are PYY (1-36) (SEQ ID NO: 1) PYY (3-36) SEQ ID NO: 2) PYY (4-36) (SEQ ID NO: 4) PYY (5- 36) (SEQ ID NO: 5), PYY (6-36) (SEQ ID NO: 6), PYY (7-36) (SEQ ID NO: 7) PYY (8-36) (SEQ ID NO: 8) , PYY9-36 (SEQ ID NO: 9) PYY (10-36) (SEQ ID NO: 10), PYY (11-36) (SEQ ID NO: 11), PYY (12-36) (SEQ ID NO: 12), PYY (13-36) (SEQ ID NO: 13), PYY (14-36) (SEQ ID NO: 14), PYY (15-36) (SEQ ID NO: 15), PYY (16-36) ) (SEQ ID NO: 16), PYY (17-36) (SEQ ID NO: 17), PYY (18-36) (SEQ ID NO: 18), PYY (19-36) (SEQ ID NO: 19) , PYY (20-36) (SEQ ID NO: 20) and PYY (21-36) (SEQ ID NO: 21).

- These peptides typically bind to the Y receptors in the brain and elsewhere, especially Y2 and / or Y5 receptors. Typically these peptides are synthesized in endotoxin-free or pyrogen-free forms although this is not always necessary. Other PYY peptides include the PYY peptides in which changes of the conservative amino acid residue have been made, eg, site-specific mutation of a PYY peptide including [Aspl5] PYY (15-36) (SEQ ID NO: 90), [ Thrl3] PYY (13-36) (SEQ ID NO: 91), [Vall2] PYY (12-36) (SEQ ID NO: 92), [Glull] PYY (ll-36) (SEQ ID NO: 93), [AsplO] PYY (10-36) (SEQ ID NO: 94), [Val7] PYY (7-36) (SEQ ID NO: 95), [Asp6] PYY (6-36) (SEQ ID NO: 96) , [Gln4] PYY (4-36) (SEQ ID NO: 97), [Arg4] PYY (4-36) (SEQ ID NO: 98), [Asn4] PYY (4-36) (SEQ ID NO: 99 ), [Val3] PYY (3-36) (SEQ ID NO: 100) and [Leu3] PYY (3-36) (SEQ ID NO: 101). Other PYY peptides include peptides in which at least two changes of conservative amino acid residues have been made including [AsplO, Aspl5] PYY (10-36) (SEQ ID NO: 102), [Asp6, Thrl3] PYY (6- 36) (SEQ ID NO: 103), [Asn4, Aspl5] PYY (4-36) (SEQ ID NO: 104) and [Leu3, AsplO] PYY (3-36) (SEQ ID NO: 105. Also included are find the analogs of a PYY for example those described in the patents of EU 5, 604,203 and 5,574,010; Balasubramaniam, et al., Peptide Research 1: 32 (1988); Japanese Patent Application 2,225,497 (1990); - Balasubramaniam, et al., Peptides 14: 1011, 1993; Grandt, et al., Reg. Peptides 51: 151, (1994); International Application of PCT 94/03380, Patents of E.U. 5, 604,203 and 5,574,010. These peptides typically bind to the Y receptors in the brain and elsewhere, especially Y2 and / or Y5 receptors. Typically these peptides are synthesized in endotoxin-free or pyrogen-free forms although this is not always necessary. PYY agonists include rat PYY (SEQ ID NO: 72) and the truncated forms of the amino terminal corresponding to the human, the pig PYY (SEQ ID NO: 73) and the truncated forms of the amino terminal corresponding to the human and the guinea pig PYY (SEQ ID NO: 74) and the truncated forms of the amino terminal corresponding to the human. In accordance with the present invention a PYY peptide also includes the free bases, acid addition salts or metal salts, such as potassium or sodium salts of the peptides, and PYY peptides which have been by such processes as amidation, modified glycosylation , acylation, sulfation, phosphorylation, acetylation, cyclization and other well-known covalent modification methods. These peptides typically bind to the Y receptors in the brain and elsewhere, especially Y2 and / or Y5 receptors. Typically these peptides are synthesized in endotoxin-free or pyrogen-free forms although this is not always - necessary. NEUROPEPTIDE AND AGONISTS NPY is another peptide binding to the Y2 reeferor. NPY peptides include NPY (1-36) of full length (SEQ ID NO: 22) as well as NPY fragments (1-36), which have been truncated at the amino terminus. To be effective at the Y2 receptor binding, the NPY agonist must have at least the last 11 amino acid residues at the carboxyl terminus, i.e., be comprised of NPY (26-36) (SEQ ID NO: 23). Other examples of NPY agonists that bind to the Y2 receptor are NPY (3-36) (SEQ ID NO: 24), NPY (4-36) (SEQ ID NO: 25), NPY (5-36) (SEQ ID NO: 26), NPY (6-36) (SEQ ID NO: 27), NPY (7-36) (SEQ ID NO: 28), NPY (8-36) (SEQ ID NO: 29), NPY (9 -36) (SEQ ID NO: 30), NPY (10-36) (SEQ ID NO: 31), NPY (ll-36) (SEQ ID NO: 32), NPY (12-36) (SEQ ID NO: 33), NPY (13-36) (SEQ ID NO: 34), NPY (14-36) (SEQ ID NO: 35), NPY (15-36) (SEQ ID NO: 36), NPY (16-36) ) (SEQ ID NO: 37), NPY (17-36) (SEQ ID NO: 38), NPY (18-36) (SEQ ID NO 39), NPY (19-36) (SEQ ID NO: 40), NPY (20-36) (SEQ ID NO 41), NPY (21-36) (SEQ ID NO. : 42), NPY (22-36) (SEQ ID NO 43), NPY (23-36) (SEQ ID NO: 44), NPY (24-36) (SEQ ID NO: 45) and NPY (25-36) ) (SEQ ID NO: 46). Other NPY agonists include rat NPY (SEQ ID NO: 75) and the truncated forms of the amino terminal of NPY (3-36) up to NPY (26-36) as in the human form, rabbit NPY (SEQ ID NO. : 76) and the truncated forms of the amino terminal - - from NPY (3-36) to NPY (26-36) as in the human form, dog NPY (SEQ ID NO: 77) and the truncated forms of the amino terminal NPY (3-36) to NPY (26-36) ) as in the human form, pig NPY (SEQ ID NO: 78) and the truncated forms of the amino terminal of NPY (3-36) up to NPY (26-36) as in the human form, cow NPY (SEQ) ID NO: 79) and the truncated forms of the amino terminal of NPY (3-36) to NPY26-36 as in the human form, sheep NPY (SEQ ID NO: 80) and the truncated forms of the NPY amino terminal (3-36) up to NPY (26-36) as in the human and guinea pig form (SEQ 81) and the truncated forms of the amino terminal of NPY (3-36) up to NPY (26-36) as in the human form . In accordance with the present invention an NPY peptide also includes the free bases, acid addition salts or metal salts, such as potassium or sodium salts of the peptides, and the NPY peptides that have been modified by such processes as amidation, glycosylation, acylation, sulfation, phosphorylation, acetylation, cyclization and other known methods of covalent modification. These peptides typically bind to the Y receptors in the brain and elsewhere, especially Y2 and / or Y5 receptors. Typically these peptides are synthesized in endotoxin-free or pyrogen-free forms although this is not always necessary. Pancreatic peptide - - The pancreatic peptide (PP) and PP agonist also bind to the Y2 receptor. Examples of PP agonists are full length PP (1-36) (SEQ ID NO: 47) and a variety of PP fragments, which are truncated at the amino terminus. To bind to the Y2 receptor the PP agonist must have the last 11 amino acid residues at the carboxyl terminus, PP (26-36), (SEQ ID NO: 48). Examples of the other PP, which binds to the Y2 receptor, are PP (3-36) (SEQ ID NO: 49), PP (4-36) (SEQ ID NO: 50), PP (5-36) (SEQ ID NO: 51), PP (6-36) (SEQ ID NO: 52), PP (7-36) (SEQ ID NO: 53), PP (8-36) (SEQ ID NO: 54), PP (9-36) (SEQ ID NO: 55), PP (10-36) (SEQ ID NO: 56), PP (11-36) (SEQ ID NO: 57), PP (12-36) (SEQ ID NO: 58), PP (13-36) (SEQ ID NO: 59), PP (14-36) (SEQ ID NO: 60), PP (15-36) (SEQ ID NO: 61), PP (16-36) (SEQ ID NO: 62), PP (17-36) (SEQ ID NO: 63), PP (18-36) (SEQ ID NO: 64), PP (19) -36) (SEQ ID NO: 65), PP (20-36) (SEQ ID NO: 66), PP (21-36) (SEQ ID NO: 67), PP (22-36) (SEQ ID NO: 68), PP (23-36) (SEQ ID NO: 69), PP (24-36) (SEQ ID NO: 70) and PP (25-36) (SEQ ID NO: 71). Other PP agonists include sheep PP (SEQ ID NO: 82) and the truncated forms of the amino terminal of PP (3-36) to PP (26-36) as in the human form, pig PP (SEQ ID NO. : 83) and the truncated forms of the amino terminal of PP (3-36) to PP (26-36) as in the human form, dog PP (SEQ ID NO: 84) and the truncated forms of the amino terminal PP (3-36) up to PP (26-36) as in human form, cat PP - - (SEQ ID NO: 85) and the truncated forms of the amino terminal of PP (3-36) to PP (26-36) as in the human form, cow PP (SEQ ID NO: 86) and the truncated forms of the amino terminal of PP (3-36) to PP (26-36) as in the human form, rat PP (SEQ ID NO: 87) and the truncated forms of the amino terminal of PP (3-36) to PP (26-36) as in the human form, (SEQ 88) mouse and the truncated forms of the amino terminal of PP (3-36) to PP (26-36) as in the human form, and guinea pig PP ( SEQ ID NO: 89). In accordance with the present invention a PP peptide also includes the free bases, acid addition salts or metal salts, such as potassium or sodium salts of the peptides, and PP peptides that have been modified by such processes as amidation, glycosylation , acylation, sulfation, phosphorylation, acetylation, cyclization, and other known covalent modification methods. These peptides typically bind to the Y receptors in the brain and elsewhere, especially Y2 and / or Y5 receptors. Typically these peptides are synthesized in endotoxin-free or pyrogen-free forms although this is not always necessary. Agents that Improve Mucosal Supply "Agents that enhance the mucosal supply" are defined as chemicals and other excipients that, when added to a formulation comprising water, salts and / or - - common buffers and the peptide binding to the Y2 receptor (the control formulation) produces a formulation that produces a significant increase in the transport of the Y2 receptor binding peptide through a mucosa as measured by the maximum concentration in blood, serum, or brain spinal fluid (Cmax) or by the area under the curve, AUC, in a graph of concentration versus time. A mucosa includes mucosal surfaces nasal, oral, intestinal, buccal, bronchopulmonary, vaginal, and rectal and in fact includes all membranes that secrete mucus that line all the cavities or passages of the body that communicate with the outside. Agents that improve the mucosal supply are sometimes called vehicles. Endotoxin-free formulation "Endotoxin-free formulation" means a formulation containing a Y2 receptor binding peptide and one or more agents that enhance mucosal delivery ie 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 decompose or die. Pyrogenic substances include thermostable substances that induce fever (glycoproteins) from the outer membrane of bacteria and other microorganisms. Both of these - - substances can cause fever, hypotension and shock if administered to humans. Producing formulations that are free of endotoxin may require special equipment, expert technicians, and may be significantly more expensive than making formulations that are not free of endotoxin. Due to the intravenous administration of NPY or PYY simultaneously with the infusion of endotoxin in rodents it has been shown to prevent hypotension and even death associated with the administration of endotoxin alone (US Patent 4,839,343), to produce endotoxin-free formulations of these agents Therapeutics would not be expected to be necessary for non-parenteral administration (not injected). Uninfused Administration "Uninfused Administration" means any method of delivery that does not involve an injection directly into an artery or vein, a method that forces or conducts (typically a fluid) into something and especially introduces into a part of the body by means of a needle, syringe or other invasive method. Uninfused administration includes subcutaneous injection, intramuscular injection, intraperitoneal injection and non-mucosal delivery injection methods. Treatment and Prevention of Obesity As noted above, the present invention provides improved and useful methods and compositions for nasal mucosal delivery of a Y2 receptor binding peptide to prevent and treat obesity in mammalian flocks. As used herein, prevention and treatment of obesity means preventing the onset or reduction of the incidence or severity of clinical obesity by reducing the absorption of food during meals and / or reducing body weight during administration or maintaining at reduced body weight after weight loss or before weight gain occurs. The present invention provides improved and useful methods and compositions for nasal mucosal delivery of the Y2 receptor binding peptide to regions of the brain, for example, the hypothalamus or arched neurons of propiomelanocortin (POMC) and NPY, to prevent and treat obesity in suejtos mammals. The peptide binding to the Y2 receptor can also be administered in conjunction with a Yl receptor antagonist such as dihiropyridine. Methods and Compositions of Delivery Improved methods and compositions for mucosal delivery of the Y2 receptor binding peptide to mammalian subjects optimize the dosing schedules of the Y2 receptor binding peptide. The present invention provides the mucosal delivery of the Y2 receptor binding peptide formulated with one or more mucosal delivery enhancing agents wherein the dosage release of the Y2 receptor binding peptide is normalized and / or substantially sustained over a period of time. effective delivery of the release ranges of the peptide binding to the Y2 receptor from approximately 0.1 to 2.0 hours; 0.4 to 1.5 hours; 0.7 to 1.5 hours; or 0.8 to 1.0 hours; after mucosal administration. Sustained release of the achieved Y2 receptor binding peptide can be facilitated by repeated administration of the exogenous Y2 receptor binding peptide using the methods and compositions of the present invention. Compositions and Methods of Sustained Release Improved compositions and methods for mucosal delivery of the Y2 receptor binding peptide to mammalian subjects optimize the dosing schedules of the Y2 receptor binding peptide. The present invention provides improved mucosal delivery (e.g., nasal) of a formulation comprising the peptide binding to the Y2 receptor. in combination with one or more mucosal supply enhancing agents and an optional agent or agents that enhance sustained release. The mucosal delivery enhancing agents of the present invention produce an effective increase in delivery, e.g., an increase in the maximum plasma concentration (Cmax) to improve the therapeutic activity of the mucosally administered Y2 receptor binding peptide. A second factor that affects the therapeutic activity of the peptide binding to the Y2 receptor in the blood plasma and CNS is the residence time (RT). Agents that enhance sustained release, in combination with agents that enhance intranasal delivery, increase Cmax and increase the residence time (RT) of the Y2 receptor binding peptide. Polymeric delivery vehicles and other agents and methods of the present invention that produce formulations that enhance sustained release, for example, polyethylene glycol (PEG), are described herein. The present invention provides an improved delivery method and dosage form of the Y2 receptor binding peptide for the treatment of symptoms related to obesity, colon cancer, pancreatic cancer, or breast cancer in mammalian cells. Within the mucosal delivery formulations and methods of the invention, the peptide binds to the Y2 receptor. it is often combined or co-ordinated with a vehicle or carrier suitable for mucosal delivery. As used herein, the term "carrier" means a solid or liquid filler, diluent or pharmaceutically acceptable encapsulating material. A liquid vehicle 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, suspending and / or increasing agents. the viscosity, tonicity agents, wetting agents or other biocompatible materials. A tabulation of ingredients listed by the previous categories can be found in U.S. Pharmacopeia National Formulary, 1857-1859, (1990). Some examples of materials that 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 waxes for suppositories; oils such as peanut oil, cottonseed oil, sunflower 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 laureate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution, ethyl alcohol and buffering solutions - of phosphate, as well as other non-toxic compatible substances used in pharmaceutical formulations. Wetting, emulsifying and lubricating agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agnes, coating agglomerates, sweeteners, flavorings and perfuming agents, preservatives and antioxidants may also be present in the compositions, according to the formulator's desire. Examples of pharmaceutically acceptable antioxidants include water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite and the like; oil-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 single dose form will vary depending on the particular mode of administration. Within the mucosal delivery compositions and methods of the invention, various supply enhancing agents that enhance delivery of the Y2 receptor binding peptide to or through a mucosal surface are employed. In this regard, delivery of the Y2 receptor binding peptide through the mucosal epithelium can occur "transcellularly" or "paracellularly". The degree to which these trajectories contribute to the total flow and bioavailability of the peptide binding to the Y2 receptor depends on the mucosal environment, the physico-chemical properties, the active agent, and the properties of the mucosal epithelium. Paracellular transport involves only passive diffusion, while transcellular transport can occur through passive, facilitated or active processes. Generally, polar, passively transported, hydrophilic solutes diffuse through the paracellular pathway, while the more lipophilic solutes utilize the transcellular pathway. Absorption and bioavailability (eg, as reflected by a permeability coefficient or physiological assay) can be easily assessed for various passive and actively absorbed solutes, in terms of both paracellular and transcellular delivery components, for any receptor binding peptide. Y2 selected within the invention. For passively absorbed drugs, the relative contribution of paracellular and transcellular trajectories for drug transport depends on the pKa, the partition coefficient, the molecular radio and the drug charge, the pH of the luminal environment in which the drug is delivered, and the area of the surface of absoción. The paracellular route represents a relatively small fraction of accessible surface area of the nasal mucosal epithelium. In general terms, cellular membranes have been reported to occupy a mucosal surface area that is a thousand times greater than the area occupied by the paracellular spaces. Thus, the smaller surface area, and the discrimination based on size and load against macromolecular permeation would suggest that the paracellular route would be a generally less favorable route than the transcellular supply for drug transport. Surprisingly, the methods and compositions of the invention provide significantly improved transport of biotherapeutics to and through the mucosal epithelia through the paracellular route. Therefore, the methods and compositions of the invention are successfully directed either by paracellular or transcellular routes, alternatively or within a single method or composition. As used herein, "mucosal delivery enhancing agents" include agents that enhance release or solubility (e.g., from a formulation supply vehicle), diffusion rate, penetration capacity and timing, absorption, residence time, stability, effective half-life, peak or sustained concentration levels, clearance and other desired mucosal supply characteristics ( eg, according to - - measured at the delivery site, or at a selected target activity site such as the blood stream or the central nervous system) of the Y2 receptor binding peptide or other biologically active compound (s) (s) Improvement of the mucosal supply can thus occur by any of a variety of mechanisms, for example by increasing the diffusion, transport, persistence or stability of the peptide binding to the Y2 receptor, increasing the fluidity of the membrane, modulating the availability or action of calcium and other ions that regulate intracellular or paracellular permeation, solubilizing the components of the mucosal membrane (e.g., lipids), changing the sulphydryl levels of protein and without protein in mucosal tissues, increasing the flow of water to Through the mucosal surface, modulating the physiology of the epithelial junction, reducing the viscosity of the mucus that overlaps the mucosal epithelium, reducing the rates of mucociliary clearance, and other mechanisms. As used herein, a "mucosally effective amount of the Y2 receptor binding peptide" contemplates effective mucosal delivery of the Y2 receptor binding peptide at a site effective for drug activity in the subject that may involve a variety of supply or transfer routes. For example, a given active agent can form its pathway through cleavage - between mucosal cells and reach an adjacent vascular wall, while by another route the agent can, either passively or actively, be absorbed into mucosal cells to act within the cells either 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 may promote translocation of active agents along one or more of such alternate routes, or may act directly on mucosal tissue or proximal vascular tissue to promote absorption or penetration of the active agent (s) . The promotion of absorption or penetration in this context is not limited to these mechanisms. As used in the present "peak concentration (Cmax) of the peptide binding to the Y2 receptor in the blood plasma", "area under concentration vs. time curve (AUC) of the Y2 receptor binding peptide in the blood plasma "," time at maximum plasma concentration (tmax) of the Y2 receptor binding peptide in the blood plasma "are known pharmacokinetic parameters for one of skill in the art. Laursen et al., Eur. J. Endocrinology, 135: 309-315, 1996. The "concentration vs.. Time curve "measures the concentration of the Y2 receptor binding peptide in a blood serum of a subject vs. time after administration of a dose of the Y2 receptor binding peptide to the subject either by intranasal, intramuscular, subcutaneous or another parenteral "Cmax" is the maximum concentration of the Y2 receptor binding peptide in the blood serum of a subject after a single dose of the Y2 receptor binding peptide to the subject, "tmax" is the time to reach the maximum concentration of the Y2 receptor binding peptide in a blood serum of a subject after administration of a single dose of the Y2 receptor binding peptide to the subject As used herein, "area under concentration vs. Time curve (AUC) of the Y2 receptor binding peptide in the blood plasma "is calculated according to the linear trapezoidal rule and with the addition of residual areas, a decrease of 23% or an increase of 30% between two doses. would detect with a probability of 90% (error type II ß = 10%). The "supply rate" or "absorption rate" is estimated by comparing the time (tmax) to reach the maximum concentration (Cmax). Both of them Cmax and tmax are analyzed using nonparametric methods. Comparisons of the pharmacokinetics of intramuscular, subcutaneous, intravenous and intranasal administrations of the Y2 receptor binding peptide were made by variance analysis (ANOVA). For peer-to-peer comparisons a sequential procedure was used Bonferroni-Hol is sequential used to evaluate the meaning. The dose-response relationship between the three nasal doses was estimated by regression analysis. P < 0.05 was the meaning considered. The results are given as mean values +/- SEM. Although the mechanism of promotion of absorption may vary with different mucosal supply enhancing agents of the invention, the reagents useful in this context will not substantially adversely affect the mucosal tissue and will be selected according to the physicochemical characteristics of the mucosal binding peptide. Y2 particular receptor or other agent active or improving the supply. In this context, agents that improve the supply that increase the penetration or permeability of mucosal tissues will often result in some alteration of the protective barrier of mucosal permeability. For such agents that enhance delivery to be of value within the invention, it is generally desired that any significant change in mucosal permeability will be "reversible within a time frame appropriate to the desired duration of drug delivery. there should be no substantial cumulative toxicity, nor changes of permanent deterioration induced in the mucosal barrier properties with long-term use .. Within certain aspects of the invention, the agents that promote absorption for coordinated administration or formulation Combination with the Y2 receptor binding peptide of the invention are selected from small hydrophilic molecules, including but not limited to, dimethyl sulfoxide (DMSO), dimethylformamide, ethanol, propylene glycol, and the 2-pyrrolidones. Long chain, for example, deacymethyl sulfoxide, azone, sodium lauryl sulfate, oleic acid, and exits bile, can be used to improve mucosal penetration of the peptide binding to the Y2 receptor. In additional aspects, the 'surfactants (e.g., polysorbates) are used as auxiliary compounds, processing agents, or additives of the formulation to improve the intranasal delivery of the peptide binding to the Y2 receptor. Agents such as DMSO, polyethylene glycol, and ethanol can, if present in sufficiently high concentrations in the delivery environment (eg, by pre-administration or incorporation into a therapeutic formulation), enter the aqueous phase of the mucosa and alter its solubilization properties, thereby improving the cleavage of the peptide binding to the Y2 receptor from the vehicle to the mucosa. Additional enhancers of the mucosal supply that are useful within the coordinated administration and methods of procuring and combinatorial formulations of the invention include, but are not limited to, mixed micelles; ena ines; nitric oxide donors (e.g., S-nitroso-N-acetyl-DL-penicillamine, N0R1, N0R4-which are preferably coadministered with a NO scrubber such as carboxy-PITO or doclofenac sodium); sodium salicylate; glycerol esters of acetoacetic acid (e.g., glyceryl-1,3-diacetoacetate or 1, 2-isopropylidene glycerin-3-acetoacetate); and other agents that promote release-diffusion or intra- or trans-epithelial penetration that are physiologically compatible for mucosal delivery. Other agents that promote absorption are selected from a variety of vehicles, bases and excipients that enhance the mucosal delivery, stability, activity or trans-epithelial penetration of the peptide binding to the Y2 receptor. These include, inter alia, cyclodextrins and β-cyclodextrin derivatives (eg, 2-hydroxypropyl-β-cyclodextrin and heptakis (2,6-di-O-methyl-β-cyclodextrin) .These compounds, optionally conjugated to one or more of the active ingredients and further optionally formulated in an oil base, improve bioavailability in the mucosal formulations of the invention Even additional absorption-enhancing agents adapted for mucosal delivery include medium chain fatty acids, including mono- and diglycerides (eg , extracts of sodium caprate- of coconut oil, Capmul), and triglycerides (eg, amylodextrin, Estaram 299, Miglyol 810).

- - The mucosal therapeutic and prophylactic compositions of the present invention can be complemented with any suitable penetration promoting agent that facilitates the absorption, diffusion, or penetration of the peptide binding to the Y2 receptor through mucosal 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 selected penetration promoting agents of sodium salicylate and salicylic acid derivatives (acetyl salicylate, colin salicylate, salicylamide, etc.); amino acids and salts thereof (eg monoaminocarboxylic acids such as glycine, alanine, phenylalanine phenylalanine, proline, hydroxyproline, etc.), hydroxy amino acids such as serine, amino acids such as aspartic acid, glutamic acid, etc., and basic amino acids such as lysine etc. ( including its alkali metal or alkaline earth metal salts), and N-acetylamino acids (N-acetylalanine, N-acetylphenylalanine, N-acetylserine, N-acetylglycine, N-acetyllysine, N-acetyl glutamic acid, N-acetylproline, N-acetylhydroxyproline, etc.) and their salts (alkali metal salts and alkaline earth metal salts) There are substances also provided as agents that promote penetration within the methods and compositions of the invention, which are generally used as emulsifiers (eg, oleyl phosphate). - sodium, sodium laurel phosphate, sodium lauryl sulfate, myristyl sodium sulfate, polyoxyethylene alkyl ethers, alkyl esters of pol ioxyethylene, etc.), caproic acid, lactic acid, malic acid and citric acid and alkali metal salts thereof, pyrrolidonecarboxylic acids, esters of alkylpyrrolidonecarboxylic acid, N-alkylpyrrolidones, proline acyl esters, and the like. Within various aspects of the invention, improved nasal mucosal delivery formulations and methods are provided that allow delivery of the Y2 receptor binding peptide and other therapeutic agents within the invention through mucosal barriers between targeted administration sites and selected . Certain formulations are specifically adapted for a selected target cell, tissue or organ, or even a particular disease state. In other aspects, the formulations and methods provide efficient, selective endo-or transitosis of the Y2 receptor binding peptide, specifically directed along a defined intracellular or intercellular pathway. Typically, the Y2 receptor binding peptide is specifically loaded at levels of effective concentration in a carrier or other delivery vehicle, and is delivered and maintained in a stabilized form, eg, in the nasal mucosa and / or during passage to through the components and intracellular membranes towards a remote target site for the action of the drug (eg, the bloodstream or a defined tissue, organ or extracellular compartment). The Y2 receptor binding peptide can be provided in a delivery vehicle or modified in some other way (eg, in the form of a prodrug), wherein the release or activation of the Y2 receptor binding peptide is activated by a stimulus. physiological (eg pH change, lysosomal enzymes, etc.). Often, the Y2 receptor binding peptide is inactivated pharmacologically until it reaches its target site for activity. In most cases, the Y2 receptor binding peptide and other components of the formulation are non-toxic and non-immunogenic. In this context, vehicles and other components of the formulation are generally selected for their ability to degrade and excrete rapidly under physiological conditions. At the same time, the formulations are chemically and physically stable in dosage form for effective storage. Mimeptic Analogs of Peptides and Proteins Included within the definition of biologically active peptides and proteins for use within the invention are peptides (comprised of two or more covalently bonded amino acids), protein peptide or protein fragments, peptide analogues or - - proteins and chemically modified derivatives or salts of active or natural or synthetic peptides or proteins, therapeutically or prophylactically active. A wide variety of useful analogs and mimetics of the Y2 receptor binding peptide are contemplated for use within the invention and can be produced and tested for their biological activity according to known methods. Frequently, the peptides or proteins of the Y2 receptor binding peptide or other biologically active peptides or proteins for use within the invention are muteins that are easily obtained by substitution, addition, or partial deletion of amino acids within a peptide or sequence of proteins that occur naturally or natively (e.g '., mutant or variant allelic that occurs naturally, wild type). Additionally, biologically active fragments of native peptides or proteins are included. Such mutant derivatives and fragments substantially retain the desired biological activity of the native peptide or proteins. In the case of peptides or proteins having carbohydrate chains, biologically active variants marked by alterations in these carbohydrate species are also included within the invention. As used herein, the term "conservative amino acid substitution" refers to the general interchangeability of amino acid residues that have similar side chains. For example, a commonly interchangeable group of amino acids having aliphatic side chains is alanine, valine, leucine, and isoleucine.; a group of amino acids having aliphatic-hydroxyl side chains is serine and trionine; a group of amino acids having side chains containing amide, is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids that have basic side chains is lysine, arginine, and histidine; and a group of amino acids having side chains containing sulfur is cistern and methionine. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another. Likewise, 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 residue such as lysine, arginine or histidine for 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 optimally aligning a peptide or protein analog with a corresponding native peptide or protein, and by using appropriate assays, eg, adhesion protein or receptor binding assays to determine a selected biological activity, it can be easily identified Peptide analogs and operable proteins for use within the methods and compositions of the invention. Analogs of operable peptides and proteins are typically immunoreactive in a manner specific to the antibodies originated in the corresponding native peptide or protein. A method for stabilizing the solid protein formulations of the invention is to increase the physical stability of the purified, e.g., lyophilized protein. This will inhibit aggregation through hydrophobic interactions as well as through covalent pathways that can increase as proteins unfold. Stabilization of 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 the structure of protein, function and stability are well known. Typically, the proteins are relatively stable in the solid state with water in volume removed. However, the therapeutic therapeutic protein formulations can be hydrated when stored at elevated humidity or during delivery from a sustained release composition or device. The stability of proteins generally falls with increased hydration. Water can also play a significant role in the aggregation of solid protein, for example, by increasing the flexibility of the protein resulting in improved accessibility of the reactive groups, by providing a mobile phase for the reactants, and by serving as a reagent in several harmful processes such as beta-elimination and hydrolysis. Protein preparations containing between about 6% to 28% water are the most unstable. Below this level, the mobility of the bound water and the internal movements of the protein are low. Above this level, the mobility of the water and the movements of the protein are in line with those of total hydration. Up to a point it has been observed in several systems, the increased susceptibility towards the solid phase aggregation with the increase of hydration. However, at higher water contents, less aggregation is observed due to the dilution effect. According to these principles, an effective method for stabilizing 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. These levels depend 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 that 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, diethylaminoethyl dextran, and carboxymethyl cellulose, which significantly increase stability and reduce aggregation in phase. solid of peptides and proteins mixed with them or linked to them. In some cases, the activity or physical stability of proteins can also be improved by various ditues to the 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 - for drying, e.g., lyophilized proteins. These additives can also be used within the invention to rotate proteins against aggregation not only during lyophilization but also during storage in the dry state. For example, sucrose and Ficoll 70 (a polymer with sucrose units) exhibit significant protection against aggregation in the peptide or protein during solid phase incubation under various conditions. These additives can also improve the stability of the solid proteins included within the polymer matrices. Still further additives, for example sucrose, stabilize the proteins against aggregation of solid state in atmospheres of humidity at elevated temperatures, as may occur in certain sustained release formulations of the invention. Proteins such as gelatin and collagen also serve as bulking or bulking agents to reduce the denaturation and aggregation of unstable proteins in this context. These additives can also be incorporated into polymeric fusion processes and compositions within the invention. For example, the polypeptide microparticles can be prepared by simply lyophilizing or spray drying a solution containing several stabilization additives described above. The sustained release of the non-aggregated peptides and proteins can be obtained by this over a prolonged period of time. Several additional preparation components and method, as well as specific formulation additives, are provided herein, which produces formulations for mucosal delivery of peptides and proteins prone to aggregation, 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 to be used within these methods and formulations. Examples of these solubilizing agents are cyclodextrins (CDs), which selectively bind to the hydrophobic side chains of polypeptides. It has been found that these CDs 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 further advantages within the methods and intranasal delivery compositions of the invention. Additional agents to be used in this context include CD dimers, trimers and tetramers with various geometries controlled by linkers that specifically block the aggregation of peptides and proteins. Still solubilizing agents and methods for the - 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 is extended to proteins through the use of peptides and peptide mimetics that similarly block protein aggregation. A wide range of methods and suitable anti-aggregation agents are available for incorporation into the compositions and methods of the invention. Agents and Methods that Modify the Load and Control the pH To improve the transport characteristics of biologically active agents (including peptide binding to the Y2 receptor, other active peptides and proteins, and macromolecular and small molecule drugs) for improved delivery Through hydrophobic mucosal membrane barriers, the invention also provides the techniques and reagents for modifying the loading of selected biologically active agents or delivery enhancing agents described herein. In this respect, the relative permeabilities of macromolecules are generally related to their division coefficient. The degree of ionization of the molecules, which depends on the pKa of the molecule and the pH on the surface of the mucosal membrane, also affects the permeability of the molecules. The permeation and division of the biologically active agents, including the Y2 receptor binding peptide and analogues of the invention, for mucosal delivery can be facilitated by altering the charge or diffusing the charge of the active agent or the active agent. Perbilization, which is achieved, for example, by altering the charged functional groups, by modifying the pH of the vehicle or delivery solution in which the active agent is delivered or by the coordinated administration of a reagent that alters the charge or the pH-with the active agent. Consistent with these general teachings, the mucosal delivery of the charged macromolecular species including the peptide binding to the Y2 receptor and other peptides and peptide proteins and biologically active proteins, within the methods and compositions of the invention is substantially improved when the active agent it is delivered to the mucosal surface in a state of substantially un-ionized or neutral electric charge. A certain binding peptide to the Y2 receptor and other components of peptides and biologically active proteins of the mucosal formulations for use within the invention will be modified from the charge to produce an increase in the density of the positive charge of the peptide or protein. These modifications also extend to the - cationization of the peptide and protein conjugates, carriers and other forms of delivery described herein. Cationization offers a convenient means to alter the biodistribution and transport properties of proteins and macromolecules within the invention. Cationization is assumed in a manner that substantially conserves the biological activity of the active agent and limits potentially adverse side effects, including tissue damage and toxicity. Inhibitory Agents and Methods of Degradative Enzyme Another excipient that can be included in a transmucosal preparation is an inhibitor of the degradative enzyme. Examples of the mucoadhesive polymer-enzyme inhibitor complexes that are useful within the formulations and mucosal delivery methods of the invention include, but are not limited to: Carboxymethylcellulose-pepstatin (with anti-pepsin activity); Poly (acrylic acid) - Bowman-Birk inhibitor (anti-chymotrypsin); Poly (acrylic acid) -chimostatin (anti-chymotrypsin); Poly (acrylic acid) -elastatinal (anti-elastase); Carboxymethylcellulose-elastatinal (anti-elastase); Polycarbophil-elastatinal (anti-elastase); Chitosan-antipain (anti-trypsin); Poly (acrylic acid) -bacitracin (anti-aminopeptidase N); Chitosan-EDTA (anti-aminopeptidase N, anti-carboxypeptidase A); Chitosan-EDTA-antipain (anti-trypsin, anti-chymotrypsin, anti-elastase). As further detail is described below, certain embodiments of the invention will optionally incorporate a new chitosan derivative or a chemically modified form of chitosan. For use within the invention one of said new derivatives is denoted as a polymer β- [1- »4] -2-guanidino-2-deoxy-D-glucose (poly-GuD). Any inhibitor that inhibits the activity of an enzyme to protect the biologically active agent (s) 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, the soybean trypsin inhibitor, the pancreatic trypsin inhibitor, the chymotrypsin inhibitor and the trypsin inhibitor, and chymotrypsin isolated from potato tubers. (Solanum tuberosum L.). A combination of mixtures of inhibitors may be employed. Additional inhibitors of proteolytic enzymes for use within the invention include the enzyme ovomucoid, gabaxate mesylate, alpha 1-antitrypsin, aprotinin, amastatin, bestatin, puromycin, bacitracin, leupepsin, alpha 2-macroglobulin, pepstatin and the trypsin inhibitor of white or soy egg. These and other inhibitors can be used alone or in combination. The inhibitor (s) can be incorporated or bound to a vehicle, eg, a hydrophilic polymer, coated on the surface of the dosage form that is to make contact with the nasal mucosa, or incorporated in the superficial surface phase , in combination with the biologically active agent or in a formulation administered separately (eg, pre-administered). The amount of the inhibitor, eg, of a proteolytic enzyme inhibitor that is optionally incorporated into 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 can reacting to introduce the ethylenic unsaturation necessary for copolymerization with the hydrogel forming monomers), and (c) the number of lectin groups, such as glycosides, which are present in the inhibitor molecule. It 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 (i.e., a separate protease inhibitor formulation or a formulation combined with the inhibitor and the biologically active agent).

In the case of trypsin inhibition, suitable inhibitors can be selected from, eg, aprotinin, BBI, soy trypsin inhibitor, chicken ovomucoid, chicken ovoinhibitor, human pancreatic trypsin inhibitor, camostate mesylate, inhibitors of flavonoids, antipain, leupeptin, p-aminobenzamidine, AEBSF, TLCK (tosylisin chloromethyl ketone), APMSF, DFP, PMSF, and poly (acrylate) derivatives. In the case of chymotrypsin inhibition, suitable inhibitors can be selected from, eg, aprotinin, BBI, soy trypsin inhibitor, chymostatin, benzyloxycarbonyl-Pro-Fe-CHO, FK-448, chicken ovoinhibitor, acid complexes sugar biphenylboronic acids, DFP, PMSF, β-phenylpropionate, and derivatives of poly (acrylate) derivatives. In the case of elastase inhibition, suitable inhibitors can be selected from, eg, elastatinal, methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone (MPCMK), BBI, soybean trypsin inhibitor, chicken ovoinhibitor, DFP, and PMSF. Additional enzyme 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 further detail below, the immobilization of these auxiliary agents to matrices or other delivery vehicles, or the development of chemically modified analogs, can be easily implemented to reduce or even eliminate the toxic effects, when found. Among this broad group of enzyme inhibitor candidates for use within the invention are organophosphorus inhibitors, such as diisopropylfluorophosphate (DFP) and phenylmethylsulfonyl fluoride (PMSF), which are potent irreversible inhibitors of serine proteases (eg, trypsin and chymotrypsin). ). The additional inhibition of acetylcholinesterase mediated by these compounds makes them highly toxic in uncontrolled supply environments. Another candidate inhibitor, 4- (2-Aminoethyl) -benzenesulfonyl fluoride (AEBSF), has an inhibitory activity comparable to DFP and PMSF, but is notably mens toxic. Hydrochloride (4-Aminophenyl) -methanesulfonyl fluoride (APMSF) is another potent trypsin inhibitor, but it is toxic in uncontrolled environments. In contrast to these inhibitors, 4- (4-isopropylpiperadinocarbonyl) phenyl 1, 2,3,4, -tetrahydro-1-naphthoate methanesulfonate (FK-448) is a substance of low toxicity, which represents a potent and specific inhibitor of Chymotrypsin Additional representatives of this non-protein group of inhibitory candidates, and which also exhibit low toxic risk, are camostate mesylate (N, N'-dimethyl carbamoylmethyl-p- (p-guanidino-benzoyloxy) phenylacetate methanesulfonate). Still another type of enzyme inhibiting agent to be used within the methods and compositions of the invention are the 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 not substantially 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 reversible, competitive inhibitors of protease enzymes. Certain modified amino acids may display a much stronger inhibitory activity. A modified amino acid desired in this context is known as a 'transition state' inhibitor. The strong inhibitory activity of these compounds is based on their structural similarity to a substrate in their transition state geometry, although they are generally selected to have a much greater 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 a-aminoboronic acid derivatives, such as boro-leucine, boron-valine and boron-alanine. The boron atom in these derivatives can form a tetrahedral boronate ion which is considered to resemble the transition state of the peptides during their hydrolysis by aminopeptidases. Amino acid derivatives are potent and reversible inhibitors of aminopeptidases and it is reported that boro-leucine is more than 100 times more effective at inhibiting enzyme 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 enzymatic activity of aminopeptidase N. The auxiliary agent also displays mucolytic properties that can be used within the methods and compositions of the invention to reduce the effects of the mucus diffusion barrier. Still other enzyme inhibitors useful for use within the methods of coordinated administration and combinatorial formulations of the invention can be selected from inhibitors of peptides and modified peptide enzymes. 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 dipeptides and tripeptides display weak non-specific inhibitory activity towards some proteases. By analogy with amino acids, their inhibitory activity can be modified by chemical modifications. For example, dipeptide analogues of phosphinic acid are also 'transition state' inhibitors with strong inhibitory activity towards aminopeptidases. It has been reported that they have been used to stabilize the enkefaliña de leucina nasally administered. Another example of a transition state analogue is the modified pentapeptide pepstatin, which is a very potent inhibitor of pepsin. The structural analysis of pepstatin, by testing the inhibitory activity of several synthetic analogues, demonstrated the main features of estrctura-función of the molecule responsible for the inhibitory activity. Another special type of modified peptide includes inhibitors with a terminal aldehyde function located in its structure. For example, the benzyloxycarbonyl-Pro-Fe-CHO sequence, which meets the primary and secondary specificity requirements of chymotrypsin, has been found to be a potent reversible inhibitor of this target proteinase. The chemical structures of additional inhibitors with a terminally localized aldehyde function, e.g. Antipain, leupeptin, chymostatin and elastatinal are known in the art, as are the structures of other known reversible modified peptide inhibitors, such as, phosphoramidon, bestatin, puromycin and amastatin. Due to their comparably high molecular mass, polypeptide protease inhibitors are more manageable than smaller compounds for concentrated delivery in a drug-vehicle matrix. Adiocinal agents for the inhibition of the protease within the formulations and methods of the invention involve the use of complexing agents. These agents mediate the inhibition of the enzyme by deprotecting the intranasal environment (or preparative therapeutic composition) of cation divalent cations, which are co-factors for many proteases. For example, the EDTA and DTPA complexing agents as coordinated or combinatorially formulated auxiliary agents, in adequate concentration, will be sufficient to inhibit selected proteases 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 keiva divalent cations, these and other complexing agents are useful within the invention as directed to agents that promote absorption. As noted in more detail elsewhere in thisIt is also contemplated to use various polymers, particularly mucoadhesive polymers, as agents that inhibit enzymes within the methods and compositions of coordinated administration, multi-processing and / or combinatorial formulation of the invention. For example, poly (acrylate) derivatives, such as poly (acrylic acid) and polycarbophil, can affect the activity of - various proteases, including trypsin, chymotrypsin. The inhivid effect of these polymers can also be based on the complexation of divalent cations such as Ca 2+. and Zn2 + .. It is further contemplated that these polymers can serve as conjugate partners or carriers for additional enzyme inhibiting agents, as described above. For example, a chitosan-EDTA conjugate that has been developed and is useful within the invention inhibits a strong inhibitory effect towards the enzymatic activity of zinc-dependent proteases. The mucoadhesive properties of the polymers after the covalent attachment of other enzyme inhibitors in this context is not expected to be substantially compromised, nor is it expected to diminish the overall utility of such polymers as a delivery vehicle for biologically active agents within the invention. On the contrary, the reduced distance between the delivery vehicle and the mucosal surface produced by the mechanism -mucoadhesive will minimize the presystemic etabolism of the active agent, while the covalently bound enzyme inhibitors remain concentrated at the drug delivery site, minimizing the unwanted dilution effects of the inhibitors as well as the toxic and other effects caused by the same . In this way, the effective amount of a co-administered enzyme inhibitor can be reduced due to the exclusion of dilution effects.

- - Exemplary mucoadhesive polymer-enzyme inhibitor complexes that are useful within the formulations and methods of the invention include, but are not limited to: Carboxymethylcellulose-pepstatin (with anti-pepsin activity); Poly (acrylic acid) - Bowman-Birk inhibitor (anti-chymotrypsin); Poly (acrylic acid) -chimostatin (anti-chymotrypsin); Poly (acrylic acid) -elastatinal (anti-elastase); Carboxymethylcellulose-elastatinal (anti-elastase); Polycarbophil-elastatinal (anti-elastase); Chitosan-antipain (anti-trypsin); Poly (acrylic acid) -bacitracin (anti-aminopeptidase N); Chitosan-EDTA (anti-aminopeptidase N, anti-carboxypeptidase A); Chitosan-EDTA-antipain (antitrypsin, anti-chymotrypsin, anti-elastase). Mucolytic and Mucus Clearance Agents and Methods The effective delivery of biotherapeutic agents through intranasal administration should take into account the rate of drug transport decreased through the protective mucus lining of the nasal mucosa, in addition to the loss of drug. due to the binding to the glycoproteins of the mucus layer. Normal mucus is a viscoelastic gel-like substance that consists of water, electrolytes, mucins, macromolecules, and discarded 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 primarily responsible for the viscoelastic nature of mucus, although other macromolecules may also contribute to this property. In the airway mucus, such macromolecules include IgA, IgM, IgE, lysozyme, and locally produced secretory broncotransferrin, which also play an important role in the host defense mechanism. The methods of coordinated administration of the present invention optionally incorporate effective mucolytic or mucus-clearing agents, which serve to degrade, thin or clear mucus from intranasal mucosal surfaces to facilitate absorption of biotherapeutic agents administered intranasally. Within these methods, a mucolytic agent or mucus-clearing agent is co-ordinated as an auxiliary compound to improve the intranasal delivery of the biologically active agent. Alternatively, an "effective amount" of a mucolytic agent or mucus-clearing agent is incorporated as a processing agent within a multi-processing method of the invention, or as an additive within a combinatorial formulation of the invention, to provide a Improved formulation that improves the intranasal supply of biotherapeutic compounds by reducing the barrier effects of intranasal mucus. A variety of mucolytic agents or mucus-clearing agents are available for incorporation into the methods and compositions of the invention. Based on their mechanisms of action, the mucic agents and mucus-clearing agents can often be classified into the following groups: proteases (e.g., pronase, papain) that divide the nucleus of the protein from mucin glycoproteins; sulfhydryl compounds that divide the mucoprotein disulfide bonds, and detergents (e.g., Triton X-100, Tween 20) that break down the non-covalent bonds within the mucus. Additional compounds in this context include, but are not limited to, bile salts and surfactants, for example, sodium deoxycholate, tauro deoxycholate, sodium glycocholate, and lysophosphatidylcholine. The effectiveness of bile salts to cause the structural decomposition of mucus is in the order of deoxycholate >; taurocholate > glycocholate Other effective agents that reduce the viscosity or adhesion of mucus to improve intranasal delivery according to the methods of the invention include, eg, short chain fatty acids, and mucolytic agents that function by chelation, such as N-acyl collagen peptides, viliary acids, and saponins (the latter work in part by chelation of Ca2 + and / or Mg2 + which plays an important role in maintaining the structure of the mucus 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 mucolytic or mucus-clearing agents make contact with the nasal mucosa, typically in a concentration range of approximately 0.2 to 20 mM, in coordination with the administration of the biologically active agent, to reduce the polar viscosity and / or mucus elasticity. intranasal Still other mucolytic or mucus-clearing agents can be selected from a range of glycosidase enzymes, which are capable of unfolding the glycosidic linkages within the mucus glycoprotein. Α-Amylase and β-amylase are representative of this class of enzymes, although their mucolytic effect may be limited. In contrast, bacterial glycosidases allow these microorganisms to permeate the mucus layers of their hosts. For combinatorial use with most of the biologically active agents within the invention, including - - Therapeutics of peptides and proteins, non-inorganic detergents are also generally useful as mucolytic agents or which clear the mucus. These agents will typically not modify or substantially impair the activity of the therapeutic polypeptides. Cilostatic Agents and Methods Because the ability of self-cleansing of certain mucosal tissues (eg, nasal mucosal tissues) by mucociliary clearance is necessary as a protective function (eg, to remove dust, allergens and bacteria), it has generally been considered 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 function, the ciliary pulsation in the passages of nasal passages and airways moves a layer of mucus along the mucosa to remove inhaled particles and microorganisms. Cryostatic agents find use within the methods and compositions of the invention to increase the residence time of the Y2 receptor binding peptide, mucosally administered analogs and mimetics (e.g., intranasally) and other biologically active agents described herein. In particular, the delivery of these agents within the methods and compositions of the invention is significantly improved in certain aspects by the coordinated administration or combinatorial formulation of one or more ciliatatic agents that function to reversibly inhibit the ciliary activity of the mucosal cells, to provide a temporary, reversible increase in the residence time of the mucosally administered agent (s). To be used within these aspects of the invention, the above ciliatic factors, either specific or indirect in their activity, are all candidates for successful use as ciliates in appropriate amounts (depending on the concentration, duration and mode of delivery) of such that they produce transient (ie, reversible) reduction or cessation of mucociliary clearance at a mucosal delivery site to enhance the delivery of the Y2 receptor binding peptide, analogs and mimetics, and other biologically active agents described herein, without unacceptable side effects. Within more detailed aspects, a specific ciliatic factor is employed in a combined formulation or administration protocol coordinated with one or more Y2 receptor binding peptide proteins, analogs and mimetics, and / or other biologically active agents described herein. Several bacterial ciliostatic factors - isolated and characterized in the literature can be employed within these embodiments of the invention. The ciliostatic factors of the bacterium Pseudomonas aeruginosa include a phenazine derivative, a pyo compound (2-alkyl-4-hydroxyquinolines), and a rhamnolipid (also known as hemolysin). The pyo compound produces ciliostasis at concentrations of 50 μg / ml and without obvious ultrastructural lesions. The phenazine derivative also inhibits ciliary motility but causes some disruption of the membrane, although at concentrations substantially greater than 400 μg / ml. Limited exposure of tracheal explants to the rhamnolipid results in ciliostasis, which was associated with altered ciliary membranes. More extensive exposures to the rhamnolipid were associated with the removal of dynein arms from the axonemes. Surface active agent and methods Within more detailed aspects of the invention, one or more membrane penetration enhancing agents can be employed within a mucosal delivery method or formulation of the invention to improve the mucosal delivery of the peptide protein of the peptide. linkage to the Y2 receptor, analogs and mimetics, and other biologically active agents described herein. The agents that improve membrane penetration in this context can be selected from: (i) a surfactant, (ii) a bile salt, (iii) a phospholipid additive, mixed micelle, liposome, or vehicle, (iv) an alcohol , (v) an enamine, (vi) a NO donor compound, (vii) a long chain amphipathic molecule (viii) a small hydrophobic penetration enhancer; (ix) sodium or a salicylic acid derivative; (x) a glycerol ester of acetoacetic acid (xi) a cyclodextrin or derivative of beta-cyclodextrin, (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 for a selected membrane component, (xvii) an inhibitor of fatty acid synthesis, or (xviii) an inhibitor of cholesterol synthesis; or (xix) any combination of the membrane penetration enhancing agents mentioned in (i) - (xix). Certain surface active agents are easily incorporated into the formulations and mucosal delivery methods of the invention as agents that improve mucosal absorption. These agents, which can be co-administered or formulated in a combinatorial fashion with peptide-binding proteins of the Y2 receptor analogs and mimetics, and other biologically active agents described herein, can be selected from a broad assembly of known surfactants. Surfactants, which generally 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 mechanism of action of these various classes of these surface active agents typically includes the solubilization of the biologically active agent. For proteins and peptides that frequently form aggregates, the active surface properties of these absorption promoters can allow interactions with proteins such that smaller units such as monomers coated with surfactant can be easily maintained in solution. Examples of other active surface agents are L-a-Phosphatidylcholine Didecanoyl (DDPC) polysorbate 80 and polysorbate 20. These monomers are presumably more transportable units than aggregates. A second potential mechanism is the protection of the peptide or protein from proteolytic degradation by proteases in the mucosal environment. Both bile salts and some fusidic acid derivatives have been reported to inhibit protein proteolytic degradation by nasal homogenates at concentrations lower or equivalent to those required to improve protein absorption. This inhibition of protease may be especially important for peptides with short biological half-lives. Enzymes and Inhibitors of Fatty Acid Degradation and - - Cholesterol Synthesis In related aspects of the invention, Y2 receptor binding peptide proteins, analogs and mimetics, and other biologically active agents for mucosal administration are formulated or administered in coordination with an agent that improves the selected penetration of an enzyme from degradation or a metabolic stimulating agent or an inhibitor of the synthesis of fatty acids, sterols or other selected epithelial barrier components, US Pat. No. 6,190,894. For example, degradative enzymes such as phospholipase, hyalunidase, neuraminidase and chondroitinase can be used to improve mucosal penetration of the Y2 receptor binding peptide protein, analogs and mimetics, and other biologically active agents without causing irreversible damage to the mucosal barrier. . In one embodiment, chondroitinase is employed within a method or composition as envisioned herein to alter the glycoprotein or glycolipid constituents of the mucosal permeability barrier, thereby improving mucosal uptake of the peptide-binding proteins. Y2 receptor, analogs and mimetics, and other biologically active agents described herein. With respect to the inhibitors and the synthesis of the mucosal barrier constituents, it is noted that the Free fatty acids account for 20-25% of epithelial lipids by weight. Two enzymes that limit the rate in the biosynthesis of free fatty acids are acetyl CoA carboxylase and fatty acid synthetase. Through a series of stages, free fatty acids are etabolized into phospholipids. Thus, inhibitors of free fatty acid synthesis and metabolism for use within 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-amilcinnamyl) amino-4-chlorobenzoic acid, bro ofenaceous bromide, monoalide, 7,7-dimethyl-5,8-eicosadienoic acid, nicergoline, cepharantin, nicardipine, quercetin , dibutyryl-cyclic 7AMP, R-24571, N-oleoylethanolamine, N- (7-nitro-2, 1, 3-benzoxadiazol-4-yl) phosphostidyl serine, cyclosporin A, topical anesthetics, including dibucaine, prenylamine, retinoids, such like all trans and 13-cis-retinoic acids, W-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, epacrine, 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. Free sterols, mainly cholesterol, account for 20-25% of epithelial lipids by weight. The rate that limits the enzyme in cholesterol biosynthesis is 3-hydroxy-3-methylglutaryl (HMG) CoA reductase. Inhibitors of cholesterol synthesis for use within the methods and compositions of the invention include, but are not limited to, competitive (HMG) CoA reductase inhibitors, such as simvastatin, lovastatin, fluindostatin (fluvastatin), pravastatin, mevastatin, as well as other HMG CoA reductase inhibitors, such as cholesterol oleate, cholesterol sulfate and phosphate, and oxygenated sterols, such as 25-OH- and 26-OH-cholesterol; squalene synthetase inhibitors; squalene epoxidase inhibitors; inhibitors of DELTA7 or DELTA24 reductases such as 22, 25-diazacholesterol, 20, 25-diazacolestenol, AY9944, and triparanol. Each of the inhibitors of fatty acid synthesis or inhibitors of sterol synthesis can be administered in coordination or formulated in a combinatorial manner with one or more peptide proteins binding to the Y2 receptor, analogs and mimetics, and other agents - biologically active inients described herein to achieve improved epithelial penetration of the active agent (s). An effective concentration range for the sterol inhibitor in a therapeutic or ancillary formulation 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 De-. Nitric Oxide Donor Methods and Methods Within other related aspects of the invention, a nitric oxide (NO) donor is selected as an agent that improves membrane penetration to improve the mucosal delivery of one or more proteins of the binding peptide. to the Y2 receptor, analogs and mimetics, and other biologically active agents described herein. Several donors are not known in the art and are useful in effective concentrations within the methods and formulations of the invention. Non-exemplary donors include, but are not limited to, nitroglycerin, nitroprusside, NOC5 [3- (2-hydroxy-1- (methyl-ethyl) -2-nitrosohydrazino) -l-propanamine], N0C12 [N-ethyl-2] - (1-ethyl-hydroxy-2-nitrosohydrazino) -etanamine], SNAP [S-nitroso-N-acetyl-DL-penicillamine], NORI and NOR4. 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 Y2 receptor binding peptide proteins, analogs and mimetics, and / or other biologically active agents. active agents described herein, in or through the mucosal epithelium. Agents for Modulating the Epithelial Binding Structure and / or Physiology The present invention provides pharmaceutical compositions containing one or more Y2 receptor binding peptide proteins, analogs or mimetics, and / or other biologically active agents in combination with improving agents. the mucosal delivery described herein formulated in a pharmaceutical preparation for mucosal delivery. The permeabilization agent reversibly improves mucosal epithelial paracellular transport, typically by modulating the epithelial junction structure and / or surface physiology. epithelial mucosal in the subject. This effect typically involves inhibition by the perothelializing agent of homotypic or herotypic binding between the epithelial membrane adhesive proteins of the surrounding epithelial cells. The target proteins for this homotypic or heterotypic binding block can be selected from several related conjunctive adhesion molecules (JAMs), occludins, or claudins. Examples of this 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 peptide permeabilization and peptide and mimetic analogs to improve mucosal epithelial cuboidal transport. Peptides and analogs of target peptides and mimetics typically function within the compositions and methods of the invention by modulating the epithelial binding structure and / or the physiology in a mammalian subject. In certain embodiments, the peptides and peptide and mimetic analogs effectively inhibit homotypic and / or heterotypic binding of an epithelial membrane adhesive protein selected from a conjunctive 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 including diarrhea in infected subjects. Fasano et al, Proc. Nat. Acad. Sci., U.S.A., 8: 5242-5246 (1991). When tested in rabbit ileal mucosa, ZOT increased intestinal permeability by modulating the structure of intercellular tight junctions. More recently it has been found that ZOT is capable of reversibly opening the tight junctions in the intestinal mucosa. It has also been reported that ZOT is capable of reversibly opening the tight junctions in the nasal mucosa. Pat of E.U. No. 5,908,825. Within the methods and compositions of the invention, ZOT, as well as various ZOT analogs and mimetics that function as agonists or antagonists of ZOT activity, are useful for improving the intranasal delivery of biologically active agents-by increasing paracellular absorption in and through the nasal mucosa. In this context, ZOT typically acts by causing a structural rearrangement of the tight junctions marked by the altered location of the ZO1 binding protein. Within these aspects of the invention, ZOT is administered coordinately or formulated combinatorially with the biologically active agent in an effective amount to produce the significantly improved absorption of the active agent, by reversibly increasing nasal mucosal permeability without adverse side effects. substantial Vasodilating Agents and Methods Still another class of absorption promoting agents that show beneficial utility within the methods and compositions of the co-ordinated administration and combinatorial formulation of the invention are vasoactive compounds, more specifically vasodilators. These compounds function within the invention to modulate the structure and physiology of the submocosal vasculature, increasing the rate of transport of the peptide binding to the Y2 receptor, analogs and mimetics, and other biologically active agents to or through the mucosal epithelium and / or tissues. or specific objective compartments (eg, systemic circulation or central nervous system). The vasodilating agents for use within the invention typically cause a submucosal blood vascular relaxation either by a decrease in cytoplasmic calcium, an increase in nitric oxide (NO) or by inhibiting the myosin light chain kinase. These are generally divided into 9 classes: calcium antagonists, potassium channel openers, ACE inhibitors, angiotensin-II receptor antagonists, α-adrenergic receptor antagonists and imidazole, ß1 adrenergic agonist, phosphodiesterase inhibitors, eicosanoids and blood donors. DO NOT. Despite the chemical differences, the pharmacokinetic properties of calcium antagonists are similar. The absorption in the systemic circulation is high and therefore these agents undergo a considerable first-pass metabolism by the liver, resulting in an individual variation in the pharmacokinetics. The half-life of calcium antagonists is short except for the new drugs of the dihydropyridine type (amlodipine, felodipine, isradipine, nilvadipine, nisoldipine and nitrendipine). Therefore, in order to maintain the effective concentration of the drug for many of these, multiple dose delivery, or controlled release formulations, as described elsewhere herein may be required. The treatment with minoxidil of opener of the potassium channel can also be limited in the form and level of administration due to its potential adverse side effects. ACE inhibitors prevent the conversion of angiotensin-I to anti-hypotensin-II, and are more effective when the production of renin is increased. Since ACE is identical to kininase II, which inactivates the potent endogenous bradykinin vasodilator, inhibition of ACE causes a reduction in bradykinin degradation. ACE inhibitors provide the added benefit of cardioprotective and cardioreparative effects by preventing and reversing cardiac fibrosis and ventricular hypertrophy in animal models. The predominant elimination pathway of most ACE inhibitors is through renal excretion. Therefore, renal deterioration is associated with reduced elimination and a reduction of 25 to 50% in the dosage is recommended in patients with moderate or severe renal deterioration. Regarding the donors of NO, these compounds are particularly useful within the invention for their additional effects on mucosal permeability. In addition to the NO donors noted above, the 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 regulate the specific enzymatic activity for NO release. NONOates release NO with a defined stoichiometry and at predictable rates that vary from <3 minutes for diethylamine / NO to approximately 20 hours for diethylenetriamine / NO (DETANE). Within certain methods and compositions of the invention, a selected vasodilating agent is administered in a coordinated manner or formulated in a combinatorial manner (eg, systemically or intranasally, simultaneously or in combinatorially effective temporal association) with one or more receptor binding peptides. Y2, analogs and mimetics, and other biologically active agent (s) to improve mucosal absorption of the active agent (s) to reach the target tissue or compartment in the subject (eg , the liver, hepatic portal vein, tissue or CNS fluid, or blood plasma). Agents and Methods that Enhance Selective Transport The compositions and methods of delivery of the invention optionally incorporate an enhancing agent - of selective transport that facilitates the transport of one or more biologically active agents. These transport enhancing agents can be used in combinatorial formulation or administration protocol coordinated with one or more Y2 receptor binding peptide proteins, analogs and mimetics described herein, to improve the coordinated delivery of one or more agent (s). ) biologically active (s) through the mucosal transport barriers, to improve the mucosal supply of the active agent (s) to reach the target tissue or compartment in the subject (eg, the mucosal epithelium, liver, tissue or fluid of the CNS, or blood plasma). Alternatively, transport enhancer agents can be employed in a combinatorial formulation or coordinated delivery protocol to directly improve the mucosal delivery of one or more of the Y2 receptor binding peptide proteins, analogs and mimetics, with or without improved delivery of an additional biologically active agent. Exemplary selective transport enhancing 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 components of the epithelial transport barrier. For example, specific "bioadhesive" ligands, including various plant lecithins and bacteria that bind to cell surface sugar residues via receptor mediated interactions, can be employed as carriers or mediators of conjugated transport for mucosal breeding, eg , the nasal delivery of biologically active agents within the invention. Certain bioadhesive ligands for use within the invention will mediate the transmission of giological signals to target epithelial cells that activate the selective uptake of the adhesive ligand by specialized cellular transport processes (endocytosis or transcytosis). Therefore, these transport mediators can be used as a "carrier system" to stimulate or direct the selective uptake of one or more peptide proteins binding to the Y2 receptor, analogs and mimetics, and other biologically active agent (s). active (s) to and / or through the mucosal epithelium. These and other transport enhancing agents significantly improve the mucosal delivery of macromolecular biopharmaceuticals (particularly peptides, proteins, oligonucleotide vectors and polynucleotides) within the invention. Lectins are plant proteins that bind to specific sugars found on the surface of glycoproteins and glycolipids of eukaryotic cells. The lecithin-concentrate solutions have a "mucotractor" effect and several studies have demonstrated rapid receptor-mediated endocytosis (SMR) of lecithins and lecithin conjugates (eg, concanavalin A conjugated to colloidal gold particles) across the surfaces mucosal Additional studies have reported that the absorption mechanisms for lecithins for intestinal target drugs can be used in vivo. In some of these studies, polystyrene nanoparticles (500nm) were covalently coupled with tomato lecithin and an improved systemic absorption performance was reported after oral administration to rats. In addition to plant lecithins, the microbial adhesion and invasion factors provide a rich source of candidates for use as adhesive / selective transport carriers within the methods and delivery compositions of the invention. Two components are necessary for the processes of bacterial adhesion, a bacterial "adhesin" (adherence factor or colonization) and a receptor on the cell surface of the host. Bacteria that cause mucosal infections need to penetrate the mucus layer before adhering themselves to the epithelial surface. This adhesion is usually mediated by bacterial fimbria or piliary structures, however other components of the cell surface can also take part in this process. Adherent bacteria colonize the mucosal epithelia by multiplying and initiating a series of biochemical reactions within the target cell through signal transduction mechanisms (with or without the help of toxins). A wide variety of bioadhesive proteins (e.g., invasin, internalin) associated with these invasive mechanisms, originally produced by various bacteria and viruses, are known. These allow the extracellular adhesion of said microorganisms with an impressive selectivity for the host species and even for objective tissues in particular. The signals transmitted by such receptor-ligand interactions direct the transport of living intact microorganisms to and eventually through epithelial cells by means of endo and transcytotic processes. Such a naturally occurring phenomenon can be exploited (eg, by complexing biologically active agents such as the Y2 receptor binding peptide with an adhesin) in accordance with the teachings herein to improve the delivery of biologically active compounds to or through mucosal epithelia and / or for other sites designated as targets for drug action. Various toxins from bacteria and plants that bind to epithelial surfaces in a specific manner similar to lectin are also useful within the methods and compositions of the invention. For example, diphtheria toxin (DT) enters the host cells rapidly by EMR. Similarly, subunit B of heat labile toxin from E. coli binds to the brush border of intestinal epithelial cells in a manner similar to highly specific lectin. The absorption of this toxin and transitosis towards the basolateral side of the enterocytes has been reported in vivo and in vitro. Other investigations have expressed the transmembrane domain of diphtheria toxin in E. coli as a maltose-binding fusion protein and chemically coupled to high-poly-L-lysine. The resulting complexes were successfully used to mediate the internalization of an in vitro reporter gene. In addition to these examples, Staphylococcus aureus produces a set of proteins (eg, Staphylococcal enterotoxin A (SEA), SEB, toxic shock syndrome toxin 1 (TSST-1) that acts both as superantigens and toxins. proteins have reported facilitated dose-dependent transitosis of SEB and TSST-I in Caco-2 cells.The viral hemagglutinins comprise another type of transport agent to facilitate the mucosal delivery of biologically active agents within the methods and compositions of the The initial stage in many viral infections is the binding of surface proteins (haemagglutinins) to mucosal cells.These binding proteins have been identified for most viruses, including rotaviruses, varicella zoster virus, semliki forest virus , adenoviruses, potato leafroll virus, and reovirus These and other exemplary viral hemagglutinins can be used in a formulation combinatorial ion (eg, a mixture or formulation of conjugate) or a coordinated administration protocol with one or more of the Y2 receptor binding peptide, analogs and mimetics described herein, to coordinately improve the mucosal delivery of one or more agent (s) additional biologically active (s). Alternatively, the viral hemagglutinins can be used in a combinatorial formulation or coordinated delivery protocol to directly improve the mucosal delivery of one or more of the Y2 receptor binding peptide protein, analogs and mimetics, with or without the improved delivery of an agent. additional biologically active A variety of endogenous, selective factors mediating the trans is also available for use within the invention. Mammalian cells have developed a variety of mechanisms to facilitate the internalization of specific substrates and direct these to defined compartments. Collectively, these processes of membrane deformations are called? endocytosis' and include phagocytosis, pinocytosis, receptor-mediated endocytosis (RME mediated by clathrin-), and potocitosis (RME not mediated by clathrin-). RME is a highly specific cellular biological process by which, as its name implies, several ligands bind to cell surface receptors and subsequently internalize and transit within the cell. In many cells the process of endocytosis is so active that the surface of the entire membrane is internalized and repositioned in less than one hour. Two classes of receptors have been proposed based on their orientation in the cell membrane; the amino terminal of Type I receptors that are located on the extracellular side of the membrane, whereas Type II receptors that have this same final part of protein in the intracellular medium. Still other embodiments of the invention utilize transferrin as a vehicle or stimulator of EMRs of mucosally delivered biologically active agents. Transferrin, a glycoprotein that carries 80 kDa iron, is efficiently absorbed into cells by EMR. Transferrin receptors are found on the surface of most proliferating cells, in high numbers in erythroblasts and in many kinds of tumors. Transferrin transidism (Tf) and transferrin conjugates have been reported to be improved in the presence of Brefeldin A (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 within the methods of the invention as agents - formulated combinatorially (eg, conjugates) and / or coordinately administered to improve receptor-mediated transport of biologically active agents , including peptide binding to Y2 receptor proteins, analogs and mimetics. Vehicles and Methods of Polymeric Delivery Within certain aspects of the invention, the peptide-binding receptor Y2 proteins, analogs and mimetics, other biologically active agents described herein, and agents that enhance delivery as described above, are found individually or combinatorially within a mucosally administered formulation (eg, nasally) that includes a compatible polymer that functions as a vehicle or base. Such polymer carriers include polymer powders, matrices or microparticulate delivery vehicles, among other polymer forms. The polymer can be of plant, animal, or synthetic origin. Frequently the polymer is crosslinked. Additionally, in these delivery systems the Y2 receptor binding peptide, analogs or mimetics, can be functionalized in such a way that it can be covalently linked to the polymer and become inseparable from the polymer by simple washing. In other embodiments, the polymer is chemically modified with an inhibitor of enzymes or other agents that can degrade or inactivate the biologically active agent (s) and / or agent (s) that improve the supply. In certain formulations, the polymer is a partially or completely water insoluble polymer but which is hydrophilic, e.g., a hydrogel. The polymers useful in this aspect of the invention are desirably interactive and / or hydrophilic in water 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 reach the balance with water. In more detailed embodiments, the polymer is a hydrogel which, when placed in contact with an excess of water, absorbs at least twice its weight of water in equilibrium when exposed to water at room temperature, US Pat. No. 6,004,583. Drug delivery systems based on biodegradable polymers are preferred in many biomedical applications because such systems are degraded either by hydrolysis or by enzymatic reaction in non-toxic molecules. The rate of degradation is controlled by manipulating the composition of the polymer-biodegradable matrix. These types of systems can therefore be used in certain environments 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 possible vehicles of 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 the Y2 receptor binding peptide, analogs and mimetics, and other biologically active agents described herein, as well as optional agents that improve delivery, these agents can be incorporated into polymer matrices, eg, polyorthoesters, polyanhydrides, or polyesters. This produces activity and sustained release of the active agent (s), e.g., 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 obstacle of polymer-based release technology is the loss of activity of the therapeutic molecules during the formation processes that frequently involve heat, sonication or organic solvents. The absorption promoting polymers contemplated for use within the invention may include derivatives and chemically or physically modified versions of the above types of polymers, in addition to other naturally occurring or synthetic polymers, gums, resins, and other agents, as well as mixtures of these materials with each other or with other polymers, so long as the alterations, modifications or mixtures do not adversely affect the desired properties, such as water absorption, hydrogel formation, and / or chemical stability for the useful application. In more detailed aspects of the invention, polymers such as nylon, acrylate and other synthetic polymers normally hydrophobic can be sufficiently modified by reaction to become hydrophilic and / or form stable gels in an aqueous medium. The polymers of the invention that promote absorption may 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 alcohols and its co- and terpolymers, polyvinylacetate, its co- and terpolymers with the monomers listed above and 2-acrylamido-2-methyl-propanesulfonic acid (AMPS®). Very useful are the copolymers of the monomers listed above with copolymerizable functional monomers such as acrylic or methacrylamide acrylate or methacrylate esters wherein the ester groups are derived from straight or branched chain alkyl, aryl having up to four aromatic rings which may contain substituents of alkyl of 1 to 6 carbons; steroids, sulfates, phosphates or cationic monomers such as N, N-dimethylaminoalkyl (meth) acrylamide, dimethylaminoalkyl (meth) acrylate, (meth) acryloxyalkyltrimethylammonium, (meth) acryloxyalkyldimethylbenzyl ammonium chloride. The additional polymers that promote 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, chitosan, pullalan, zooglan, alginates and modified alginates such as "Kelcoloid" (a modified polypropylene glycol alginate) gellan gums such as "Kelocogel", Xanatan gums such as "Keltrol", estastine, alpha hydroxy butyrate and their copolymers, hyaluronic acid and its derivatives , polylactic and glycolic acids. A very useful class of polymers applicable within 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 an olefinic double bond that functions readily in the polymerization due to its presence in the monomer 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-cyanoacrylic acid, beta-methylacrylic acid (crotonic acid), alpha-phenyl acrylic acid, beta-acryloxypropionic acid, acid cinnamic, p-chloro cinnamic acid, l-carboxy-4-phenyl butadiene-1,3 acid, 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 polycarboxylic acids and anhydride acids, such as maleic anhydride, wherein the anhydride group is formed by the removal of a water molecule from two carboxyl groups located therein. carboxylic acid molecule. Representative acrylates useful as agents that promote absorption within 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. Greater alkyl acrylic esters are decyl acrylate, isodecyl methacrylate, lauryl acrylate, stearyl acrylate, behenyl acrylate and melisyl acrylate and methacrylate versions thereof. Mixtures of two or more long-chain acrylic esters or trees can be polymerized successfully with one of the carboxylic monomers. Other comonomers include olefins, including alpha olefins, vinyl ethers, vinyl esters, and mixtures thereof. Other vinylidene monomers, including acrylic nitriles, can also be used as the agents that promote absorption within the methods and compositions of the invention to improve the delivery and absorption of one or more of the receptor binding peptide proteins. Y2, analogs and mimetics, and other biologically active agent (s), including to enhance delivery of the active agent (s) to a target tissue or compartment in the subject (eg , liver, hepatic portal vein, tissue or CNS fluid, or blood plasma).

- - Useful alpha, beta-olefinically unsaturated nitriles are preferably monoolefinically unsaturated nitriles having from 3 to 10 carbon atoms such as acrylonitrile, methacrylonitrile, and the like. Most preferred are acrylonitrile and methacrylonitrile. Acrylic amides containing from 3 to 35 carbon atoms including monoolefinically unsaturated amides can also be used. Representative amides include acrylamide, methacrylamide, Nt-butyl acrylamide, N-cyclohexyl acrylamide, higher alkyl amides, wherein the alkyl group in the nitrogen contains from 8 to 32 carbon atoms, acrylic amides including N-alkylol amides of alpha, beta-olefinically unsaturated carboxylic acids including those having from 4 to 10 carbon atoms such as N-methylol acrylamide, N-propanol acrylamide, N-methylol methacrylamide, N-methylol maleimide, esters of N-methylol maleamic acid, N-methylol-p-vinyl benzamide, and the similar. Still further useful promoting materials for absorption 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 alil esters such as vinyl acétate; vinyl aromatics such as styrene, methyl styrene and chloro-styrene; vinyl and allyl ethers and ketones such as vinyl methyl ether and methyl vinyl ketone; - chloroacrylates; cyanoalkyl acrylates such as alpha-cyanomethyl acrylate, and the alpha-, beta-, and gamma-cyanopropyl acrylates; alkoxy acrylates such as methoxy ethyl acrylate; haloacrylates such as chloroethyl acrylate; vinyl halides 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) vinyl phosphonate and the like as is known to those skilled in the art. Copolymers wherein the carboxy-containing monomer is a mirror constituent, and the other vinylidene monomers present as the main components are easily prepared according to the methods described herein. When the hydrogels are used as agents promoting absorption within the invention, these may be composed of synthetic copolymers of the group of acrylic and methacrylic acids, acrylamide, methacrylamide, hydroxyethylacrylate (HEA) or methacrylate (HEMA), and vinylpyrrolidones which are interactive with water and hydrophilic. Exemplary illustrative examples of useful polymers, especially for the delivery of peptides or proteins, are the following types of polymers: (meth) acrylamide and from 0.1 to 99 wt. % (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 7? MPS.RTM. (trademark of Lubrizol Corp.); (meth) acrylamide and from 0 to 30% by weight of alkyl (meth) acrylamides and 0.1-75% by weight of AMPS. TM; (meth) acrylamide and 0.1-99 wt. % of HEMA; (metb) acrylamide and from 0.1 to 75% by weight of HEMA and from 0.1 to 99% of (meth) acrylic acid; (meth) acrylic acid and 0.1-99% by weight HEMA; 50 mol% of vinyl ether and 50 mol% of maleic anhydride; (meth) acrylamide and from 0.1 to 75% by weight of (meth) acryloxyalkyl dimethyl benzylammonium chloride; (meth) acrylamide and from 0.1 to 99% by weight of vinyl pyrrolidone; (meth) acrylamide and 50% by weight of vinyl pyrrolidone 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 Cl to C30, preferably Cl to C22, linear and branched and from C4 to C16 cyclic; where (met) is used, it means that the monomers with and without the methyl group are included. Other very useful hydrogel polymers are hydrophilic, but insoluble versions of poly (vinyl pyrrolidone) starch, carboxymethyl cellulose and polyvinyl alcohol.

Additional polymeric hydrogel materials useful within the invention include hydrogels (poly) hydroxyalkyl (meth) acrylate: anionic and cationic: poly (electrolyte) complexes; poly (vinyl alcohols) having a low acetate residual: a crosslinked crosslinked agar and crosslinked carboxymethyl cellulose: a sponge composition comprising methyl cellulose mixed with an economically crosslinked agar; a hydrophilic copolymer produced by a dispersion of the finely divided copolymer of maleic anhydride with styrene, ethylene, propylene, or isobutylene; a hydrophilic polymer of N-vinyl lactams; Sodium salts sponge of carboxymethyl cellulose; and the similar. Other gellable fluids, which inhibit and retain useful polymers to form the hydrophilic hydrogel for mucosal delivery of biologically active agents within the invention include pectin; polisacids such as agar, acacia, Baraya, tragacanth, algin and guar and their cross-linked versions; acrylic acid polymers, copolymer derivatives and salts, polyacrylamides; polymers of maleic anhydride of indene hydrophilic; starch graft copolymers; acrylate-type polymers and copolymers with water absorbency of approximately 2 to 400 times their original weight; polyglucan diesters; a mixture of poly (vinyl alcohol crosslinked) and poly (N-vinyl-2-pyrrolidone); polyoxybutylene-polyethylene block copolymer gels; carob gum; polyester gels; poly urea gels; polyether gels; polyamide gels; polyimide gels; polypeptide gels; polyamino acid gels; poly cellulosic gels; cross-linked maleic anhydride indene-acrylate polymers; and polysaccharides. The synthetic hydrogel polymers for use within the invention can be made by an infinite combination of several monomers in various proportions. The hydrogel can crosslink and generally has the ability to inhibit and absorb fluid and increase in volume or expand to an enlarged state of equilibrium. The hydrogel typically increases in volume or expands upon delivery to the nasal mucosal surface, abominably abetting 2-5, 5-10, 10-50, up to 50-100 or more times its water weight. The optimum degree of bulking capacity for a given hydrogel will be determined by different biologically active agents depending on factors such as molecular weight, size, solubility and diffusion characteristics of the active agent ported or trapped or encapsulated within the polymer, and the space specific and movement of the cooperative chain associated with each individual polymer. The hydrophilic polymers within the invention are insoluble in water but hydrophilic. Such polymers increased in volume by water typically refer - - as hydrogels or gels. Such gels can conveniently be produced from the 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 conditions of pH, temperature and / or ionic concentration definition, according to methods known in the art. Typically the polymers are crosslinked, i.e., crosslinked to the extent that the polymers possess good hydrophilic properties, have improved physical integrity (in comparison to uncrosslinked polymers of the same or similar type) and exhibit improved ability to retain within the gel network both the biologically active agent of interest and the additional compounds for co-administration therewith such as a cytosine or the enzyme inhibitor, while retaining the ability to release the active agent (s) at the appropriate location and time. Generally the hydrogel polymers for use within the invention are crosslinked with crosslinking in the amount of from 0.01 to 0.25 percent by weight, based on the weight of the monomers forming the copolymer, and more preferably from 0.1 to 20 percent. by weight and more frequently from 0.1 to 15 percent by weight of the crosslinking agent. Another useful amount of a crosslinking agent is 0.1 to 10 percent by weight. The tri, tetra or multifunctional crosslinking agents can also be used. When such reagents are used, smaller amounts may be required to achieve the equivalent crosslink density, ie, the degree of crosslinking, or network properties that are sufficient to effectively contain the biologically active agent (s). . The crosslinks can be covalent, ionic or hydrogen bonding with the polymer having the ability to increase in volume in the presence of water containing fluids. Such crosslinkers and crosslinking reactions are known to those skilled in the art and in many cases depend on the polymer system. Thus a network of crosslinking can be formed by the copolymerization of the free radical of unsaturated monomers. Polymeric hydrogels can also be formed by crosslinking by 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, e.g. decadiene or trivinyl cyclohexane; acrylamides, such as N, N-methylene-bis (acrylamide); polyfunctional acrylates, such as trimethylol propane triacrylate; or - - polyfunctional vinylidene monomer containing at least 2 terminal groups CH2 < , which include, for example, divinyl benzene, divinyl naphthine, allyl acrylates 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 there is an olefinic double bond attached to a group. of terminal methylene (eg, 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 a varying number 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 solvents per molecule are used. Other crosslinking monomers include, for example, diallyl esters, dimetalyl ethers, allyl or methallyl acrylates and acrylamides, tetravinyl silane, polyalkenyl methanes, diacrylates, and dimethacrylates, divinyl compounds such as divinyl benzene, polyallyl phosphate, diallyloxy compounds and phosphite esters and the like. Typical agents are allyl pentaerythritol, allyl sucrose, trimethylolpropane triacrylate, 1,6-hexanediol diacrylate, trimethylolpropane diallyl ether, pentaerythritol triacrylate, tetramethylene dimethacrylate, ethylene diacrylate, ethylene dimethacrylate, triethylene glycol dimethacrylate, and the like. Allyl pentaerythritol, trimethylolpropane diallyl ether and allyl sucrose provide suitable polymers. When the crosslinking agent is present, the polymer blends commonly contain between about 0.01 to 20 percent by weight, e.g., 1%, 5%, or % or more by weight of crosslinking monomer based on the totall of carboxylic acid monomer, plus other monomers. In more detailed aspects of the invention, the mucosal delivery of the Y2 receptor binding peptide, analogs and mimetics and other biologically active agents described herein, are enhanced by retaining the active agent (s) in a slow release carrier or carrier. or enzymatically or physiologically protective, for example a hydrogel that protects the active agent from the action of the degrading enzymes. In certain embodiments, the active agent is chemically linked to the carrier or vehicle, to which additional agents such as enzyme inhibitors, cytokines, etc. can also be mixed or bound. The active agent can be immobilized alternatively through - Sufficient physical atrophy within the carrier or vehicle, e.g., a polymer matrix. Polymers such as hydrogels useful within the invention may incorporate linked functional agents such as glycosides chemically incorporated in the polymer to improve the intranasal bioavailability of active agents formulated therewith. 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 indica. There are several ways in which a typical glycoside can be attached 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 of 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 the condensation of acetobromoglucose with colest-5-en-3-beta-ol in a maleic acid copolymer. The N-substituted polyacrylamides can be synthesized by the reaction of activated polymers with omega-aminoalkyl glycosides: (1) (carbohydrate spacer) (n) -polyacrylamide, "pseudopolysaccharides N; (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 absorption of the (1) biologically active agent (s), eg, a peptide binding to the Y2 receptor encapsulated in polymer. of more detailed aspects of the invention, one or more Y2 receptor binding peptides, analogs and mimetics and / or other biologically active agents, described herein, which optionally include secondary active agents such as protease inhibitor (s), cytokine (s), additional modulator (s) of the intercellular binding physiology, etc., are modified and linked to a polymeric vehicle or matrix, for example, this can be carried out by linking chemistry. a peptide or protein active agent and other optional agent (s) within a crosslinked polymer network. It is also possible to chemically modify the polymer separately with an interactive agent such as a glycoside-containing molecule. In certain aspects, the biologically active agent (s) and the optional secondary active agent (s) can be functionalized, ie, where an appropriate reactive group is identified or chemically added to the agent (s) (s) active (s). 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 solution polymerization (commonly in water), emulsion, suspension or dispersion polymerization. Frequently, the functionalizing agent is provided with a high enough concentration of functional or polymerizable groups to ensure that several sites in the active agent (s) are functionalized. For example, in a polypeptide comprising 16 amine sites, it is generally desired to functionalize at least 2, 4, 5, 7, and up to 8 or more of the sites. After functionalization, the functionalized active agent (s) 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 binding agent (s). The polymer is then washed with water or other suitable and purified solvents to remove traces of non-reactive impurities and, if necessary, grind or grind by physical means such as by physical means such as by agitation, force it through a mesh, ultrasound or other suitable means for a desired particle size. The solvent, usually water, is then removed in such a way that it is not denatures or otherwise degrades the active agent (s). A desired method is lyophilization (freeze drying) but other methods (e.g., vacuum drying, air drying, spray drying, etc.) are also available and can be used. To induce polymerizable groups in peptides, proteins and other active agents within the invention, it is possible to react the amino, hydroxyl, thiol and other available reactive groups with electrophiles containing unsaturated groups. For example, unsaturated monomers containing N-hydroxy succinimidyl groups, active carbonates such as p-nitrophenyl carbonate, trichlorophenyl carbonates, tresylate, oxycarbonylimidazoles, epoxide, isocyanates and aldehyde and unsaturated carboxymethyl azides and unsaturated ortopyridyl disulphide belong to this category of reagents. Illustrative examples of unsaturated reactants are allyl glycidyl ether, allyl chloride, allylbromide, allyl iodide, acryloyl chloride, allyl isocyanate, allyl sulfonyl chloride, maleic anhydride, maleic anhydride copolymers 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 cysteine if the local environment improves the nucleophilicity of these groups.

The functionalized reagents containing aldehyde are specific for lysine. These types of reactions with available groups of plants, cysteines, tyrosine have been documented extensively in the literature and are known from experience in the field. 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 and introduce the polymerizable acrylic group in 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, binds to the polymer and remains attached thereto. In additional aspects of the invention, biologically active agents, including peptides, proteins, nucleosides and other molecules that are bioactive in vivo, are stabilized by conjugation by covalently binding one or more active agent (s) to a polymer that is it incorporates as an integral part thereof both a hydrophilic residue, eg, a linear polyalkylene glycol, a lipophilic residue (see, eg, US Patent No. 5,681,811). In one aspect, a biologically active agent is covalently coupled to a polymer comprising (i) a linear polyalkylene glycol residue and (ii) a lipophilic residue, wherein the agent - - active, the linear polyalkylene glycol residue and the lipophilic residue are installed conformationally in relation to each other so that the therapeutically active agent has an improved in vivo resistance to enzymatic degradation (ie, in relation to its stability under similar conditions in a non-toxic manner). null conjugate of the polymer coupled to it). In another aspect, the formulation stabilized by conjugation has a three-dimensional conformation comprising the biologically active agent covalently coupled to a polysorbate complex which (i) a linear poly-alkylene glycol residue and (ii) a lipophilic residue, wherein the active agent, the linear polyalkylene glycol residue and the lipophilic residue are installed conformationally relative to each other so that (a) the lipophilic residue it is available externally in the three-dimensional conformation and (b) the active agent in the composition has an improved resistance in vivo to enzymatic degradation. In a related further aspect, a conjugated multiligand complex comprising a biologically active agent covalently coupled to a residual triglyceride structure through a polyalkylene glycol separation group attached to a carbon atom of the tri-structure residue is provided. -glyceride and at least one covalently bound fatty acid residue either - directly to a carbon atom of the triglyceride structure residue or covalently bound through a polyalkylene glycol spacer residue (see, e.g., U.S. Patent No. 5,681,811). In such conjugate multiligand therapeutic agent complex, the alpha and beta carbon atoms of the bioactive triglyceride residue may have bound fatty acid residues and covalently linked either directly thereto or covalently linked indirectly therein through the spacing residues of polyalkylene glycol. Alternatively, a fatty acid residue can be covalently linked either directly or through a polyalkylene glycol spacer residue to the alpha and beta carbons of the residues of the triglyceride structure, with the therapeutically bioactive agent covalently coupled to the carbon atom of the residue. of triglyceride structure, either covalently linked directly to it or indirectly bound to the moiety through a polyalkylene spacer residue. It is recognized that a wide variety of structural, compositional and conformational forms are possible for the multiligand conjugated therapeutic agent complex comprising the triglyceride structure residue, within the scope of the invention. It should be further noted that in such a multiligand conjugated therapeutic agent complex, the biologically active agent (s) can be covalently coupled advantageously with the modified triglyceride structure residue via alkyl or alkyl spacer groups. alternatively other acceptable spacer groups, within the scope of the invention. As used in such a context, the acceptability of the spacer group refers to spherical compositional and ultimately the use of application-specific acceptability characteristics. In still further aspects of the invention, a complex conjugated stabilized comprising a polysorbate complex comprising a polysorbate residue including a triglyceride structure covalently coupled to the alpha, alpha 'and beta carbon atoms of the same groups is provided. of functionalization including (i) a fatty acid group; and (ii) a polyethylene glycol group having a biologically active agent or residue covalently attached thereto, e.g., linked to an appropriate functionality of the polyethylene glycol group. Such a covalent bond can be either direct, eg, to a hydroxy terminal functionality of the polyethylene glycol group or alternatively, the covalent bond can be inactive, eg, by reactive blocking of the hydroxy teminal of the polyethylene glycol group with a group carboxy terminal functionality spacer, so that the resulting capped polyethylene glycol group has a carboxy terminal functionality to which the biologically active agent or residue can be covalently linked. In still further aspects of the invention, there is provided a stable, water-soluble stabilized conjugation complex comprising one or more peptide proteins binding to the Y2 receptor, analogs and mimetics and / or other biologically active agent (s) ( s) + described herein covalently coupled to a modified glycolipid residue of a physiologically compatible polyethylene glycol (PEG). In such a complex, the biologically active agent (s) can be covalently coupled to the glycolipid residue of physiologically compatible PEG by a labile covalent bond to a free amino acid group of the active agent, wherein the covalent bond labile is separable in vivo by biochemical hydrolysis and / or proteolysis. The physiologically compatible PEG-modified glycolipid residue may advantageously comprise a polysorbate polymer, e.g., a polysorbate polymer comprising fatty acid ester groups selected from the group consisting of monopalmitate, dipalmitate, monolaurate, dilaurate, trilaurate, monoleate, dioleate, trioleate, monostearate, distearate, and tristearate. In such a complex, the physiologically compatible PEG-modified glycolipid residue may suitably comprise a polymer selected from the group consisting of polyethylene glycol fatty acid ethers, and polyethylene glycol fatty acid esters, wherein the fatty acids for example comprise a fatty acid selected from the group consisting of lauric, palmitic, oleic, and stearic acids. Storage of Material In certain aspects of the invention, combinatorial formulations and / or methods of co-ordinated administration herein incorporate an effective amount of peptides and proteins that 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 absorption of the polypeptide or protein in a glass container. In still further aspects of the invention, a kit for treatment of a mammalian subject comprises a pharmaceutically stable composition of one or more Y2 receptor binding peptide compound (s) formulated for mucosal delivery to the mammalian subject where the The composition is effective in alleviating one or more symptoms of obesity, cancer or malnutrition or cancer related exhaustion in said subject without unacceptable adverse side effects. The equipment also comprises a pharmaceutical reagent bottle to contain the one or more compounds - of the peptide binding to the Y2 receptor. The pharmaceutical reagent bottle is composed of pharmaceutical grade polymer, glass or other suitable material. The pharmaceutical reagent bottle is, for example, a silanized glass bottle. The equipment 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 delivery 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 a temperature higher than that of the surfaces to be silanized. The method provides reproducible surfaces with homogeneous and functional stable layers of silane having characteristics of a monolayer. The silanized surfaces prevent the binding to the glass of the polypeptides or agents that improve the mucosal supply of the present invention. The method is useful for preparing silanized pharmaceutical reagent bottles to contain the Y2 receptor binding peptide compositions of the present invention. The glass trays are cleaned by rinsing with double distilled water (ddH20) before being used. The silane tray is then rinsed with 95% EtOH and the acetone tray is rinsed with acetone. The pharmaceutical reagent bottles are sonicated in acetone for 10 minutes. After sonication of the acetone, the reagent bottles are washed in trays of ddH20 at least twice. The reagent bottles are sonicated in 0.1M NaOH for 10 minutes. While the reagent bottles are sonicated in NaOH, the silane solution is made under a cover. (Silane solution: 800 ml of 95% ethanol, 96 1 of glacial acetic acid, 25 ml of glycidoxypropyltrimethoxy silane). After sonication with NaOH, the reagent bottles are washed in the ddH20 tray at least twice. The reagent bottles are sonicated in silane solution for 3 to 5 minutes. The reagent bottles are washed in a tray of 100% EtOH. The reagent bottles are dried with gas purified N2 gas and stored in an oven at 100 ° C for at least 2 hours before being used. Vehicles and Methods of Bioadhesive Supply In certain aspects of the invention, combinatorial formulations and / or methods of co-ordinated administration herein incorporate an effective amount of a non-toxic bioadhesive as a compound or auxiliary carrier to improve the mucosal delivery of one or more biologically active agent (s). The bioadhesive agents in this context exhibit adhesion - general or specific to one or more components or surfaces of the target mucosa. The bioadhesive maintains a desired concentration gradient of the biologically active agent in or through the mucosa to insure the penetration of even larger molecules (e.g., peptides and proteins) into or through the mucosal epithelium. Typically, the use of a bioadhesive within the methods and compositions of the invention results in an increase of times from two to four, often from five to ten in permeability for the peptides and proteins to or through the mucosal epithelium. This improvement of the epithelial penetration frequently allows the effective transmucosal delivery of large macromolecules, for example to the basal portion of the nasal epithelium or in the adjacent extracellular compartments or the blood plasma or tissue or CNS fluid. This improved delivery provides greatly improved effectiveness of the supply of peptides, proteins and other bioactive macromolecular therapeutic species. These results will depend on the hydrophilicity of the compound, through which greater penetration with hydrophilic species will be achieved compared to water-insoluble compounds. In addition to these effects, the use of bioadhesives to improve the persistence of the drug on the mucosal surface can elicitar a reserve mechanisms for the supply of drug protected, whereby - compounds not only penetrate through the mucosal tissue but also diffuse back towards the mucosal surface once the material on the surface is exhausted. A variety of bioadhesives suitable in the art for oral administration are described, US Pat. 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; Patent of E.U. Reissue 33,093, which finds use within the new methods and compositions of the invention. The potential of various bioadhesive polymers such as a mucosal, eg, the nasal delivery platform within the methods and compositions of the invention can be easily assessed by determining their ability to retain and release the peptide binding to the Y2 receptor, as well as by its capacity to interact with the mucosal surfaces after the incorporation of the active agent in them. In addition, well-known methods will be applied to determine the biocompatibility of the selected polymers with the tissue at the site of mucosal administration. When the target mucosa is covered by mucus (i.e., in the absence of mucolytic treatment or mucus cleansing), it can serve as a connecting link to the underlying mucosal epithelium. Therefore, the term "bioadhesive" as used herein also covers useful mucoadhesive compounds to improve the mucosal delivery of biologically active agents within the invention. However, adhesive contact to the mucosal tissue mediated through adhesion to a mucus gel layer can be limited by incomplete or transient binding between the mucus layer and the underlying tissue, particularly on nasal surfaces where rapid clearance of the mucosa occurs. mucus. In this regard, the mucin glycoproteins are secreted continuously and immediately after their release from the cells or glands form a viscoelastic gel. However, the luminal surface of the adherent gel layer is continuously weathered by mechanical, anzymatic and / or ciliary ation. Where such activities are more prominent or where longer adhesion times are desired, the methods of coordinated administration and combinatorial formulation methods of the invention may also incorporate mucolytic and / or ciliates methods or agents as described hereinabove. Typically, the mucoadhesive polymers for use within the invention are natural or synthetic macromolecules that 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 incorporating bioadhesives that adhere directly to a cell surface, rather than to mucus, by means of - - Specific interactions, including receptor-mediated. An example of bioadhesives that work in this specific form is the group of colloids known as lectins. These are glycoproteins with an ability to specifically recognize and bind sugar molecules, e.g. glycoproteins or glycolipids, which are part of intranasal epithelial cell membranes and can be considered as "lectin receptors". In certain aspects of the invention, bioadhesive materials for improving the intranasal delivery of biologically active agents comprise a matrix of a hydrophilic, eg, polymer or a mixture of water-soluble or water-absorbing polymers, which can adhere to a surface of wet mucus. . These adhesives can be formulated as ointments, hydrogels (see above) thin films and other forms of application. Frequently, these adhesives have the biologically active agent mixed therewith to effect the 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, e.g., into the circulatory system of the individual. Several polymers, both natural and synthetic, show significant binding to the epithelial surfaces of mucus and / or mucosal surfaces under physiological conditions.

- The resistance of this interaction can be measured easily by mechanical detachment or cutting tests. When applied to a wet mucosal surface, many dry materials will adhere spontaneously, at least lightly. After such initial contact, some hydrophilic materials begin to attract water by absorption, volume or capillarity forces, and if this water is absorbed from the underlying substrate or from the polymer-tissue interface, adhesion may be sufficient to achieve the objective. of improving the mucosal absorption of biologically active agents. Such "adhesion by hydration" can be quite strong, but the formulations adapted to use this mechanism must respond to the increase in volume which continues as the dose is transformed into a hydrated mucilage. This is protected by many hydrocolloids useful within the invention, especially some cellulose derivatives, which are generally non-adhesive when applied in the pre-hydrated state. However, bioadhesive drug delivery systems for mucosal administration are effective within the invention when such materials are applied in the form of a dry powder, microsphere, or film-like delivery form. Other polymers adhere to mucosal surfaces not only when applied dry, but also in fully hydrated state and in the presence of excessive amounts of water. The selection of a mucoadhesive thus requires due consideration of the physiological as well as physiochemical conditions under which the contact with the tissue will be formed and maintained. In particular, the amount of water or moisture commonly present in the proposed sites of adhesion and the prevalent pH is known to greatly affect the mucoadhesive binding strength of different polymers. Several polymeric bioadhesive drug delivery systems have been manufactured and studied in the past 20 years, not always successfully. However a variety of such carriers are currently used in clinical applications that involve dental, orthopedic, ophthalmological and surgical applications. For example, acrylic based hydrogels have been used extensively for bioadhesive devices. Acrylic-based hydrogels are very suitable for bioadhesion due to their flexibility and non-abrasive characteristics in the partially increased volume state, which reduces the friction that causes damage to the tissues in contact. In addition, its high permeability in the state of volume increase allows unreacted monomer, uncrosslinked polymer chains and the initiator to wash out of the chalking after polymerization, which is an important characteristic for - the selection of bioadhesive materials to be used within the invention. The acrylic-based polymeric devices exhibit very high adhesive bond strength. For controlled mucosal delivery of peptide and protein dorgas, the methods and compositions of the invention optionally include the use of vehicles, eg, polymeric delivery vehicles, which function in part to protect the biologically active agent from proteolytic decomposition, while At the same time they provide improved penetration of the peptide or protein into or through the nasal mucosa. In this context, bioadhesive polymers have shown considerable potential to improve the oral drug supply. As an example, the bioavailability of 9-desglicinamide, 8-arginine vasopressin (DGAVP) administered intraduodenally to rats together with a 1% (w / v) saline dispersion of the polycarbophil derivative of mucoadhesive poly (acrylic acid), was increased 3-5-fold 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 enzymatic 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 metallo-proteins, such as trypsin and chymotrypsin. By depriving the proteases of their cofactors by means of polyacrylic acid, it was reported that they induce irreversible structural changes of the enzymatic proteins that were accompanied by a loss of enzymatic activity. At the same time, other mucoadhesive polymers (e.g., some cellulose derivatives and chitosan) can not inhibit proteolytic enzymes under certain conditions. In contrast to other enzyme inhibitors contemplated for use within the invention (eg aprotinin, bestatin), which are relatively small molecules, the trans-nasal absorption of inhibitory polymers is probably minimal in light of the size of these molecules and is eliminated by this the possible adverse side effects. Thus, mucoadhesive polymers, particularly of the poly (acrylic acid) type, can serve both as an adhesive that promotes absorption and an enzyme protective agent to improve the controlled delivery of peptide and protein drugs, especially when safety concerns are considered. . In addition to protecting against enzymatic degradation, bioadhesives and other agents that promote polymeric or non-polymeric absorption for use within the invention can directly increase mucosal permeability to biologically active agents. To facilitate the transport of large and hydrophilic molecules, such - as peptides and proteins, through the nasal epithelial barrier, mucoadhesive polymers and other agents have been postulated to produce improved permeation effects beyond what is considered by the prolonged premucosal residence time of the delivery system. The time course of plasma concentrations with drug have been reported suggesting that bioadhesive microspheres cause an acute but transient increase in insulin permeability through the nasal mucosa. Other mucoadhesive polymers for use within the invention, for example guitosan, have been reported to improve 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 acidic hyaluronic acid and ester derivatives thereof. A particularly useful bioadhesive agent within the co-ordinated administration, and / or the 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 pharmaceutical and medical applications due to its favorable properties of low toxicity and good biocompatibility. It is a natural polyaminessaccharide prepared from chitin by N-deacetylation with alkali. As used within the methods and compositions of the invention, chitosan increases the retention of peptide proteins binding to the Y2 receptor, analogs and mimetics, and other biologically active agents described herein at a mucosal application site. 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 within the invention is denoted as a polymer of β- [1-4] -2-guanidino-2-deoxy-D-glucose (poly-GuD). The qosan is the N-deacetylated product of chitin, a naturally occurring polymer that has been used extensively to prepare microspheres for oral and intra-nasal formulations. The chitosan polymer has also been proposed as a soluble vehicle for the delivery of parenteral drug. Within one aspect of the invention, o-methylisourea is used to convert a chitosan aamine to its guanidinium residue. The guanidinium compound is prepared, for example, by the reaction between equi-normal solutions of chitosan and o-methylisourea at pH above 8.0. The guanidinium product is polymer of - [14] -guanidino-2-deoxy-D-glucose. This is abbreviated as Poli-GuD in this context (Monomer F. of Amina in Quitosan = 161; Monomer F.W. of Guanidinio in Poli-GuD = 203). An exemplary Poli-GuD preparation method for use within the invention involves the following protocol. Solutions: Preparation of 0.5% Acetic Acid Solution (0.088N) Pipette of 2.5 ml of glacial acetic acid in a 500 ml volumetric flask, diluted to volume with purified water. Preparation of 2N NaOH Solution: Transfer pellets of approximately 20 g of NaOH to a beaker with approximately 150 ml of purified water. Dissolve and cool to room temperature, transfer the solution to a 250 ml volumetric flask, dilute to volume with purified water. Preparation of Sulfado O-metilsourea (equivalent to the 0.4N urea group): Transfer approximately 493 mg of O-methyl sulfate sulfate in a 10 ml volumetric flask, dissolve and dilute to volume with purified water. The pH of the solution is 4.2 Preparation of Barium Chloride Solution (0.1M): Approximately 2.086 g of Barium Chloride is transferred into a 50 ml volumetric flask, dissolved and diluted to volume with purified water - Preparation of Chitosan Solution (equivalent to 0.06N amine): Approximately 100 mg of Chitosan is transferred into a 50 ml beaker, 10 ml of 0.5% Acetic Acid (0.088N) are added. Shake to remove completely The pH of the solution is 4.5 Preparation of O-Methylsourea Chloride Solution (equivalent to the 0.2N urea group): Pipette 5.0 ml of 0-methylsourea sulphate solution (equivalent to the 0.4 N urea group) and 5 ml of a barium chloride solution in a beaker. A precipitate forms. The solution is continued mixing for 5 minutes. The solution is filtered through a 0.45m filter and the precipitate is discarded. The concentration of O-methylsourea chloride in the supernatant solution is equivalent to the 0.2 N urea group. The pH of the solution is- 4.2 Procedure: Add 1.5 ml of 2N NaOH to 10 ml of the chitosan solution (equivalent to 0.06 N amine). ) prepared as described in Section 2.5. The pH is adjusted to the solution with 2N NaOH from about 8.2 to 8.4. The solution is stirred for an additional 10 minutes. 3.0 ml of methylsourea chloride solution (equivalent to the urea group) is added 0. 2 N) prepared as described above. The solution is stirred overnight. The pH of the solution is adjusted to 5.5 com 0.5% Acetic Acid (0.088 N). The solution is diluted to a final volume of 25 ml using purified water. The concentration of Poly-GuD in the solution is 5 mg / ml, equivalent to 0.024 N (guanidium group). Additional compounds classified as bioadhesive agents for use within the present invention. They act by mediating specific interactions, typically they are classified as "ligand-receptor interactions" between complementary structures of the bioadhesive compound and a component of the mucosal epithelial surface. Many natural examples illustrate this form of specific binding, as exemplified by the lecithin-sugar interactions. Lecithins are proteins (glyco) of non-immune origin that bind to polysaccharides or glycoconjugates. Several plant lecithins have been investigated as agents that promote pharmacological absorption. A lecithin of Phaseolus vulgaris hemagglutinin (PHA) plant exhibits high oral bioavailability of more than 10% after feeding rats. Tomato lecithin (TL) (Lycopersicon esculeutum) seems safe for several models of - administration . In summary, the above bioahesive agents are useful in the combinbational formulations and methods of coordinated administration of the present invention, which optionally incorporate an effective amount of the form of a bioadhesive agent to prolong the persistence or otherwise increase the mucosal absorption of one. of proteins, analogs and mimetics of the Y2 receptor binding peptide and other biologically active agents. The bioadhesive agents can be administered co-ordinately as adjuncts or as additives within the combinational formulations of the invention. In certain modalities, the bioadhesive agent acts as a "pharmaceutical glue", conring that in other modalities the adjunctive supply or combinational formulation of the bioadhesive agent serves to identify contacts of the biologically active agent with the nasal mucosa, in some cases by promoting the interactions of the receptor-specific ligand with "recipient" epithelial cells, and in others by increasing the permeability of the epithelium to significantly increase the concentration gradient of the drug measured at a target delivery site (e.g., liver, blood plasma, or tissue of the CNS or fluid). Still further bioadhesive agents for use within the invention act as enhancers of the enzyme (eg, protease) to improve the stability of biotherapeutic agents administered to the mucosa coordinately supplied or in a combination formulation with the bioadhesive agent. . Liposomal and Micellar Delivery Vehicles The coordinated delivery methods and combinatorial formulations of the present invention optionally incorporate lipid or fatty acid-based vehicles, processing agents, or delivery vehicles, to provide improved formulations for mucosal delivery of the peptide protein binding to the Y2 receptor, analogs and mimetics, and other biologically active agents. For example, a variety of formulations and methods are provided for the mucosal delivery comprising one or more of these active agents, such as a peptide or protein, mixed or encapsulated, or co-ordinated with, a mixed liposome, micellar carrier, or emulsion, to improve chemical and physical stability and increase the half-life of biologically active agents (eg, by susceptibly reducing proteolysis, chemical modification and / or denaturation) to 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 from natural, biodegradable, non-toxic and non-immunogenic lipid molecules, and can be efficiently trapped or bound to drug molecules, including peptides and proteins, in or on their membranes. This attractiveness 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 liposome membrane protects them from the proteolysis and other factors of destabilization. Although all methods of liposome preparation known for the encapsulation of peptides and proteins are not feasible due to their unique physical and chemical properties, several methods allow the encapsulation of these macromolecules without susceptor deactivation. A variety of methods are available to prepare liposomes for use within the invention, U.S. Pat. No. 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 vesicle or lipid, or attached to the out of the vesicle. Similar to liposomes, long-chain unsaturated fatty acids, which have also improved activity for mucosal absorption, can form vesicles with bistratified-like structures (so-called "ufasomes"). These can be formed, for example, by using oleic acid to treat peptides and proteins for mucosal delivery, e.g., intranasal, within the invention. Other delivery systems for use within 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 controlled through the use of covalent crosslinking agents and the addition of antifibrinolytics to the fibrin polymer. More specifically, the delivery systems for use within the invention include the use of cationic lipids as delivery support vehicles, which can be effectively employed to provide an electrostatic interaction between the lipid carrier and such biologically active agents as polyanionic proteins and nucleic acids. . This allows efficient packaging of the drugs in a form suitable for mucosal administration and / or subsequent delivery to the systemic compartments. Additional supply vehicles for use within the invention include long and medium chain fatty acids, as well as micelles mixed with surfactant with fatty acids. Natural lipids in the form of esters have important implications with respect to their own transport through mucosal surfaces. The free fatty acids and their monoglycerides having polar pooled groups have been shown in the form of mixed micelles to act on the intestinal barrier as penetration enhancers. This discovery of the function of free fatty acids that modifies the barrier (carboxylic acids with a long chain that varies from 12 to 20 carbon atoms) and their polar derivatives have stimulated extensive research in the application of these agents as enhancers of the mucosal absorption. For use within the methods of the invention, long chain fatty acids, especially fusogenic lipids (unsaturated fatty acids and monoglycerides such as oleic acid, linoleic acid, linoleic monoolein acid, etc.) provide useful vehicles for improving the mucosal delivery of the peptide binding to the Y2 receptor, analogs and mimetics, and other biologically active agents described herein. Medium chain fatty acids (C6 to C12) and monoglycerides have also been shown to have improved activity in intestinal absorption of the drug and can be adapted for use within the formulations and mucosal delivery methods of the invention. In addition, the sodium salts of medium and long chain fatty acids are effective delivery vehicles and agents that improve the absorption for mucosal delivery of biologically active agents within the invention. Thus, the fatty acids can be used in soluble forms of sodium salts or by the addition of non-toxic surfactants, e.g., hydrogenated polyoxyethylated castor oil castor oil, sodium taurocholate, etc. Other fatty acids and mixed micelle preparations which are useful within 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 within the invention include chemical modification of biologically active peptides and proteins by cavalent attachment of polymeric materials, for example dextrans, polyvinyl pyrrolidones, glycopeptides, polyethylene glycol and polyamino acids. The resulting peptides and conjugated proteins retain their biological activities and solubility for mucosal administration. In alternative modalities, the analog and mimetic proteins of the Y2 receptor binding peptide, and other biologically active peptides and proteins, are conjugated to polyalkylene oxide polymers, particularly polyethylene glycols (PEG). Patent of E.U. No. 4,179,337. PEG polymers that react with amine for use within the invention include SC-PEG with molecular masses of 2000, 5000, 10000, 12000, and 20 000; U-PEG-10000; NHS-PEG-3400-biotin; T-PEG-5000; T-PEG-12000; and TPC-PEG-5000. PEGylation of biologically active peptides and proteins can be achieved by modification of the carboxyl sites (e.g., aspartic acid or glutamic acid groups in addition to the carboxyl terminals). The utility of PEG-hydrazide in the selective modification of carboxyl groups of carbodiimide-activated protein under acidic conditions has been described. Alternatively, the bifunctional modification of PEG of biologically active peptides and proteins can be employed. In some methods, charged amino acid residues that include lysine, aspartic acid, and glutamic acid have a marked tendency to be accessible to solvents or protein surfaces. Other Stabilization Modifications of Active Agents In addition to PEGylation, biologically active agents such as peptides and proteins for use within the invention can be modified to improve the circulating half-life by protecting the active agent through conjugation to other known stabilizing or protective compounds, for example by creating of fusion proteins with an active peptide, protein, analogs or mimetics linked to one or more carrier proteins, such as one or more inmuglobin chains. Formulation and Administration The mucosal delivery formulations of the present invention comprise analogs and mimetics of the Y2 receptor binding peptide, typically combined together with one or more pharmaceutically acceptable carriers and, optionally, other therapeutic ingredients. The vehicle (s) must be "pharmaceutically acceptable" in the sense of being compatible with other ingredients of the formulation and not producing an unacceptable harmful effect on the subject. Such vehicles are described hereinbefore or are otherwise well known to those of experience in the pharmacology art. Desirably, the formulation should not include substances such as enzymes or oxidizing agents whereby the biologically active agent administered is known to be incompatible. The formulations can be prepared by any of the methods well known in the art of pharmacy. Within the compositions and methods of the invention, the proteins, analogs and mimetics of the Y2 receptor binding peptide, and other biologically active agents described herein can be administered to subjects through a variety of modes of administration, including oral delivery. , rectal, vaginal, intranasal, intrapulmonary, or transdermal, or by topical delivery to the eyes, skin, or other mucosal surfaces. Optionally, the proteins, analogs and mimetics of the Y2 receptor binding peptide, and other biologically active agents described herein are administered in a coordinated or adjunct manner by non-mucosal pathways, including by intramuscular, subcutaneous, intravenous, intra-atrial, intra-articular routes. , intraperitoneal, or parenteral. In other alternative embodiments, the biologically active agent (s) can be administered ex vivo by direct exposure to the cells, tissues or organs of a mammalian subject, for example as a component of a treatment formulation of an exogenous tissue or organ. vivo 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 such as a nasal or pulmonary asperion and can be administered in the form of a spray by a variety of methods known to those of skill in the art. Preferred systems for administering liquids as a nasal spray are described in the U.S. Patent. No. 4,511,069. The formulations can be presented in - - multi-dose containers, for example in the sealed delivery system described in the U.S. Patent. No. 4,511,069. Additional forms of aerosol delivery may include, eg, compressed air, jet, ultrasonic, and piezoelectric nebulizers, which deliver the biologically active or suspended agent in a pharmaceutical solvent, eg, water, ethanol, or a mixture thereof. The nasal or pulmonary spray solutions of the present invention typically comprise the drug or drug to be administered, optionally formulated with a surfactant, such as a nonionic surfactant (e.g., 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 3.0 and 6.0, preferably 5.0 + 0.3. Shock absorbers suitable for use within these compositions are as described above or as otherwise known in the art. Other components can be added to improve or maintain the guimic stability, including preservatives, surfactants, dispersants, or gases. Suitable preservatives include, but are not limited to, phenol, methyl paraben, paraben, m-cresol, thiomersal, chlorobutanol, benzylalkonium chloride, and the like. Suitable surfactants include, but are not limited to, oleic acid, sorbitan trioleate, polysorbates, lecithin, phosphotidyl cholines, and various long chain diglycerides and phospholipids. Suitable dispersants include, but are not limited to, ethylenediaminetetraacetic acid, and the like. Suitable gases include, but are not limited to, nitrogen, helium, chlorofluorocarbons (CFCs), hydrofluorocarbons (HFCs), carbon dioxide, air, and the like. Within alternative embodiments, the mucosal formulations are administered as dry powder formulations comprising the biologically active agent in a dry form, normally lyophilized, of an appropriate particle size, or within a range of appropriate particle size, for delivery intranasal The minimum particle size appropriate for deposition within the nasal or pulmonary passageway is frequently of approximately 0.5 μm mean aerodynamic diameter of the average mass (MMEAD), commonly of approximately 1 μMMEAD, and more typically of approximately 2 μMEMAD. The maximum appropriate particle size for deposition within the nasal passage is frequently of approximately 10 μMMEAD, commonly of approximately 8 μMEMAD, and more typically of approximately 4 μMEMAD. Intranasal the respirable powders within these size ranges can be produced by a variety of techniques - conventional, such as jet grinding, spray drying, solvent precipitation, supercritical fluid condensation and the like. These suitable MMEAD dry powders can be administered to a patient through a conventional dry powder inhaler (DPI), which is found in the patient's breath, to lung or nasal inactivation, to disperse the powder in a spray amount. Alternatively, the dry powder can be administered through air-assisted devices that use external powder sources to disperse the powder in an aerosolized amount, eg, a piston pump. Dry powder devices typically require a mass of dust in the range of about 1 mg to 20 mg to produce a single aerosol dose ("puff"). If a dose is required or desired or if the dose of a biologically active agent is less than this amount, the powdered active agent will typically be commingled with a powder in dry pharmaceutical volume to provide the total polka mass required. Preferred dry bulk powders include sucrose, lactose, dextrose, mannitol, glycine, trehalose, human serum albumin (HSA), and starch. Other suitable dry bulk powders include cellobiose, dextrans, maltotriose, pectin, sodium citrate, sodium ascorbate, and the like. To formulate compositions for the supply - mucosal within the present invention, the biologically active agent can be combined with various pharmaceutically acceptable additives, as well as a base or vehicle for the dispersion of the active agent (s). The desired additives include, but are not limited to, pH control agents, such as arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid, etc. In addition, local anesthetics (eg, benzyl alcohol), isotonization agents (eg, sodium chloride, mannitol, sorbitol), absorption inhibitors (eg, Tween 80), solubility improving agents (eg, cyclodextrins and derivatives of the same), stabilizers (eg, serum albumin), and reducing agents (eg, glutathione). When the composition for the mucosal delivery is a liquid, the tonicity of the formulation, as measured by reference to the tonicity of 0.9% (w / v), the physiological saline solution taken as a unit, is typically adjusted to a value at which no harm will be induced to the substantial, irreversible tissue in the nasal mucosa at the site of administration. Generally, the tonicity of the solution is adjusted to a value of approximately 1/3 to 3, more typically 1/2 to 2, and more frequently 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 capability to disperse the active agent and any desired additive. The base can be selected from a wide range of suitable vehicles, including but not limited to copolymers of polycarboxylic acids or salts thereof, carboxylic anhydrides (eg maleic anhydride) with other monomers (eg methyl (meth) acrylate, acrylic acid, etc.). ), hydrophilic vinyl polymers such as polyvinyl acetate, polyvinyl alcohol, polyvinyl pyrrolidone, cellulose derivatives such as hydroxymethyl cellulose, hydroxypropyl cellulose, etc., and natural polymers such as chitosan, collagen, sodium alginate, gelatin, hyaluronic acid, and metal salts non-toxic of the same. Frequently, a biodegradable polymer is selected as a base or carrier, for example, polylactic acid, poly (lactic acid-glycolic acid) copolymer, polyhydroxybutyric acid, poly (hydroxybutyric acid-glycolic acid) copolymer and mixtures thereof. Alternatively or additionally, the synthetic fatty acid esters such as polyglycerin fatty acid esters, sucrose fatty acid esters, etc. they can be used as vehicles. Hydrophilic polymers and other vehicles can be used alone or in combination, and improved structural integrity can be imparted to the vehicle by partial crystallization, ionic bonding, crosslinking and the like. The vehicle can be provided in a variety of ways, including fluid or viscose solutions, gels, pastes, powders, microspheres and films for direct application to the nasal mucosa. The use of a vehicle selected in this context may result in the promotion of the absorption of the biologically active agent. The biologically active agent can be combined with the base or carrier according to a variety of methods, and the release of the active agent can be by diffusion, disintegration of the vehicle, or associated formulation of the water channels. In some circumscriptions the active agent is dispersed in microcapsules (microspheres) or nanocapsules (nanospheres) prepared from a suitable polymer, eg, isobutyl 2-cyanoacrylate and dispersed in a biocompatible dispersion medium applied to the nasal mucosa, which produces sustained delivery and biological activity over a prolonged time For the further improved mucosal delivery of pharmaceutical agents within the invention, the formulations comprising the active agent also contain a low molecular weight hydrophilic compound as a base or excipient. Such a hydrophilic low molecular weight compound provides a means of passage through which a soluble active agent agent in water, such as an active physiological peptide or protein, can diffuse through the base of the body surface where the agent is absorbed. active. The hydrophilic low molecular weight compound optionally absorbs moisture from the mucosa or atmosphere of administration and dissolves the water-soluble active peptide. The molecular weight of hydrophilic low molecular weight compound is generally not more than 10,000 and preferably no more than 3,000. The exemplary hydrophilic low molecular weight compound includes polyol compound, 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 the low molecular weight hydrophilic compounds useful as carriers within the invention include N-methylpyrrolidone, and alcohols (eg oligovinyl alcohol, ethanol, ethylene glycol, propylene glycol, etc.). These low molecular weight hydrophilic compounds can be used alone. or in combination with a different or with other active or inactive component of the intranasal formulation. The composition of the invention may alternatively contain as pharmaceutically acceptable carriers substances that are required for approximate physiological conditions, such as pH adjusting agents and buffers, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, lactate - - Sodium chloride, 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 the similar. Therapeutic compositions for administering the biologically active agent may also be formulated as a soluton, microemulsion, or other ordered structures suitable for high concentration of the active ingredients. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity for the solutions can be maintained, for example, by the use of a coating such as lecithin., by maintaining 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 carried out approximately by including in the composition an agent to challenge the absorption, for example, salts of monostearate and gelatin. In certain embodiments of the invention, the biologically active agent is administered in a release formulation over time, for example in a composition that includes a slow release polymer. The active agent can be prepared with vehicles that will protect against rapid release, for example a controlled release vehicle such as a polymer, microencapsulated delivery system or bioadhesive gel. The controlled delivery active agent, in various compositions of the invention, can be brought close to by and include in the composition agents that retard absorption, for example, aluminum monostearate hydrogels and gelatin. When controlled release formulations of the biologically active agent are desired, the 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 agent biologically active. Such numerous materials are known in the art. Useful controlled release binders are materials that are metabolized slowly under physiological conditions after their intranasal delivery (e.g., 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 are inserted to surround the tissues, and do not cause significant adverse side effects such as nasal irritation, immune response, inflammation, or the like. They are not metabolized into metabolic products that are not compatible and are easily eliminated from the body. Exemplary polymeric materials for use in this context include, but are not limited to, derivatized polymer matrices of copolymeric and homopolymeric polyesters having water-soluble ester linkages. Several of these are known in the art to be biodegradable and to lead to degradation products that have low or no toxicity. Exemplary polymers include polyglycolic acids (PGA) and polylactic acids (PLA), poly (DL-lactic acid-co-glycolic acid) (DL PLGA), 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-acid) glycolic), 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. Generally many methods are known to prepare such formulations by those of experience in the art. Other useful formulations include e.g., controlled release compositions, microcapsules, U.S. Patent. No. 4,652,441 and 4,917,893, copolymers of lactic acid-glycolic acid useful for making microcapsules and other formulations, US Pat. No. 4,677,191 and 4,728,721, and sustained release compositions for water soluble peptides, US Pat. No. 4,675,189. 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. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the other ingredients required from those listed above. In the case of sterile powders, the preparation methods include vacuum drying and freeze drying which produces a powder of the active ingredient more than any desired additional 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 mucosal administration according to the invention allows the self-administration of the treatment by the patients, provided that the sufficient security is placed to control and monitor the dose and the side effects. The administration in mucous membranes also overcomes certain disadvantages of other administration forms. , such as injections, which are very painful and expose the patient to possible infections and may present bioavailability problems of the drug. For the nasal and pulmonary supply, controlled aerosol systems that are administered from therapeutic liquids such as a spray are well known. In one embodiment, the measured doses of the active ingredient are supplied by means of a specially constructed mechanical pump valve. Patent of E.U. No. 4,511,069. Dosage For prophylactic and treatment purposes, the biologically active agent (s) described in the present can be administered to the subject in the presence of a single bolus, through continuous supplies (eg, transdermal continuous supply, mucosal, or intravenous) through a period of time or in a protocol of repeated administration (eg, protocol of administration of repetition per hour, daily or weekly). In this context, a therapeutically effective dose of the Y2 receptor binding peptide may include repeated doses within a prophylaxis or treatment regimen that will produce clinically meaningful results to alleviate one or more symptoms or detectable conditions associated with a disease or objective condition such as stablece up. The determination of effective doses in this context is typically based on studies of an animal model followed by clinical trials in humans and is guided by the determination of effective dose and administration protocols that significantly reduce the occurrence or severity of the symptoms or condition of the objective disease in the subject. Suitable models in this regard include, for example, murine, rat, porcine, feline, non-human primate, and other accepted animal model subjects known in the art. Alternatively, the effective dose can be determined using in vitro models (e.g., immunological and histopathological assays). Using such models, only ordinary calculations and adjustments are typically required - to determine an appropriate concentration and dose for administering a therapeutically effective amount of the biologically active agent (s) (eg, amounts that are intranasally effective, transdermally effective, intravenously effective or intramuscularly effective to produce a desired response ). The actual dose of the biologically active agents will vary with the factors such as the indication of the disease and the particular state of the subject (eg, age, size, good health, degree of symptoms, susceptibility factors of the subject, etc.), time and route of administration, other drugs or treatments that are being administered concurrently, as well as the pharmacology specifies the biologically active agent (s) to produce the desired activity or biological response in the subject. Dosage regimens can be adjusted to provide an optimal prophylactic or therapeutic response. A therapeutically effective amount is also in which any toxic or deleterious collateral effect of the biologically active agent is weighted in clinical terms by beneficial therapeutic effects. A non-linear range for a therapeutically effective amount of a Y2 agonist within the methods and formulations of the invention is 0.7 μg / kg to about 25 μg / kg. To promote weight loss, an intranasal dose of the Y2 receptor binding peptide is administered at a sufficiently large dose to promote satiety but low enough not to induce any unwanted side effects such as nausea. A preferred intranasal dose of PYY3-36 is approximately 1 μg-10 μg / kg per patient weight, more preferably from approximately 1.5 μg / kg to approximately 3 μg / kg per patient weight. In a standard dose a patient will receive from 50 μg to 1600 μg, more preferably from approximately 75 μg to 800 μg, more preferably 100 μg, 150 μg, 200 μg to approximately 400 μg. Alternatively, a non-linear range for a therapeutically effective amount of a biologically active agent within the methods and formulations of the invention is between about 0.001 pmol to about 100 pmol per kg of body weight, between about 0.01 pmol to about 10 pmol per kilogram of body weight. kg of body weight, between about 0.1 pmol to about 5 pmol per kg of body weight, or between about 0.5 pmol to about 1.0 pmol per kg of body weight. Dosages within this range can be achieved by multiple or single administrations, including, e.g., multiple administrations per day, administrations daily or weekly. For administration, it is desirable to administer at least one microgram of the biologically active agent (eg, one or more proteins, analogs and mimetics of the Y2 receptor binding peptide, and other biologically active agents), more typically between about 10 μg and 5.0 mg , and in certain modalities between approximately 100 μg and 1.0 or 2.0 mg to an average human subject. For certain oral applications, doses as high as 0.5 mg per kg of body weight may be necessary to achieve adequate plasma levels. It is further noted that for each particular subject, the specific dose regimens should be evaluated and adjusted over time according to the individual needs and professional judgment of the person administering or supervising the administration of the perbeabilization peptide (s) and others. biologically active agent (s). An intranasal dose of a PYY will vary from 50 μg to 1600 μg of PYY, preferably from 75 μg to 800 μg, more preferably 100 μg to 400 μg with a more preferred dose being between 100 μg to 200 μg with 150 μg being the dose considered to be highly effective The repeated intranasal dosage with the formulations of the invention, in a program ranging from approximately 0.1 to 24 hours between doses, preferably between 0.5 and 24.0 hours between doses, will maintain normalized, sustained therapeutic levels of the Y2 receptor binding peptide to maximize clinical benefits while minimizing the risks of excessive exposure to side effects. This dose can be administered several times a day to promote satiety, preferably half an hour before a meal or when you feel hungry. The aim is to administer in the mucosa an amount of the peptide binding to the Y2 receptor sufficient to raise the concentration of the peptide binding to the Y2 receptor in the plasma of an individual that mimics the concentration that should normally occur after the meal, ie, after that the individual has finished eating. The dose of Y2 agonists such as PYY may be varied by the attending physician or patient, if self-administered a dosage form greater than the amount, to maintain a desired concentration at the target site. In an alternative embodiment, the invention provides compositions and methods for the intranasal delivery of the Y2 receptor binding peptide, wherein the compound (s) of the Y2 receptor binding peptide is administered repeatedly through the effective intranasal dose that includes multiple administrations of the Y2 receptor binding peptide to the subject during a daily or weekly schedule to maintain a high and less than therapeutically effective pulsatile level of the Y2 receptor binding peptide, during an extended dose period. The compositions and methods provide the Y2 receptor binding peptide compound (s) that are self-administered by the subject in a nasal formulation - between one and six times a day to maintain a high pusaatil level and lower therapeutically effective level of the peptide binding to the Y2 receptor during an extended dosing period of 8 hours to 24 hours extended dosing period. Equipment The present invention also includes products, packages and multi-container units containing the compositions, active ingredients, and / or pharmaceutical means described above, for the administration thereof for use in the prevention and treatment of diseases and other conditions in sleep. mammals In summary, these kits include a container or formulation containing one or more of proteins, analogs or mimetics of the peptide binding to the Y2 receptor, and / or other biologically active agents in combination with agents that enhance the mucosal delivery described in the present formulation. in a pharmaceutical preparation for mucosal delivery. The intranasal formulations of the present invention can be administered using any spray bottle or syringe. An example of a nasal spray bottle is found in, "Nasal Spray Pump w / Safety Clip, Pfeiffer SAP # 60548, which supplies a dose of 0. lml by spray and has a tube depth length of 36.05 mm. Acquired from Pfeiffer of America of Princeton, NJ The intranasal dose of a Y2 receptor binding peptide such as PYY may vary from O.lμg / kg to approximately 1500 μg / kg. When administered as a nasal spray, it is preferable that the particle size of the spray is between 10-100 μm (microns) in size, preferably 20-100 μm in size.To promote weight loss, an intranasal dose of a Y2 PYY receptor binding peptide is administered doses high enough to promote satiety but low enough so as not to induce any unwanted side effects such as nausea A preferred intranasal dose of a Y2 receptor binding peptide such as PYY (3-36) is about 3 μg - 10 μg / kg by weight of the patient, more preferably approximately 6 μg / kg by weight of the patient. In a standard dose, the patient will receive from 50 μg to 800 μg, more preferably from approximately 100 μg to 400 μg, more preferably 150 μg to approximately 200 μg. A peptide binding to the Y2 receptor such as PYY (3-36) is preferably administered at least ten minutes to one hour before eating to avoid nausea but no more than about twelve to twenty-four hours before eating. The patient is dosed at least once a day preferably before each meal until the patient has lost a desired amount of weight. The patient then receives maintenance doses at least once a week preferably daily to maintain weight loss. "- 72 As shown by the data of the following examples, when administered intranasally to humans using the Y2 receptor binding peptide formulation of the present invention, PYY (3-36) was found to reduce appetite. The examples also show that for a first time the physiological post-meal levels of a peptide PYY can be achieved through an intranasal route using the peptide formulations of the Y2 receptor binding of the present invention in which PYY (3-36) was the peptide binding to the Y2 receptor. Nasal Administration in PYY Spray We have discovered that binding peptides. The Y2 receptor can be administered intranasally using a nasal spray or aerosol. This is surprising because many proteins and peptides have been shown to be shared or denatured due to the mechanical forces generated by the actuator when producing the spray or aerosol. In this area the following definitions are useful. IX Aerosol - A product that is packaged under pressure and contains therapeutically active ingredients that are released upon activation of an appropriate valve system. Measured aerosol - A pressurized dose form comprises a dose metering valve, which allows the supply of a uniform quantity of the spray at each activation. Powdered Spray - A product that is packaged under pressure and contains therapeutically active ingredients in the form of a powder, which is released upon activation of an appropriate valve system. Spray aerosol - An aerosol product that uses a compressed gas as the propellant to provide the force necessary to expel the product as a wet spray; it is generally applied to solutions of medicinal agents in aqueous solvents. Spray - A liquid that is minutely divided as if by a jet or air stream. Nasal spray drug products contain therapeutically active ingredients dissolved or suspended in solutions or mixtures of excipients in non-pressurized suppliers. Measured spray - A non-pressurized dosage form consisting of valves that allow the delivery of a specific amount of spray at each activation. Dewdrop - A liquid preparation containing solid particles dispersed in a liquid vehicle and in the form of drops in the direction or as finely divided solids. The dynamic characterization of aerosol spray fluid is emitted by measured nasal spray pumps - as a drug delivery device ("DDD"). The characterization of the spray is an integral part of the regulatory subjection necessary for the approval by the Food and Drug Administration ("FDA") of the research and development procedures, quality commitment and stability tests for new and existing spray pumps. Through the characterization, it has been found that the geometry of the spray is the best indicator of the total performance of the nasal spray pumps. In particular, measurements of the divergence angle of the spray (boom geometry) as it exits the device; the transverse ellipticity of the spray, the uniformity and distribution of the particles / drops (spray pattern); and the evolution of the dew time that develops has been found to be the best representative performance quantities in the characterization of a nasal spray pump. During the quality commitment and the stability test, the measurements of the boom geometry and the spray pattern are key identifiers to verify the consistency and conformation with the criteria of the approved data for nasal spray pumps. Definitions Boom height - the measurements from the tip of the actuator to the point at which the angle of the boom becomes non-linear due to the breaking of the linear flow. With - - based on the visual examination of digital images, and to establish a measurement point for the width ie consistent with the furthest measurement point of the spray pattern, a height of 30 mm is defined for this study. Main Axis - the longest rope that can be removed within the adapted spray pattern that crosses the COMw in base units (mm). Proportion of Ellipticity - the smallest rope that can be extracted within the adapted spray pattern that crosses the COMw in base units (mm). Proportion of Ellipticity - the proportion of the major axis to the minor axis. Dio - the diameter of the droplets for which 10% of the total liquid volume of samples consisting of droplets of a smaller diameter (μm). D5n - the diameter of the droplets for which 50% of the total liquid volume of samples consisting of droplets of a smaller diameter (μm), also known as the medi-mass diameter. D90 - the diameter of the droplets for which 90% of the total liquid volume consists of samples consisting of droplets of a smaller diameter (μm).

Path - measurement of the deviation width, the smallest value, the narrowest distribution. The path (D90-D10) is calculated as ° 50% RSD - standard deviation in relation to the percentage, the standard deviation divided by the average of the series and multiplied by 100, also known as% CV. Figures 21A and 21B show a nasal spray device 10 before the clutch (Figure 21A) and after the clutch (Figure 21B). The nasal spray bottle 10 is comprised of a bottle 12 in which the formulation of the peptide binding to the Y2 receptor is placed, and an actuator 14, which when the forces act or clutch a spray pen, 16, of the binding peptide the receiver Y2 leaves the spray bottle, 12, through the actuator, 14. A spray pattern is determined by taking a photograph of the cross section of the spray head 16 above a predetermined height, 18, of the pen . The spray boom also has an ejection angle, 20, as it leaves the actuator, 14. A spray pattern of the spray boom 16 is shown in Figure 22. The spray pattern 22, is elliptical and has a major axis, 24, and a minor axis 26. The following examples are provided by way of illustration, without limitation - - EXAMPLE 1 A formulation that exemplifies the improved nasal mucosal delivery of peptide YY following the techniques of the present specification was prepared and evaluated as follows: Table 1: Composition of the YY peptide formulation - - EXAMPLE 2 Nasal mucosal delivery - Permeation kinetics and cytotoxicity 1. Organotypic Model The following methods are generally useful for evaluating the parameters, kinetics and side effects of the nasal mucosal delivery for the YY peptide within the formulations and method of the invention, as well as to determine the efficiency and characteristics of various enhancing agents the intranasal delivery described herein for the combinational formulation or the coordinated administration with the YY peptide. The permeation and cytotoxicity kinetics are also useful for determining the efficiency and characteristics of the various mucosal supply enhancing agents described herein for the combinational formulation or the. coordinated administration with agents that improve the mucosal supply. In an exemplary protocol, the permeation kinetics and lack of unacceptable cytotoxicity are demonstrated for an intranasal delivery enhancing agent as described above in combination with a biologically active therapeutic agent, exemplified by the YY peptide.

The EpiAirway System was developed by MatTek Corp (Ashland, MA) as a model of the pseudostratified epithelium that lines the respiratory tract. Epithelial cells develop in cell cultures supported by porous membrane inserts at an air-liquid interface, which results in cell differentiation for a highly polarized morphology. The apical surface is ciliated with a microvellose ultrastructure and the epithelium produces mucus (the presence of mucin has been confirmed by immunoblotting). The inserts have a diameter of 0.875 cm, providing a surface area of 0.6 cm2. The cells are plated on inserts at the factory approximately three weeks before shipment. A "team" consists of 24 units. A. Upon arrival, the units are placed on sterile supports in 6-well microplates. Each well receives 5 ml of patented culture medium. This medium based on DMEM is free of serum but is complemented with epidermal growth factor and other factors. The medium is always tested for the endogenous levels of any cytokine or growth factor, which is being considered for intranasal delivery, but has been free of all cytokines and factors studied to date except insulin. The volume of 5 ml is just enough to provide contact with the bottom of the - - units or their supports, but the apical surface of the epithelium is allowed to remain in direct contact with the air. Sterile tweezers are used in this stage and in all subsequent stages that include the transfer of units to wells containing liquid to ensure that air is not trapped between the bottom of the units and the medium. B. The units are kept in their plates at 37 ° C in an incubator in a 5% C02 atmosphere in air for 24 hours. At the end of this time the medium is replaced with fresh medium and the units are returned to the incubator for another 24 hours. 2. Experimental Protocol - Permeation kinetics A. A "team" can be routinely used 24 EpiAirway Units to evaluate five different formulations, each of which is applied to wells in quadruplicate. Each well is used for the determination of permeation kinetics (4 time points), transepithelial resistance, mitochondrial reductase activity as measured by MTT reduction, and cytolysis as measured by LDH release. An additional set of wells is used as controls, which are treated in a simulated manner during the determination of permeation kinetics, but otherwise handle in an identical manner to the units containing the test sample for transepithelial resistance determinations and viability. The determinations on the controls are also routinely done in quadrupled units, but occasionally we have used units in triplicate for the controls and we have dedicated the remaining four units in the equipment for measurements of transepithelial resistance and viability on the untreated units or we have frozen and Thawed units for determinations of total LDH levels to serve as a 100% reference for cytolysis. B. In all experiments, the nasal mucosal supply formulation to be studied is applied to the apical surface of each unit in a volume of 100 μl, which is sufficient to cover the entire apical surface. An appropriate volume of the test formulation in the concentration applied to the apical surface (generally no more than 100 μl is required) is set aside for the subsequent determination of concentration of the active material by ELISA or other designated assays. C. Units are placed in 6-well plates without support for the experiment: each well contains 0.9 ml of medium which is sufficient to contact the bottom of the porous membrane of the unit but does not generate any significant upward hydrostatic pressure on unit D. To minimize potential sources of error and avoid any formation of concentration gradients, the units are transferred from a well that contains 0.9 ml to another at each time point in the study. These transfers are made at the following time points, based on a zero point at which the volume of 100 μl of the test material was applied to the apical surface: 15 minutes, 30 minutes, 60 minutes, and 120 minutes. E. Between the time points the units in their plates are kept in the 37 ° C incubator. Plates containing 0.9 ml of medium per well are also maintained in the incubator so that a minimum change in temperature occurs during the brief periods when the plates are removed and the units are transferred from one well to another using sterile forceps F At the end of each time point, the medium is removed from the well from which each unit was transferred, and aliquots are made in two tubes (one tube receives 700 μl and the other 200 μl) to determine the concentration of the Permeate test material and, for the case that the test material is cytotoxid, by the release of the cytosolic enzyme, lactic dehydrogenase, from the epithelium. These samples are kept in the refrigerator if the tests will be conducted within 24 hours, or the samples will be sub-aliquots and kept frozen at -80 ° C until they are thawed for the test. Repeated freeze-thaw cycles are avoided.

- G. In order to minimize errors, all tubes, plates and wells are pre-labeled before starting an experiment. H. At the end of the 120 minute time point, the units are transferred from the last of the 0.9 ml wells to the 24 well microplates, containing 0.3 ml of medium per well. This volume is again sufficient to put the lower part of the units in contact, but does not exert upward hydrostatic pressure on the units. The units are returned to the incubator before transepithelial resistance measurement. 3. Experimental protocol - Transepithelial resistance A. Respiratory air epithelial cells form tight junctions in vivo as well as in vitro, restricting the flow of solutes through the tissue. These junctions confer a transepithelial resistance of several hundred ohms x cm2 in extracted aerial tissues; in EpiAirway MatTek units, the transepithelial resistance (TER) is claimed by the manufacturer to be routinely around 1000 ohms x cm2. We have found that the TER of the EpiAirway control units that have been exposed in simulation during the sequence of the stages in the permeation study is sometimes lower (700-800 ohms x cm2), but, since the permeation of the molecules small is proportional to the inverse of TER, this value is still high enough to provide a greater sweep for permeation. The units of the lower part of the porous membrane without cells, provide inversely only a minimum transmembrane resistance (5-20 ohms x cm2). B. Exact TER determinations require that the ohmmeter electrodes be placed on a significant surface area above and below the membrane, and that the distance of the electrodes from the membrane be controlled in a reproducible manner. The method for the determination of TER recommended by MatTek and used for all experiments, employ an "EVOM" ™ epithelial voltometer and a tissue resistance measuring chamber "ENDOHM" ™ from World Precision Instruments, Inc., Sarasota. , FL. C. The chamber is initially filled with Dulbecco's phosphate buffered saline (PBS) for at least 20 minutes before the TER determinations in order to balance the electrodes. D. The TER determinations are made with 1.5 ml of PBS in the chamber and 350 μl of PBS in the unit -supported in the membrane that is being measured. The upper electrode is adjusted to a position just above the membrane of a unit that does not contain cells (but which contain 350 μl of PBS) and is then fixed to ensure reproducible placement. The resistance of a cell-free unit is typically 5-20 ohms x cm2 ("background resistance"). E. Once the chamber is prepared and bottom resistance is recorded, the units in a 24-well plate that have only been used in the permeation determinations are removed from the incubator and placed individually in the chamber for determinations of TER. F. Each unit is transferred first to a Petri dish containing PBS to ensure that the bottom of the membrane becomes wet. An aliquot of 350 μl of PBS is added to the unit and then carefully aspirated into a labeled tube to raise the apical surface. A second wash of 350 μl of PBS is applied to the unit and aspirated in the same collection tube. G. The unit is gently immuno-transferred free of excess PBS on its outer surface just before being placed in the chamber (containing a fresh aliquot of 1.5 ml PBS). An aliquot of 350 μl PBS is added to the unit before the top electrode is placed on the chamber and the TER is read on the EVOM meter. H. After the TER of the unit in the ENDOHM chamber is read, the unit is removed, the PBS is aspirated and stored, and the unit is returned with an air interface on the apical surface to a plate of 24- wells containing 0.3 ml of medium per well. I. The units are read in the following sequence: all the controls treated in simulation followed by all the samples treated with the formulation, followed by a second reading of TER of each of the controls treated in false. After all TER determinations are completed, the units in the 24-well microplate are returned to the incubator for road determination by MTT reduction. 4. Experimental protocol - Viability by MTT reduction MTT is a cell-permeable tetrazolium salt that is reduced by the activity of mitochondrial dehydrogenase to an insoluble for azan colored by viable cells with intact mitochondrial function or by the activity of non-mitochondrial dehydrogenase NAD (P) H of the cells capable of generating a respiratory burst. Formazan formation it is a good indicator of the viability of epithelial cells since these cells do not generate a significant respiratory burst. We have used an MTT reagent prepared by MatTek Corp for its units in order to assess viability. A. The MTT reagent is supplied as a concentrate and diluted in a patented DMEM based diluent on the viability day for. analyzed (typically in the afternoon of the day in which the permeation kinetics and TER were determined in the morning). The insoluble reagent is removed by brief centrifugation after use. The final concentration of MTT is 1 mg / ml. B. The final MTT solution is added to the wells of a 24-well microplate at a volume of 300 μl per well. As noted above, this volume is sufficient to contact the membranes of the EpiAirway units but does not impose significant positive hydrostatic pressure on the cells. C. The units are removed from the 24 well plate in which they were placed after the TER measurements and after removing any excess liquid from the outer surface of the units, these are transferred to the plate containing the MTT reagent . The units in the plate are then placed in an incubator at 37 ° C in an air atmosphere with 5% C02 for 3 hours. D. At the end of 3 hours of incubation, the units containing the viable cells will have visibly become purple. The insoluble formazan must be extracted from the cells in their units to quantify the degree of MTT reduction. The extraction of the formazan is carried out by transferring the units to a 24-well microplate containing 2 ml of extraction solution per well, after removing the excess liquid from the outer surface of the units as before. This volume is sufficient to completely cover both the membrane and the apical surface of the units. The extraction is allowed to proceed - l í overnight at room temperature in a light-tight chamber. MTT extractions traditionally contain high concentrations of detergent and destroy the cells. E. At the end of the extraction the fluid from within each unit and the fluid in its surrounding wells is combined and transferred to a tube to make subsequent aliquots in a 96-well microplate (the 200 μl aliquots are optimal) and the determination of absorbance at 570 nm in a VMax multi-well microplate spectrophotometer. To ensure that the turbidity of the debris coming from the extracted units does not contribute to the absorbance, the absorbance at 650 nm is also determined for each well in the VMax and is automatically subtracted from the absorbance at 570 nm. The "control" for formazan absorbance determinations is an aliquot of 200 μl of the extract to which no unit has been exposed. This absorbance value is assumed to constitute zero viability. F. Two units of each 24-Unit EpiAirway unit are left untreated during the determination of permeation and TER kinetics. These units are used as the positive control for 100% cell viability. In all the studies we have conducted, there have been no statistically significant differences in the viability of the cells in these untreated units vs. the cells in the control units that have been treated in simulation for the permeation kinetics and in which have made the TER determinations. The absorbance of all units treated with the test formulations is assumed to be linearly proportional to the percent viability of the cells in the units at the time of incubation with MTT. It should be noted that this test is typically carried out no less than four hours after the introduction of the test material to the apical surface, and subsequent to the washing of the apical surface in the units during the determination of TER. 5. Determination of Feasibility by releasing LDH Although measurements of mitochondrial reductase activity by reducing MTT is a test sensitive to cell viability, the assay necessarily destroys the cells and can therefore be carried out only at end of each. When the cells undergo necrotic lysis, their cytosolic contents are poured into the surrounding medium, and cytosolic enzymes such as lactic dehydrogenase (LDH) can be detected in this medium. An assay for LDH in the medium can be performed on samples of the medium removed at each time point from the determination of every two hours of the permeation kinetics. Thus, the cytotoxic effects of the formulations that do not develop can be detected until a significant time has elapsed, as well as the effects of the formulations that induce cytolysis in the first minutes of exposure to the aerial epithelium. A. The recommended LDH test to evaluate the cytolysis of the EpiAirway Units is based on the conversion of lactate to pyruvate with the generation of NADH from NAD. The ADH is then re-oxidized together with the simultaneous re-oxidation of the salt with INT tetrazolium, catalyzed by a crude preparation of "diaphorase". The formed formazan of the 1NT reduction is soluble, so that the complete assay for LDH activity can be carried out in a homogeneous aqueous medium containing lactate, NAD, diaphorase, and INT. B. The analysis for LDH activity is carried out in 50 μl aliquots of the samples from the "supernatant" medium surrounding an EpiAirway unit and collected at each time point. These samples are stored either for no longer than 24 hours in the refrigerator or thawed after being frozen within a few hours after collection. Each EpiAirway unit generates samples of the supernatant medium collected at 15 min, 30 min, 1 h, and 2 h after the application of the test material. The aliquots are all transferred to a 96-well microplate. C. An aliquot of 50 μl medium that has not been exposed to a unit serves as a "control" or negative control of 0% cytotoxicity. We have found that the apparent level of "endogenous" .LDH present after the reaction of the mixture of the analyzed reagent with the unexposed medium is the same within the experimental error as the apparent level of LDH released by the control units treated in simulation. in the 2-hour full-time course required to conduct a study of permeation kinetics. A) Yes, within the experimental error, these units treated in simulation do not show cytolysis of the epithelial cells in the course of time of the measurements of the permeation kinetics. D. To prepare a sample of supernatant medium that reflects the level of LDH released after 100% of the cells in a unit have been lysed, a unit which has not been subjected to any previous manipulation is added to a well of a 6-well microplate containing 0.9 ml of medium as in the protocol for the determination of permeation kinetics, the plate containing the unit is frozen at -80 ° C, and the contents of the well are then allowed to thaw. This freeze-thaw cycle effectively smooths the cells and releases their cytosolic contents, including LDH, into the supernatant medium. An aliquot of 50 μl of the frozen and thawed cell medium is added to the 96 well plate - - as a positive control that reflects 100% cytotoxicity. E. To each well containing an aliquot of the supernatant medium, a 50 μl aliquot of LDH assay reagent is added. The plate is then incubated for 30 minutes in the dark. F. The reactions are terminated by the addition of a 1 M acetic acid "stop" solution, and within one hour of the addition of the stop solution, the absorbance of the plate at 490 nm is determined. G. Computation of the percent of cytolysis is based on the assumption of a linear relationship between absorbency and cytolysis, with the absorbance obtained from the medium only that serves as a reference for 0% cytolysis and the absorbance obtained from the surrounding medium the frozen and thawed unit that serves as a reference for 100% cytolysis. 6. ELISA Determinations Methods for determining the concentrations of biologically active agents as test materials for evaluating the improved permeation of active agents in conjunction with the coordinated administration of mucosal delivery enhancing agents or the combination formulation of the invention are generally is described above and in accordance with known methods and - - specific instructions of the manufacturer of ELISA equipment used for each particular analysis. The permeation kinetics of the biologically active agent is generally determined by taking measurements at multiple time points (eg, 15 min, 30 min, 60 min, and 120 min) after the biologically active agent comes into contact with the biologically active agent. cellular surface of the apical epithelium (which may be simultaneous with, or subsequent to, the exposure of the apical cell surface to the agent (s) that improves the mucosal supply). The methods for determining the concentrations of peptide YY neuropeptide Y, and pancreatic serum in blood serum, tissues or fluids of the central nervous system (CNS), brain spinal fluid (CSF), or other tissues or fluids of a mammalian subject can be determined by the immunological assay for peptide YY neuropeptide Y, and pancreatic peptide. The methods for determining Y peptide neuropeptide Y concentrations, and pancreatic serum as test materials to evaluate the improved permeation of active agents in conjunction with the coordinated administration of mucosal delivery enhancing agents or the combinational formulation of the invention are generally is described above and in accordance with known methods and manufacturer-specific instructions for radioimmunoassay reagents (RIA), enzyme immunoassay (EIA), and antibody for immunohistochemistry or immunofluorescence for peptide YY neuropeptide Y, and pancreatic peptide. Bachem AG (King of Prussia, PA). EpiAirway ™ tissue membranes are grown in phenol red free medium and hydrocortisone (MatTek Corp., Ashland, MA). The tissue membranes are cultured at 37 ° C for 48 hours to allow the culture to equilibrate. Each tissue membrane is placed in an individual well of a 6-well plate containing 0.9 ml of serum-free medium. 100 μl of the formulation (test sample or control) is applied to the apical surface of the membrane. Samples in triplicate or quadruplicate of each test sample (agent that improves the mucosal supply in combination with a biologically active agent, peptide YY) and control (biologically active agent, peptide YY, alone) are evaluated in each assay. At each time point (15, 30, 60 and 120 minutes) the tissue membranes move to new wells containing fresh medium. Samples of the underlying 0.9 ml medium are harvested at each time point and stored at 4 ° C for use in the ELISA and lactate dehydrogenase (LDH) assays. The ELISA kits are typically two-stage stratified ELISAs: first, the immunoreactive form of the agent being studied is captured by an antibody immobilized in a 96-well microplate and after washing the unbound material from the wells, a "detection" antibody is allowed to react with the active immunoreactive agent. This detection antibody is typically conjugated to an enzyme (most commonly horseradish peroxidase) and the amount of enzyme bound to the plate in the immune complexes is then measured by analyzing its activity with a chromogenic reagent. In addition to samples of the supernatant medium collected at each time point in the permeation kinetics studies, appropriately diluted samples of the formulation (ie, containing the biologically active test agent being treated) that was applied The apical surface of the units at the beginning of the kinetic study are also analyzed on the ELISA plate, together with a set of standards provided by the manufacturer. Each sample of supernatant medium is generally analyzed in duplicate wells by ELISA (it will be recalled that the units are used for each formulation in a determination of permeation kinetics, which generate a total of sixteen samples of supernatant medium collected through the four time points). A. It is not common for the apparent concentrations of the active test agent in the samples of supernatant medium or in the diluted samples of the applied material - to the apical surface of the units that are outside the range of the concentrations of the standards after of the completion of an ELISA. No concentration of the material present in the experimental samples is determined by extrapolation beyond the concentrations of the standards; preferably, the samples are appropriately rediluted to generate concentrations of the test material that can be more accurately determined by interpolation between standards in a repeated ELISA. B. The ELISA for a biologically active test agent, for example, peptide YY, is unique in its design and recommended protocol. Unlike most equipment, the ELISA employs two monoclonal antibodies, one for capture and the other directly towards a determinant not overlaid with the biologically active test agent, eg, peptide YY, as the detection antibody (this antibody is conjugated to horseradish peroxidase). As the YY peptide concentrations below the upper limit of the assay are present in the experimental samples, the assay protocol can be used according to the manufacturer's instructions, which allow the incubation of the samples in the ELISA plate with both antibodies simultaneously present. When the levels of the YY peptide in a sample are significantly greater than its upper limit, the levels - of the immunoreactive YY peptide may exceed the amounts of the antibodies in the incubation mixture, and some YY peptides that have no antibody binding detection are they will capture on the plate, while some YY peptides that have the detection antibody bound can not be captured. This leads to a serious under-estimation of the levels of peptide YY in the sample (it will be apparent that the levels of peptide YY in each sample are significantly below the upper limit of the assay). To eliminate this possibility, the test protocol has been modified: B.l. The diluted samples are first incubated in the ELISA plate containing the immobilized capture antibody for one hour in the absence of any detection antibody. After one hour of incubation, the wells are washed to release the unbound material. B.2. The detection antibody is incubated with the plate for one hour to allow the formation of immune complexes with all the captured antigen. The concentration of the detection antibody is sufficient to react with the maximum level of peptide YY that has been bound by the capture antibody. This plate is then washed again to remove any unbound detection antibody. B.3. The peroxidase substrate is added to the plate and incubated for fifteen minutes to allow it to have 1 place the development of color. B.4. The "high" solution is added to the plate, and the absorbance is read at 450 nm as well as at 490 nm on the VMax microplate spectrophotometer. The absorbance of the colored product at 490 nm is much lower than at 450 nm, but the absorbance at each wavelength is still proportional to the concentration of the product. The two readings ensure that the absorbance is linearly related to the amount of YY peptide bound through the worked ransa of the instrument or VMax (we routinely restrict the range from 0 to 2.5 OD, although the instrument reports being accurate across a range of 0 to 3.0 OD). The amount of peptide YY in the samples is determined by interpolation between the OD values obtained from the different standards included in the ELISA. Samples with OD readings out of range obtained by the redilidas standards and run in a repeated ELISA. RESULTS Measurement of resistance, transepithelial by TER assay: time points of the assay, membranes were placed in individual wells of a 24-well culture plate in 0.3 ml of clean medium and transepithelial electrical resistance (TER) was measured using the EVOM Epithelial Voltometer and an Endohm camera (World Precision Instruments, Sarasota, FL). The upper electrode was adjusted - to be closer but not in contact with the upper surface of the membrane. The tissues were removed, one at a time, from their respective wells and the basal surfaces were rinsed by immersion in CLEAN PBS. The apical surfaces were gently rinsed twice with PBS. The tissue unit was placed in the Endohm chamber, 250 μl of PBS was added to the insert, the upper electrode was relocated and the resistance was measured and recorded. After the measurement, the PBS was decanted and the tissue insert was returned to the culture plate. All TER values are reported as a function of the surface area of the tissue. The final numbers were calculated as: TER of the cell membrane = (Resistance (R) of the Insert with R-membrane of Control Insert) Area X of membrane (0.6 cm2). Formulation P exemplifies the YY peptide formulation, showing the greatest decrease in cell membrane resistance. (Table 2). The results indicate that the exemplified formulation (e.g., Formulation P) reduces the resistance of the membrane to less than 1% of the control at the concentrations tested. The values show the average of three replicates of each formulation. Formulations A and B are controls prepared by reconstituting the YY peptide (Bachem AG, King of Prussia, PA) containing 60 μg of peptide Y3-36 in 100 ml buffered saline-phosphate (PBS) at H 7.4 or 5.0 . Peptide YY without mucosal supply enhancers does not decrease resistance. The results indicate that an exemplified formulation for improved intranasal delivery of the YY peptide (e.g., Formulation P) decreases the cell membrane resistance and significantly decreases the cellular permeability of the mucosal epithelium. The exemplified formulations will improve the intranasal delivery of the YY peptide to the blood serum or to the tissue or fluid of the central nervous system. The results indicate that these exemplified formulations when in contact with a mucosal epithelium produce significant increases in the cellular permeability of the mucosal epithelium for the YY peptide. Table 2: Influence of the Pharmaceutical Formulations comprising the YY Peptide and the Agents that Improve the Intranasal Delivery in the Transepithelial Resistance (TER) of the EpiAirway Cell Membrane - - Permeation kinetics as measured by the ELISA assay: The effect of the pharmaceutical formulations of the present invention comprising the YY peptide and the intranasal delivery enhancing agents on the permeation of the peptide YY through the EpiAirway Cell Membrane (mucous epithelial cell layer) is measured as described above. The results are shown in Table 3. Permeation of peptide YY through the Cell Membrane ,, T, EpiAirway is measured by the ELISA test.

- For the exemplified intranasal formulations (eg, Formulation P) of the present invention, the greatest increase in permeation for peptide YY occurs in Formulation P as shown in Table 3. The procedure uses an ELISA assay to determine the concentration of the Biologically active YY peptide that has permeated the epithelial cells in the surrounding medium through the multiple time points. The results show the increased permeation of peptide YY in Formulation P compared to formulation A or B (peptide control formulation YY; 60 μg of peptide YY3-36 in 100 ml of phosphate buffered saline; (PBS) at pH 7.4 or 5.0; Bachem AG, King of Prussia, PA). The cumulative average permeation at 120 minutes using the exemplified intranasal formulation Formulation P is approximately 1195 times greater than Control Formulations A or B.

Table 3: Influence of the Pharmaceutical Formulations comprising the YY Peptide and the Improving Agents of the Intranasal Supply on the Peptide YY Permeation through the EpiAirway Cell Membrane by ELISA.

MTT test: MTT were performed using MatTek MTT-100 equipment. 300 ml of the MTT solution was added to each well. The tissue inserts were rinsed gently with clean PBS and placed in the MTT solution. The samples were incubated at 37 ° C for 3 hours. After incubation, the cell culture inserts were then immersed with 2.0 ml of the extracted solution per well to fully complete each insert. The extraction plate was covered and sealed to reduce evaporation. The extraction proceeded overnight at room temperature in the dark. After the extraction period is complete, the extracted solution is mixed and pipetted into 96-well microtiter plates. Triplicates of each sample were loaded, as well as the extracted controls. The optical density of the samples was then measured at 550 nm on a plate reader (Molecular Devices) (molecular devices). The MTT assay in an exemplified formulation for improved nasal mucosal delivery of the YY peptide following the teachings of the present specification (e.g., Formulation P) compared to the control formulation (Formulations A or B) is shown in Table 4. The results for the formulations comprising the YY peptide and one or more of the intransal delivery enhancing agents, eg, Formulation P (experiment performed on three replicates) indicates that there is a minimal toxic effect of this exemplified modality on the viability of the mucosal epithelial tissue. Table 4 Influence of Pharmaceutical Formulations Comprising the YY Peptide and Agents that Improve the Intranasal Delivery on the Viability of EpiAirway Membrane Cell as shown by% MTT LDH Assay: The LDH assay in an exemplified formulation for improved nasal mucosal delivery of the YY peptide following the teachings of the present specification (eg, Formulation P) are shown in Table 5. The results of three replications of Formulation P they indicate that there is a minimum toxic effect of this exemplified modality on the viability of the mucosal epithelial tissue. Table 5 Influence of the Pharmaceutical Formulations comprising the YY Peptide and Improving Agents of the Intranasal Delivery on the Feasibility of the EpiAirway Cell Membrane as shown by the% of Smoothed Cells (LDH Assay).

H n-Capric Sodium Acid 1.3 (0.075% w / v) Chitosan (0.5% w / v) 0.7 J Didecanil La- 1.2 Phosphatidylcholine (3.5% w / v) K S-Nitroso-N-Acetyl- 0.7 Penicillamine (0.5% p / v) Palmotoil-DL-Carnitine (0.02% 0.8 w / v) Pluronic-127 M (0.3% w / v) 1.0 N Sodium Nitroprusside (0.3% 0.6 w / v) O Sodium Glicocolate (1% w / v) 0.8 Fl: Gelatine, DDPC, MBCD, 2.0 EDTA EXAMPLE 3 Formulation P (Peptide YY) of the present invention in combination with Acetonide Triamcinolone Corticosteroid that Improves Cell Viability The present example provides an in vitro study to determine the permeability and reduction in inflammation of the epithelial mucosa of a YY peptide administered in a manner intranasal, for example, the human YY peptide, in combination with a steroid composition, for example, triamcinolone acetonide, and further in combination with one or more intranasal delivery enhancing agents. The study includes the determination of epithelial cell permeability by the TER assay and the reduction of epithelial mucosal inflammation as measured by cell viability in an MTT assay by applying a modality comprising the peptide YY and the acetonide triamcinolone . Formulation P (see Table 1 above) is combined in a formulation with triamcinolone acetonide at a dose of 0.5, 2.0, 5.0, or 50 μg. The normal dose of acetonide triamcinolone, (Nasacort®, Aventis Pharmaceuticals) for seasonal allergic rhinitis, is 55 μg per spray. Formulation P in combination with the corticosteroid acetonide triamcinolone improves cell viability as measured by the MTT assay, while maintaining epithelial cell permeability as measured by the TER and ELISA assays. According to the methods and formulations of the invention, the measurement of the permeability of Formulation P in the presence or absence of acetonide triamcinolone is carried out by transepithelial electrical resistance (TER) tests on an EpiAirway ™ cell membrane. The TER assays of Formulation P plus the triamcinolone acetonide at a concentration of 0.5, 2.0, 5.0, or 50 μg per spray indicate that the permeability of the peptide YY does not decrease and was equal to the permeability of Formulation P alone. Formulation P plus acetonide triamcinolone at a concentration of acetonide triamcinolone between 0 and 50 μg per spray is typically at least 10-fold to 100-fold greater than the permeability of Formulations A or B (control peptide YY). In accordance with the methods and formulations of the invention, the measurement of the permeability of Formulation P in the presence or absence of acetonide triamcinolone is carried out by the ELISA assay on an EpiAirway ™ cell membrane. Similar to the previous TER assay, the ELISA assay of Formulation P plus acetonide triamcinolone at a concentration of 0.5, 2.0, 5.0, or 50 μg per spray indicates that the permeability of peptide YY does not decrease and was equal to the permeability of Formulation P alone. . Formulation P plus acetonide triamcinolone at a concentration of acetonide triamcinolone between 0 and 50 μg per spray is typically greater than the permeability of Formulations A or B (control peptide YY). According to the methods and formulations of the invention, the MTT assay measures the cellular viability of Formulation P in the presence or absence of acetonide triamcinolone. Typically, the addition of triamcinolone acetonide (at a concentration of 0.5, 2.0, 5.0, or 50 μg by spray) to Formulation P improves cell viability as compared to Formulation P in the absence of acetonide triamcinolone. The addition of acetonide triamcinolone to the Formulation P increases cell viability and maintains epithelial permeability as measured by the TER assay comparable to Formulation P in the absence of acetonide triamcinolone. The reduction in mucosal epithelial inflammation of a YY peptide administered intranasally is carried out with an intranasal formulation of the peptide YY in combination with one or more steroid or corticosteroid compound (s), typically compounds or high potency formulations, but also in certain compound cases or formulations of medium power, low power. The total power is given (equivalent doses) of high, medium and low potency steroids. Typically, an intranasal formulation of the YY peptide in combination with a high potency steroid combination includes, but is not limited to, betamethasone (dose of 0.6 to 0.75 mg), or dexamethasone (dose of 0.75 mg). In an alternative formulation, an intranasal formulation of the YY peptide in combination with a medium potency steroid composition includes, but is not limited to, methylprednisolone (4 mg dose), triamcinolone (4 mg dose), or prednisolone (5 mg dose). mg). In a further alternative formulation, a formulation formulation of the peptide YY in combination with a low potency steroid composition includes, but is not limited to, hydrocortisone (dose of 20 mg) or cortisone (dose of 25 mg). EXAMPLE 4 Preparation of a PYY Formulation Free of a Stabilizer that is a Protein A PYY formulation suitable for intranasal administration of PYY, which was substantially free of a stabilizer which is a protein was prepared having the formulation listed below. 1. Approximately water was added to a beaker and stirred with a stir bar on a stir plate and the sodium citrate was added until completely dissolved. 2. EDTA was then added and stirred until completely dissolved. 3. Citric acid was then added and stirred until completely dissolved. 4. Methyl-β-cyclodextrin was added and stirred until completely dissolved. 5. DDPC was then added and stirred until completely dissolved. 6. Lactose was added and stirred until completely dissolved. 7. Sorbitol was then added and stirred until completely dissolved. - 8. Chlorobutanol was then added and stirred until completely dissolved. 9. Add PYY 3-36 and stir gently until dissolved 10. Check pH to be sure at 5.0 ± 0.25. Dilute HCl or diluted NaOH is added to adjust the pH. Water is added for the final volume. Table 6 Formulation pH 5 +/- 0.25 Osmolarity ~ 250 EXAMPLE 5 A second formulation is prepared as above, except that the concentration of PYY 3-36 was 15 mg / ml as shown below in Table 7 Table 7 - Formulation pH 5 +/- 0.25 EXAMPLE 6 Determination of Optimal pH of PYY Determination of the stability of PYY3_36 V s pH at 40 ° C for 5 days A. Protocol for formulating PYY (3-36) / pH stability study samples Osmolarity: 250mM Target Using a Citrate / Sodium citrate, tri-basic cushion, lOmM Osmolarity = No. particles x molarity = (1 + 4) X lOmM = 50mM Therefore the osmolarity is taken to 250mM with lOOmM NaCl (2 particles) B. Prepare stability samples as follows (3500pl) Final Concentration 1400μl 25mM citrate buffer (of the required final pH) lOmM PYY 700μl 1.5mg / ml 300ug / ml Chlorobutanol 350μl 2.5%, 0.25% NaCl, 350μl l.OM, 100mM Verify the pH and adjust it if required Q.S. for 3500 μl with water Procedure: A sample of 120μl in 200 ul sialylated inserts in autosampler vials 3 pulis / time point Samples incubated at 40 ° C for 5 days C. Comparison of the objective and current pH of the stability mixtures pH objective; LVO current pH 3.0 2.99 3.5 3.47 4.0 3.90 4.5 4.42 5.0 4.90 7.0 7.38 D. HPLC Procedure Column: C18 Bondapak Waters lOμm 4.6 x 300mm HPLC System: Alliance 2690 Waters 2487 Waters Detector Dual wavelength at 220nm Flow rate Iml / mim Injection volume 30μl Temp. of column 30 ° C Mobile phases: Shock absorber A: 0.1% TFA, 1% acetonitrile in water Shock absorber B: 0.11% TFA in acetonitrile Gradient: Time (mins)% A% B 0 75 25 17 42 58 19 75 25 28 75 25 E. Results and Conclusions The results indicate that under the particular conditions used in this study, that the optimum pH for stability is 4.90. There is an increase in stability from 76 to 87%, increasing the pH from 2.99 to 4.90. The higher the pH, i.e. 7.38 there is a large drop in the stability of PYY (3-36) with only 15% remaining zero time. EXAMPLE 7 Development of Intranasal Formulation Peptides and proteins are relatively fragile molecules in comparison to low molecular weight therapeutics. The objective of the development phase of the formulation was to identify a suitable candidate formulation for intranasal delivery. In order to achieve this goal, numerous candidates were tested to identify a formulation with drug stability, nasal delivery through the nasal mucosa, toxicity and conservation effectiveness. Initially, the effect of pH was examined. The Figure 1 shows the stability of PYY 3_36 at high temperature (40 ° C) at various pHs from 3.0 to 7.4. At physiological pH, there were substantial losses of drug at elevated temperature. The best stability was achieved at approximately pH 5.0. This pH was selected for further optimization of the formulation. To optimize the additional stability, various stabilizing agents were tested for their ability to facilitate the passage of the drug through the nasal mucus. The breeders tested were selected based on their ability to open tight joints with limited cellular toxicity. To do this, a first human epithelial cell model was used (EpiAirway, MatTek, Inc., Ashland MA). This cell line forms a columnar pseudo-stratified columnar cell layer with similar tight junctions - to the respiratory epithelium found in the nose. The drug formulations were placed on the apical side of the tissue layer, and the quantification of the drug was carried out by the basal medium. The degree of opening of the hermetic joints was measured by the decrease in transepithelial electrical resistance (TEER). Cell viability and cytotoxicity were monitored by MTT and LDH assays, respectively. The data from a representative exploration experiment are shown in Figures 2-5. Figure 2 shows the data for TEER. In some cases there was little or no decrease in TEER compared to control, indicating tight connections that remained closed. In other cases there was a substantial drop in TEER indicating the opening in hermetic joints. The results show that the cell model in vitro is able to discriminate the ability of different formulations to open the hermetic joints. In the candidate candidate formulations the cell viabilities (Figure 3, MTT) were good and the cytotoxicities (Figure 4; LDH) were low. In total, more than 200 different formulations were tested, reflecting the high production nature of the in vitro systematic screening model. Using all the available data, a multivariate analysis was conducted to determine the effect of each component of the formulation used in each of the 7 variables produced (drug permeability, osmolarity, stability under refrigerated and accelerated conditions, TEER, and MTT assays). and LDH). Multivariate analyzes consist of an initial analysis of each formulation component for some level of correlation with the production parameters (p <0.1). with the identified subset, either a linear regression or logistic selection in stages was used. The results suggest that one excipient correlates osmolarity and toxicity (r2 = 0.91 and 0.27, respectively), two correlate to the permeation of PYY3_36 (r2 = 0.44), three affect the stability (r2 = 0.24), and five impact the paracell resistance (r2 = 0.55). The best formulations determined by this process increase at least 30-75 times the transmporte of PYY 3-36 compared to the simple buffered solutions. Based on these analyzes, an optimized PYY 3_36 formulation was selected for further development. This optimized formulation contained two stabilizers, two permeation enhancers, a chelating agent, and a preservative in a sodium acetate buffer, pH 5.0. This formulation passed the USP conservation effectiveness test. The synergistic contributions of the various components on the permeation of the drug are presented in Figure 5. Compared to simple formulations of amorgiguador in the same osmolarity, the optimized formulation exhibits more than 100-fold increase in the permeation of the drug . Finally, pre-clinical and clinical groups of the optimized formulation were prepared and placed in stability at 5 ° C and 25 ° C in the final product package. The preliminary data, shown in Table 8, reveal that storage for up to two months at either 5 ° C or 25 ° C results in 90% or better retention of the peptide.

TABLE 8 In summary, our formulation development process has produced a PYY_36 formulation with adequate ladroga stability, delivery through the nasal mucosa, toxicity and preservation effectiveness, which allows delivery of a 4 kD peptide.

Preclinical Studies To date, a series of six preclinical studies in rats, rabbits and dogs was completed. Plasma levels of PYY3-36 were determined in all species by a validated patented radioimmunoassay method. The bioavailability (the mole fraction of the drug identified in plasma divided by the amount administered nasally) in rats was determined to be approximately 6%, and in rabbits of approximately 8%. These values may underestimate the true bioavailability, as well as any degradation of the peptide in plasma before sampling, or degradation after sampling despite the presence of a proteinase inhibitor, will decrease the measured bioavailability. Nasal toxicity has been calculated in rat and rabbit models for up to 14 consecutive days at doses of 50x the expected human clinical dose on a mg / kg basis. There were no obvious microscopic or pathological findings related to the test article. There were no clinical observations.

- Systemic toxicity after intravenous administration was evaluated in the rat and rabbit models. At IV doses up to approximately 160x the expected human dose (400 ug / kg in the rat and 205 ug / kg in the rabbit) there was no test article related to microscopic or macroscopic findings. The cardiovascular toxicity was assessed in the anesthetized dog model in a dose that varied in the study design. The highest dose, an infusion of PYY3_36 up to 24ug / kg for 60 minutes corresponded to 33x the expected human dose on a body surface area basis. The resulting plasma levels, 30 ng / ml, were approximately 380x the level in canine basal plasma. At this level in plasma, there was no effect on arterial blood pressure, femoral blood flow, or QTc and only minor changes in heart rate were noted (average increase from 123 to 148 bpm) and respiratory rate (decreased from 54 to 36) . The pharmacokinetic data were collected in these preclinical studies. From a study in rats, plasma levels after intranasal administration at various doses are shown in Figures 6, 7 and 8. Figure 6 shows that PYY3_36 is observed in the plasma within 5 minutes, the concentrations of Peak plasma (Tmax) are reached in 10-15 minutes, and the terminal elimination half-life is approx. 15 minutes. Both Cmax and AUCo-t are linear with respect to the intranasal dose. Clinical Studies A clinical trial has been initiated varying the dose with the goal of establishing the safety, PK, and bioavailability of the intranasal formulation of PYY3_36. To date, patients have been enrolled in the first two of the five subsequent doses. One patient reported a taste in the back of his throat; there were no other adverse events to date. Conclusion In the formulation, preclinical and initial clinical studies were started in an intranasal formulation of PYY3_36. The procedure for the formation resulted in more than a hundred fold increase in the transmembrane permeability of this peptide 4 kD an increase in cell toxicity. Preclinical studies have shown a considerable margin of safety for nasal, cardiovascular, and systemic toxicity for PYY3_36. Based on clinical studies that vary the ongoing dose, weight loss studies are planned by chronic administration. EXAMPLE 8A CLINICAL PROTOCOL NASAL ABSORPTION OF THE PEPTIDE YY3-36 INTRASAL (PYY3-36) IN SUBJECTS - HEALTHY HUMANS The objective of the present study was to evaluate the absorption of PYY3_36 administered intranasally into the bloodstream from the nose. This was a phase one, in a clinical trial, of a single dose, of stepped dose, including male volunteers and healthy, normal females in fasting. Increasing doses of PYY3-36 intranasal between 20 μg to 200 μg were evaluated to assess the safety, nasal tolerance and absorption of PYY3-36. The assessment of the appetite sensation in each individual was also evaluated. PYY3_36 was administered to 15 healthy humans divided into 5 groups of 3 individuals each. Group I The first group was administered by an intranasal spray 20 μg of PYY3_36 in 0.1 ml of solution.

Group II The second group received 50μg of PYY3_36 intransally in 0.1ml of solution. Group III The third group received lOOμg of PYY3_36 intransally in 0.1 ml of solution. Group IV The fourth group received 150μg of PYY3_36 intransally in 0.1ml of solution.

- - Group V The fifth group received 200μg of PYY3_36 intransally in 0.1ml of solution. Blood samples were collected and plasma concentrations of PYY were determined at 0 (i.e., pre-dose), 5, 7.5, 10, 15, 20, 30, 45, 60 minutes postdose. The subjects were then fed and a blood sample was taken and the concentration of PYY was determined 30 minutes after the meal. Plasma concentrations of PYY3-36 were determined using a validated analytical procedure. For each subject, the following PK parameters were calculated, whenever possible, based on the plasma concentrations of PYY3-36, according to the procedure independent of the model: Cmax Maximum observed concentration. tmax Maximum concentration time. AUC0-t Area under the concentration-time curve from time 0 to the time of the last measurable concentration, calculated by the linear trapezoidal rule. The following parameters were calculated when the data allowed the exact estimation of these parameters: AUCo-8 Area under the concentration-time curve extrapolated to infinity, calculated under the following formula: Ct AUCo-8 = AUCo-t + Ke where Ct is the last measurable concentration and Ke is the constant of the apparent terminal phase rate. Ke Constant of the apparent terminal phase rate, where Ke is the magnitude of the linear regression of the logarithmic concentration versus the time profile during the terminal phase. t? / 2 Average life of the apparent terminal phase (when possible), where t? / 2 = (In2) / Ke. The PK calculations were performed using commercial software such as WinNonlin (Pharsight Corporation, Version 3.3, or higher). The results are shown in the graphs below. Discussion and Conclusion Background: Each dosed group included three subjects who were dosed intranasally once with a formulation of this invention containing a specific dose of pyrogen-free synthetic human PYY3_36. The five dosed groups were organized, with staggered doses of PYY ~ 36 - in the formulation. Blood samples were collected at specific intervals in blood collection tubes containing lithium heparin (to inhibit coagulation) and aprotinin (to preserve PYY 3_36). The plasma of each blood sample was collected by centrifugation and stored in frozen aliquots. A frozen aliquot of each blood sample was sent to Nastech Analytical Services and arrived frozen. Each sample was stored frozen until analyzed for the concentration of PYY by radioimmunoassay (RIA). Observation: Group 1: This group of subjects was dosed with 20 micrograms of PYY 3_36. The concentrations of PYY in plasma for the subjects ranged from a minimum of "less than 20 pg / ml" (below the lower limit of radioimmunoassay quantification) to a maximum of 159 pg / ml. Trends The observed concentrations are not consistent with the significant absorption of the drug in the blood of the subjects studied. Group 2: This group of subjects was dosed with 50 micrograms of PYY 3-36- The concentrations of PYY in plasma for the subjects varied from a minimum of 50 pg / ml "up to a maximum of 255 pg / ml. of the observed concentrations are consistent with the significant absorption of the drug in the blood of the subjects - studied Group 3: To this group of subjects were dosed with 100 micrograms of PYY 3-36- The concentrations of PYY in plasma for the subjects ranged from a minimum of 87 pg / ml "up to a maximum of 785 pg / ml. The trends of the observed concentrations are consistent with the significant absorption of the drug in the blood of the subjects studied. Group 4: To this group of subjects, they were dosed with 150 micrograms of PYY 3-36- The concentrations of PYY in plasma for the subjects varied from a minimum of 45 pg / ml "up to a maximum of 2022 pg / ml.The trends of the observed concentrations are consistent with significant absorption of the drug in the blood of the subjects studied Group 5: This group of subjects was dosed with 200 micrograms of PYY -3e- The concentrations of PYY in plasma for the subjects ranged from a minimum of 48 pg / ml "up to a maximum of 1279 pg / ml. The trends of the observed concentrations are consistent with the significant absorption of the drug in the blood of the subjects studied. These results are consistent with dose-dependent absorption of PYY3_36. Observations and additional data: Summary of findings: • In the intranasal dose of 50 ug - 200 ug, there is an absorption of PYY in plasma dependent on the dose.

• The duration of elevated plasma concentrations is considerably longer than predicted, with an elimination half-life calculated at 55 minutes. • Cmax and AUC 0-t show good linearity with the dose. • There is considerable variability between subjects at a given dose. • Surprisingly, this study failed to detect the postprandial elevation of PYY despite the fact that previously ingested food was not measured and if it ate too little this could explain the observations. • On a visual-analogous scale, hunger issues suggest decreased hunger with increased doses of PYY. • Nausea and dizziness seem to be related to very high concentrations of PYY. Notes: • In some cases pMol / 1 is used as the PYY measurement units; in other analyzes, pg / ml is used. The conversion factor is pmol / l * 4.05 = pg / ml. • In some cases, the 150-minute time point is displayed in the graphs. Strictly speaking, this is a point of the postprandial data, and therefore may confuse the evaluation of PK. However, an unexpected finding described in more detail below is that the 30-minute postprandial time point is not different from the value of the baseline. Concentrations of PYY in plasma: The PYY assay described in this specifiaction has been validated by its own plasma PYY concentration assay. Using this assay, samples from each time point were tested in triplicate. Note that out of data points 180, 3 (1.6%) seems to be biologically unlikely "outlier." The data through this preliminary analysis uses a data set in which these three point data were removed. Table 9 Descriptive parametersPK: The calculated PK parameters include: Examination of the plotted PK means suggests a dose response of 50 - 200 ug per dose, but that the 20 μg dose is in the nose. Therefore, many of the subsequent analyzes will be based only on data from 50-200 ug doses. We also propose that, because PYY is an endogenous molecule, the AUC 0-t is more relevant than AUC 0- inf. Tmax and Tl / 2 (elimination half-life): The Tmax of 24 minutes is typically for a nasal product. The elimination half-life of 55 minutes is considerably longer than would have been expected. The literiary references- indicate a t 2 typically of 5-10 minutes. The elimination half-life can also be affected by some continuous absorption of the nasal mucosa that occurs after Tmax and by the components of the formulation that effect the metabolism of the peptides. Alternatively, because the assay described in this specification employs an extraction procedure, the assay will capture both free PYY and protein bound, while an assay that does not use an extraction can mainly test the free fraction. From this analysis of the change in average VAS of the baseline (average values of 10, 30, and 60 minutes minus the baseline) vs dose, it is observed: For VAS Ql "How hungry do you feel?" the subjects were less hungry after receiving a higher dose of PYY. For VAS Q3, "How much do you think you can eat?" Subjects considered that they could eat less after receiving a higher dose of PYY. However, for VAS Q2"How satisfied do you feel?" subjects felt less satisfied after receiving higher doses of PYY. This suggests that the feeling after administration of PYY does not include satisfaction, inflammation or gastric hypercontractility.

EXAMPLE 8B Administration to humans of PYY and Weight Loss The following formulation of nasal PPY was elaborated - Formulation pH 5 +/- 0.25 One or two daily sprays were administered to a human subject over a period of 10 days and a weight loss of 2.5 pounds was recorded. During periods that varied from 10 minutes to 12 hours after administration, the subjects registered reduced hunger. EXAMPLE 9 Buccal formulation of PYY3-36 (Prophetic) Bistratified tablets were prepared in the following manner. An adhesive layer was prepared weighing 70 parts by weight of polyethylene oxide (Poliox 301N; Union Carbide), 20 parts by weight of polyacrylic acid (Carbopol 934P; B.F. Goodrich), and 10 parts by weight of a charge of xylitol / compressible carboxymethyl cellulose (Xylitab 200; Xyrofin). These ingredients were mixed by turning in a jar for 3 minutes. The mixture was then transferred to an evaporation vessel and rapidly wet granulated with absolute ethanol to a semi-paste-like consistency. This mass is immediately and rapidly forced through a 14-mesh stainless steel screen (1.4 mm opening), to which the wet granules adhere. The screen was covered with perforated aluminum foil, and the humid granules were dried overnight at 30 ° C. The dried granules were removed from the screen and then passed through a 20 mesh screen (0.85 mm opening) further reduce the size of the granules. The particles that did not pass through the 20 mesh screen were briefly crushed with a mortar and crushed to minimize the amount of fines and then passed through the 20 mesh screen. The remaining granules were then placed in a taro and 0.25 parts by weight of stearic acid and 0.06 parts by weight of mint flavor (Universal Flavors) were added and mixed with the granules. The final percentages by weight of the ingredients are thus 69.78% polyethylene oxide, 9.97% load of xylitol / carboxymethyl cellulose, 19.94% polyacrylic acid, 0.25% stearic acid, and 0.06% mint flavor. An amount of 50 mg of the mixture was placed in a 0.375 inch diameter die and pre-compressed to a Carver Press Model C with compression of 0.25 metric tons for a residence time of 3 seconds to form the adhesive layer. The active layer was prepared by weighing 49.39 parts by weight of mannitol, 34.33 parts by weight of hydroxypropyl cellulose (Klucel 1 F, Aqualon, Wilmington, Del.) And 15.00 parts by weight of sodium taurocholate (Aldrich, Milwaukee, Wis.) , and mixed by turning in a jar for 3 minutes. The mixture was then transferred to an evaporation vessel and rapidly wet granulated with absolute ethanol to a semi-paste-like consistency. This mass was immediately and rapidly forced through a 14-mesh stainless steel screen to which the wet grains adhered. The sieve was covered with perforated aluminum foil and the granules were dried at 30 ° C. The dried granules were then sequentially passed through 20, 40 sieves. (0.425 mm opening), and 60 (0.25 mm opening) meshes to further reduce the particle size. Particles that did not pass through a screen were briefly ground with a mortar and crushed to minimize the ends and then passed through the screen. The screened particles were weighed and then 0.91 parts by weight of PYY3-36 and 0.06 parts by weight of yellow FD &C # 6HT of aluminum lake dye were mixed sequentially with the dry-granulation by geometric dilutions. The dried granulation was then placed in a mixing jar and mixed with 0.25 parts by weight of magnesium stearate (lubricant) and 0.06 parts by weight of mint flavor when rotated for 3 minutes. A 50 mg sample of this material was placed on top of the partially compressed adhesive layer and both layers were then compressed to 1.0 ton pressure for a residence time of 3 seconds to produce a dextratified tablet suitable for buccal delivery. This procedure resulted in a gingival tablet wherein the active layer contains 0.91% by weight of PYY3-36, 15% by weight of NaTC, and 84.09% by weight of filler, lubricant, colorant, formulation aids, or flavoring agents. . EXAMPLE 10 A study was conducted comparing the capacity of endotoxin-free PYY (3-36) (SEQ ID NO: 2) vs. PYY (3-36) not free of endotoxin to permeate the bronchial epithelium according to the procedure of Example 1 It was determined that approximately twice the amount of endotoxin-free PYY (3-36) permeated the bronchial epithelium as compared to the formulation PYY (3-36) containing endotoxin. Both formulations contained Chlorobutanol 2.5mg / ml, 2.mg / ml DDPC, 10 mg / ml albumin, Img / ml EDTA (disodium edetate) and 45 mg / ml M-B-CD. One formulation contained PYY (3-36) free of endotoxin and the other formulation contained 70 EUs or greater of endotoxin. The average MTT of the formulation of PYY (3-36) containing endotoxin was 91.72% while the formulation of PYY (3-36) free of endotoxin had an average MTT of 100.16%. The average permeation of the formulation of PYY (3-36) containing endotoxin was 5.36%, while the average permeation of the formulation of PYY (3-36) free of endotoxin was 10.75%. Various known mucosal delivery enhancer excipients can be efficiently combined with peptides that bind to the endotoxin-free Y2 receptor, especially endotoxin-free PYY3-36, and can be used to improve non-infused formulations especially for oral delivery. Such excipients are contained in the following patent applications which are incorporated by reference: U.S. patent applications. 20030225300, 20030198658, 20030133953, 20030078302, 20030045579, 20030012817, 20030012817, 20030008900, 20020155993, 20020127202, 20020120009, 20020119910, 20020065255, 20020052422, 20020040061, 20020028250, 20020013497, 20020001591, 20010039258, 20010003001.

- Oral Formulation of a Y2 Receptor Binding Peptide An oral formulation of a Y2 receptor binding peptide can be prepared according to the following procedure. A preferred formulation for an oral delivery contains approximately 0.5 mg / kg endotoxin-free PYY and between 100 and approximately 200 mg / kg of one or more mucosal supply enhancing excipients.

(Prophetic) EXAMPLE 11 Preparation of Aldehyde N-cyclohexanoylphenylalanine: Methyl phenylalanine ester (1 g, 0.0046 mole) was dissolved in 5 ml pyridine. Cyclohexanoyl chloride (0.62 ml) was added and the mixture was stirred for 2 hours. The reaction mixture was poured into hydrochloric acid (IN) and triturated on ice. The aqueous mixture was extracted twice with toluene. The combined toluene extracts are concentrated in vacuo to give 1.1 g of crude methyl N-cyclohexaneylphenylalanine ester. Methyl N-cyclohexaneylphenylalanine ester was dissolved (0.5 g) in dimethyl ethylene glycol ether (20 ml). The solution was cooled to 70 ° C and diisobutylaluminum hydride (2.04 ml of a 1.5M toluene solution) was added. The resulting reaction mixture was stirred at -70 ° C for 2 hours. The reaction was quenched by the dropwise addition of 2N hydrochloric acid. The mixture was extracted with cold ethyl acetate. The ethyl acetate solution was washed with brine and dried over sodium sulfate. The concentration to the vaio was provided with a soft solid, which was purified by silica gel chromatography. aH NMR (300 MHz, DMSO-dd): 9.5 (s, 1H), 8.2 (d, 1H), 7.2 (m, 5H), 4.2 (, 1H), 3.2 (d, 1H), 2.7 (d, 1H ), 2.1 (, 1H), 1.6 (br.m, 4H), 1.2 (br.m, 6H). R (KBr): 3300, 3050, 2900, 2850, 2800, 1700, 1600, 1500 cm-1. Spec. Mass: M + l m / e 261. EXAMPLE 12 Preparation of N-acetylphenylalanine Aldehyde Methyl N-Acetylphenylalanine ester (4.2 g, 19 mmol) was dissolved in dimethyl ethylene glycol ether. The solution was cooled to -70 ° C and diisobutylaluminum hydride (25.3 ml of a 1.5M toluene solution, 39 mmol) was added. The resulting reaction mixture was stirred at -70 ° C for 2 hours. The reaction was warmed by the addition of 2n hydrochloric acid. The mixture was extracted 4 times with cold ethyl acetate and 4 times with toluene. The combined extracts were washed with brine and dried over magnesium sulfate. Concentration in vacuo followed by silica gel chromatography provided 2.7 g of white solid. The NMR is as reported in the literature, Biochemistry, 18: 921-926 (1979). EXAMPLE 13 Preparation of 3-acetamido-4- (p-hydroxy) phenyl-2-butanone (N-acetyl-tyrosinone) A mixture of tyrosine (28.9 g, 16 mmol), acetic anhydride (97.9 g, 96 mmol) and pyridine ( 35 g, 16 mmol) was heated at 100 ° C for 1 hour. The reaction mixture was concentrated in vacuo to provide a yellow oil. The oil was distilled under reduced pressure to provide 29.9g or an oil. 2H NMR (DMSO-d6): NMR (d6-DMSO); 8.2 (d, 1H), 7.3 (d, 2H), 7.0 (d, 2H), 4.4 (m, 1H), 3.1 (dd, 1H), 2.7 (dd, 1H), 2.3 (s, 3H), 1.8 (s, 3H) EXAMPLE 14 Preparation of 3-acetamido-7-amino-2-butanone (N-acetyllysinone) Following the procedure of Example 3, the lysine was converted to N-acetillisinone. XH NMR (DMSO-d6): 8.1 (d, 1H), 7.8 (br.m. 1H), 4.1 (m, 1H), 3.0 (, 2H), 2.0 (s, 3H), 1.9 (s, 3H) and 1.3 (br.m, 6H EXAMPLE 15 Preparation of 3-acetamido-5-methyl-2-butanone (N-acetylleucinone) Following the procedure of Example 3, leucine was converted to N-acetylleucinone. 1 H NMR (DMSO-d 6): 8.1 (d, 1 H), 4.2 (m, 1 H), 2.0 (s, 3 H), 1.8 (s, 3 H), 0.8 (d, 6 H).

EXAMPLE 16 Modification of 4- (4-Aminophenyl) butyric acid using Sulfonyl Benzene Chloride 4- (4-Aminophenyl) butyric acid (20 g 0.11 mol) was dissolved in 110 ml of 2N aqueous sodium hydroxide solution. After being stirred for about 5 minutes at room temperature, sulfonyl benzene chloride (14.2 ml, 0.11 mol) was added dropwise to the amino acid solution over a period of 15 minutes. After stirring for about 3 hours at room temperature the mixture was acidified to pH 2 by the addition of hydrochloric acid. This provided a clear brown precipitate which was isolated by filtration. This precipitate was washed with warm water and dried. The melting point is 123-25 ° C. If necessary, the purified amino acids can be purified by recrystallization and / or chromatography.

EXAMPLE 17 Modification of 4-aminobenzoic acid using sulfonyl benzene chloride Following the procedure of Example 6, 4-aminobenzoic acid was converted to 4- (phenylsulfonamide) benzoic acid.

EXAMPLE 18 - Modification of 4-Aminophenylacetic Acid, 4-Aminohippuric Acid and 4-Aminomethyl Benzoic Acid Following the procedure of Example 6, 4-aminophenyl acetic acid, 4-aminohippuric acid and 4-aminomethylbenzoic acid were converted to 4- (phenylsulfonamide) phenylacetic acid, 4-phenylsulfonamide hippuric acid and 4- (phenylsulfonamide methyl) benzoic acid.

EXAMPLE 19 Modification of Amino Acids with Chloride of Sulfonyl Benzene A mixture of sixteen amino acids was prepared before the chemical modification. The constituents of the mixture are summarized in the Table below. 65 grams of the amino acid mixture (total group concentration [-NH2] = 0.61 moles) was dissolved in 760 ml of 1N sodium hydroxide solution (0.7625 eguivalents) at room temperature. After being stirred for 20 minutes, sulfonyl benzene chloride (78 ml, 1 eq.) Was added over a period of 20 minutes. The reaction mixture was then stirred for 2.5 hours without heating. As some precipitation may occur, additional NaOH solution can be added (2N) to the solution until pH 9.3 is reached. The reaction mixture was stirred overnight at room temperature.

Afterwards, the mixture was acidified using hydrochloric acid - - diluted (38%, 1: 4) and a cream colored material was precipitated. The resulting precipitate was isolated by decantation and dissolved in sodium hydroxide (2N). This solution was then reduced in vacuo to give a yellow solid, which was dried in the lyophilizer. Table 10 Composition of Amino Acids No. of moles of NTo. d, e poop Moles of -Amino Acid Weight (g)% of Total Amino Acid Weight (xio "2) [" NH2] Thr 2.47 3.8 2.07 2.07 Ser 2.25 3.46 2.1 2.1 Wing 4.61 7.1 5.17 5.17 Val 4.39 6.76 3.75 3.75 Met 0.53 0.82 0.35 0.35 lie 2.47 3.8 0.36 0.36 Leu 3.86 5.94 2.95 2.95 Tyr 1.03 1.58 0.56 0.56 Phe 4.39 6.76 0.27 0.27 His 2.47 3.8 1.6 3.2 Lys 4.94 7.6 3.4 6.8 Arg 5.13 7.9 2.95 5.90 Glutamine 9.87 15.18 6.76 13.42 Acid 9.87 15.18 .6.70 6.70 Glutamic Asparagine 3.32 5.11 2.51 5.02 Acid 3.32 5.11 2.50 2.50 Aspartic EXAMPLE 20 Modification of a mixture of five Amino Acids using Sulfonyl Benzene Chloride A mixture of amino acids 86.1 g (0.85 moles NH2) (see Table below) was dissolved in 643 ml (1.5 eq.) Of 2N aqueous sodium hydroxide solution. After being stirred for 30 minutes at room temperature, sulfonyl benzene chloride (108 ml, 0.86 mole) was added portionwise at the amino acid solution for a period of 15 minutes. After stirring for 2.5 hours at room temperature, the pH of the reaction mixture (pH 5) was adjusted to pH 9 with 2N sodium hydroxide solution plus 2N sodium hydroxide solution. The reaction mixture was stirred overnight at room temperature. After, the pH of the reaction mixture was adjusted to pH 2.5 by the addition of dilute aqueous hydrochloric acid solution (4: 1, H20: HCl) and a precipitate of modified amino acids was formed. The top layer was discarded and the resulting yellow precipitate was isolated by decantation, washed with water and dissolved in 2N sodium hydroxide (2N). The solution was reduced in vacuo to give a yellow solid, which was leophilized overnight. Table 11 Amino Acid Mole Moles of [-NH2] Amino Acid xlO "(10 ^) Valine 7.5 7.5 Leucine 10.7 10.5 Phenylalanine 13.4 13.4 Lysine 21.0 42.0 Arginine 6.0 12.0 EXAMPLE 21 Modification of a Mixture of Five Amino Acids using Benzoyl Chloride A mixture of 86 g amino acids (0.85 moles of NH 2) (see Table in Example 20) was dissolved in 637 ml (1.5 eq.) Of 2N aqueous sodium hydroxide solution. After stirring for 10 minutes at room temperature, benzoyl chloride (99 ml, 0.85 mole) was added portionwise in the amino acid solution over a period of 10 minutes. After stirring for 2.5 hours at room temperature, the pH of the reaction mixture (pH 12) was adjusted to pH 2.5 using dilute hydrochloric acid (4: 1, H20: HCl) and a precipitate of modified amino acids was formed. After sitting for 1 hour, the resulting precipitate was isolated by decantation, washed with water and dissolved in sodium hydroxide (2N). This solution was then reduced in vacuo to give a crude modified amino acid as a white solid (expected yield 220.5 g).

EXAMPLE 22 Modification of L-valine Using Sulfonyl Benzene Chloride L-Valine (50 g, 0.43 mol) was dissolved in 376 ml (0.75 eq.) Of 2N aqueous hydroxide member by stirring at room temperature for 10 minutes. Sulfonyl benzene chloride (68.7 ml, 0.38 mol, 1.25 eq.) Was then added to the solution amino acid solution for a period of 20 minutes at room temperature. After stirring for 2 hours at room temperature, a precipitate appears. The precipitate is dissolved by adding 200 ml of additional 2N sodium hydroxide solution. After stirring for an additional 30 minutes, the aqueous solution of hydrochloric acid dluído (4: 1, H20: HCl) is added until the pH of the reaction mixture reaches 2.6. A precipitate of modified amino acids was formed and recovered by decantation. This material was dissolved in 2N sodium hydroxide and dried in vacuo to give a white solid. The expected yield of the crude modified amino acids is 84.6 g, 77%).

EXAMPLE 23 Modification of Methyl Phenylalanine Ester Using Hippuril Chloride Methyl p-phenylalanine ester hydrochloride (15 g, 0.084 mol) was dissolved in dimethylformamide (DMF) (100 ml) and pyridine (30 ml) was added thereto. A solution of hippuryl chloride chloride (16.6 g, 0084 moles in 100 ml DMF) was immediately added to the amino acid ester solution in two portions. The reaction mixture was stirred at room temperature overnight. The reaction mixture was then reduced in vacuo and dissolved in IN aqueous sodium hydroxide. The solution was heated at 70 ° C for 3 hours in order to hydrolyze the methyl ester to a free carboxyl group. Then, the solution was acidified to pH 2.25 using dilute aqueous hydrochloric acid solution (1: 3 HC1 / H20). A rubber-like precipitate formed and this was recovered and dissolved in 1N sodium hydroxide. The solution was reduced in vacuo to obtain an expected 18.6 g of crude modified amino acid product. After recrystallization from acetonitrile, pure modified phenylalanine was recovered (expected yield 12 g) as a white powder m.p. 223-225 ° C.

EXAMPLE 24 Dose Preparation of PYY Solutions (3-36) In a 568 mg test tube were added acetyl phenylalanine aldehyde, 132 mg carbomethoxy phenylalanilleucine and 100 mg acetyl-Phe-Leu-Leu-Arg aldehyde. 2.9 ml of 15% ethanol. The solution was stirred and NaOH (1.0 N) was added until the pH was raised to 7.2. Water was added to bring the total volume to 4.0 ml. The sample had a vehicle concentration of 200 mg / ml. PYY (3-36) (800 μg) was added to the solution. The concentration of PYY3-36 is 200 μg / ml. Following a similar procedure, a second solution was prepared having 668 mg of acetyl phenylalanine aldehyde and 132 mg of carbomethoxy phenylalanilleucine as the composition of the vehicle and a third solution having acetyl phenylalanine as the vehicle. Each solution - had a concentration of PYY (3-36) free of endotoxin of 200 μg / ml. EXAMPLE 25 Preparation of Modified Amino Acid Compositions / PYY (3-36) Preparation of Modified Amino Acid Microspheres Containing PYY3-36 Endotoxin-Free Encapsulation The mixture of modified amino acids, prepared according to Example 9, was dissolved at 40 ° C in distilled water (pH 7.2) at a concentration of 100 mg / ml. The solution was then filtered with a 0.2 micro filter and the temperature was maintained at 40 ° C. PYY3-36 (Bachem) was dissolved in an aqueous solution of citric acid (1.7N) and gelatin (5%) at a concentration of 150 mg / ml. This solution was then heated to 40 C. The two hot solutions were then mixed 1: 1 (v / v). The resulting microsphere solution was then filtered with glass fiber and concentrated for 50 minutes at 1000 g. The pellet was resuspended with 0.85N citric acid at a volume 5 to 7 times lower than the original volume. The concentration of PYY3-36 of the resuspended pellet was determined by HPLC. Additional microspheres were made according to the above procedure without PYY3-36. These "empty microspheres" were used to dilute the preparation in microspheres of PYY3-36 encapsulated salmon to a final dose suspension, if necessary. (b) Preparation of a Vehicle of a Solubles Modified Amino Acid System / PYY3-36 A soluble amino acid dose preparation containing PYY3-36 was prepared by dissolving the natural amino acid material in distilled water (pH 8) to an appropriate concentration. The solution was heated to 40 ° C and then filtered with a 0.2 micron filter. PYY3-36, also dissolved in distilled water, was then added to the modified amino acid solution above for oral administration. PYY3-36 (Prophetic) Pulmonary Administration Vehicle compounds, praparados as described below, can be used directly as a delivery vehicle by simply mixing one or more compounds or salts, poly amino acids or peptides with a peptide binding to the Y2 receptor free of charge. endotoxin for pulmonary administration. The administration mixtures are prepared by mixing an aqueous solution of the vehicle with an aqueous solution of the active ingredient, just before administration. Alternatively, the vehicle and the biological or gumanically active ingredient can be mixed - during the manufacturing process. The solutions may optionally contain additives such as phosphate buffer salts, citric acid, acetic acid, gelatin, and acacia gum. Several known methods of pulmonary administration can utilize peptides that bind to the receptor to the Y2 receptor free of endotoxin, especially PYY3-36, to improve the administration of PYY to the lungs. The following non-limiting patent applications are incorporated herein by reference for pulmonary administration: U.S. Patent Application. 20030223939, 20030215514, 20030215512, 20030209243, 20030203036, 20030198601, 20030183228, 200301885765, 20030150454, 20030124193, 20030094173. EXAMPLE 26 Preparation of Vehicles Preparation of 2- (4- (N-salicyloyl) aminophenyl) propionic acid (Vehicle B) A mixture of 58.6 g (0.355 mol) of 2- (4-aminophenyl) propionic acid and 500 ml of methylene chloride are treated with 90.11 ml (77.13 g, 0-710 mol) of trimethylsilyl chloride and heated at reflux for 120 min. The reaction mixture is cooled to 0 ° C. and it comes with 184.44 ml (107.77 g, 1.065 mol) of triethylamine. After stirring for 5 minutes, this mixture is treated with a solution of 70.45 g (0.355 mol) of O-acetylsalicyloyl chloride and 150 ml of methylene chloride. The reaction mixture is heated to 25 ° C and stirred for 64 hours. The volatiles are removed in vacuo. The residue was stirred in 2N aqueous sodium hydroxide for one hour and acidified with 2M aqueous sulfuric acid. The solid was recrystallized twice from ethanol / water to give a light brown solid. Isolation by filtration resulted in an expected yield of 53.05 g (52% yield) of 2- (4- (N-salicyloyl) aminophenyl) propionic acid. Properties Solubility: 200 mg / m: 200 mg + 350 .μl 2N NaOH + 650 .μl H20-pH-7.67. Analysis: C, 67.36; H, 5.3; N, 4.91. . Preparation of sodium 2- (4- (N-salicyloyl) aminophenyl) propionate (Sodium salt of Vehicle B) A solution of 53.05 g of acid (0.186 mol) of 2- (4- (N-salicyloyl) aminophenyl-) propionic acid and 300 ml of ethanol was treated with 7.59 g (0.190 mol) of NaOH dissolved in 22 ml of water. The reaction mixture was stirred for 30 min at 25 ° C and for 30 min at 0 ° C. The resulting pale yellow solid was isolated by filtration to give 52.61 g of sodium 2- (4- (N-salicyloyl) aminophenyl) propionate. Properties Solubility: 200 mg / ml clear solution; pH = 6.85. Analysis C, 60.45; H, 5.45; N, 3.92; Na, 6.43. Melting point 236-238 ° C. Preparation of Sodium Salt from Vehicle C A 2 1 round bottom flask equipped with an agitator and a backflow condenser was charged with a suspension of 3- (4-aminophenyl) propionic acid (15.0 g 0.084 moles, 1.0 equiv. ) in dichloromethane (250 ml). Chlorotrimethylsilane (18.19 g, 0.856 moles, 2.0 equiv.) Was added in one portion, and the mixture was heated to reflux for 1.5 h under argon. The reaction was allowed to cool to room temperature and placed in an ice bath (internal temperature <10 ° C). The reflux condenser was recoloured with an addition funnel containing triethylamine (25.41 g, 0.251 mol, 3.0 equiv.). Triethylamine was added dropwise over 15 min, and a yellow solid formed during the addition. The funnel was replaced by another addition funnel containing a solution of 2,3-dimethoxybenzoylchloride (I 8.31 g, 0.091 mol, 1.09 equiv.) In dichloromethane (100 ml). The solution was added dropwise during 30 min. The reaction was stirred in the ice bath for another 30 min and at room temperature for 3 h. The dicholoromethane was evaporated in vacuo to give brown oil. The brown oil was cooled in an ice bath, and an ice-cooled solution of saturated sodium bicarbonate (250 ml) was added. The ice bath was removed and the reaction was stirred 1 h to gauge a light brown solution. The solution was acidified with concentrated HCl and stored at ca SC for 1 hour. The mixture was extracted with dichloromethane (3.times.100 ml), dried over sodium sulfate, the salts were filtered and the dichloromethane was removed in vacuo. The resulting solid was recrystallized from 50% ethyl acetate / water (v / v) to gauge the acid of Vehicle C as off-white needles (25.92 g, 90%). Analysis for C? GH21N05: C, 66.46; H, 6.16; N, 4.08. mp = 99-102 ° C. 12 grams of the acid of Vehicle C were dissolved in ethanol, 75 ml, with heating. To this solution was added 8.5 M sodium hydroxide solution (1.02 molar equivalents, 1426 grams in 4.5 ml of water). The mixture was stirred for 15 minutes. Approximately three ethanol batches were removed in vacuo and 100 ml of n-heptane was added to the resulting oil causing a precipitate to form. The solids were dried in vacuo at 50 ° C. Analysis: C? 9H20NO5Na0.067H2O: C, 62.25; H, 5.54; N, 3.82; Na, 6.27. Preparation of N- (4-methylsalicyloyl) -8-aminocaprylic acid (Vehicle D) (a) Preparation of Oligo (4-metilsalicylate) Acetic anhydride (32 ml, 34.5 g, 0.338 mol, 1.03 eq), 4-methylsalicylic acid (50 g, 0.329 mmol, 1.00 eq), and xylenes were added. (100 ml) to a four-necked flask of 1 1, adapted with a magnetic stir bar, a thermometer and a condenser. The flask was placed in a sand bath and heating of the cloudy white mixture was started. The reaction mixture was clarified to a yellow solution around 90 ° C. The majority of the volatile organic compounds (xylenes and acetic acid) were distilled in the Dean-Stark separator for three hours (135-146 ° C). The distillation was continued for another hour (a total of 110 ml distillates), during which the temperature of the vessel slowly rose to 204 ° C and the distillate decreased to a trickle. The residue was emptied while still hot in an aluminum container. Upon cooling, a yellow, chewable crystal formed. The solid was ground to a fine powder. The reciprocal oligo (4-methylsalicylate) was used without further purification. (b) Preparation of N- (4-methylsalicyloyl) -8-aminocaprylic acid A solution of potassium carbonate (45 ml, 43.2 g, 0.313 mol, 0.95 eq), 8-aminocaprilic acid (41.8 g, 262 mol, 798 eq), and water (20 ml) to a 1 1 round bottom flask equipped with a magnetic stir bar, condenser and addition funnel. The cloudy white mixture was treated with an oligo (4-methylsalicylate) solution (44.7 g, 0.329 mmol 1.0 eq) and dioxane (250 ml) was added, for thirty minutes. The reaction mixture was heated at 90 ° C for 3 hours (at which time the reaction was determined to have ended, by HPLC). The light orange reaction mixture was cooled to 30 ° C. and acidified to pH = 2 with 50% aqueous sulfuric acid (64 g). The resulting solid was isolated by filtration. The white solid was recrystallized from 1170 ml of 50% ethanol-water. The solid was recovered by filtration and dried for 18 hours in a vacuum oven at 50 ° C. The N- (4-methylsalicyloyl) -8-aminocaprylic acid was isolated as a white solid (30.88 g, 52%); mp = 113-114 °. Analysis: C6H23N04: C, 65.51; H, 7.90; N, 4.77. An aqueous solution of PYY (3-36) was then prepared and mixed with one or more of the carriers to produce a PYY (3-36) composition, which can then be atomized into the lungs. A suitable concentration of PYY3-36 for the resulting composition should be about 400 μg / ml. See the U.S. Patent Application. No. 20030072740. EXAMPLE 27 Radioimmunoassay of Total Extraction for the Determination of the Concentration of PYY in Plasma 1.0 Introduction: A radioimmunoassay was developed to measure the concentration of Human Peptide YY (3-36) (hPYY) in human plasma. Samples were collected with anti-aging agent (EDTA) and protease inhibitor (aprotinin) and frozen. The trial is a four day process. Samples, controls, and standards were extracted in alcohol and dried on Day 1. All samples were reconstituted and mixed with a rabbit polyclonal antiserum directed against hPYY on Day 2. iodinated hPYY was harvested on Day 3. they added specific precipitation agents (Anti-Rabbit Rabbit IgG and Normadle Rabbit Serum) were added on Day 4. Trace traces were separated from the free traces by centrifugation, and traces joined in the gamma counter were counted. The concentration was calculated by interpolation of a standard curve and the performance of the test was controlled with Quality Control samples.

Materials: 2.1 PYY Peninsula Team (Peninsula Laboratories, Cat. No. S-2043-0001) 2.2 Reactive Alcohol (Fisher Inc., Cat. No. A995-4) (or equivalent) 2.3 Naked Plasma of Human (with Lithium Heparin, solid, agglomerated) Golden West Biologics Inc. (Cat. No., SD1020-H) (Analytical SOP # A-003) 2.4 Ice Baths (Fisher, Cat No. 11-676-36) (or equivalent) 2.5 Disposable pipettes of 10 ml (Fisher Cat. No. 13-678-11E) (or equivalent) 2.6 PYY Human Synthetic -Standard of Nastech QC (3-36) (Bachem Cat. No. H8585) 2.7 Distilled water (Mílli-Q Millipore, Cat. No. ZMQ56VFT1) (or equivalent) -25 - 2. 8 Triton X-100 (Sigma, Cat. No. T-9284) (or equivalent) 2.9 Aluminum Sheet (Fisher, Cat. No. 01-213-3) (or equivalent) 2.10 Aprotinin (ICN Biomedicals Inc. Cat. No. 190779) (or equivalent) 2.11 12x75 mm tubes (Evergreen Scientific, Cat. No. 214-2023-010) (or equivalent) 2.12 12x75 mm Tube Caps (Evergreen Scientific, Cat. No. 300-2912-G20) (or equivalent) ) 2.13 Microfuge tubes 1.5 ml (Fisher, Cat. No. 05- 402-25) (or equivalent) 2.14 3. 0 Instruments: 3. 1 Wallac WIZARD 1470 Automatic Gamma Counter (Perkin Elmer, Model No. 1470-002) (or equivalent) 3.2 Isothermal Basic Freezer, -70 ° C (Kenμdro Laboratory Products, Model No. C90-3A31) (or equivalent) 3.3 CentriVap Concentrator (Labconco, Cat. No. 7810000) (or equivalent) 3.4 Multi-tube agitator VX-2500 (VWR, Cat. No. 58816- 115) (or eguivalent) 2 3. 5 Marathon 21000R Centrifuge (Fisher, Cat. No. 04- 977-21000R) (or equivalent) 3.6 Swinging Bucket Rotor (Fisher, Cat. No. 04-976-006) (or equivalent) 3.7 Motorized Pipette Auxiliary (Fisher , Cat. No. 13-681-15E) (or equivalent) 3.8 Eppendorf Micropipette 3.8.1 2 μl - 20 μl (Fisher, Cat. No. 21-371-6) (or equivalent) 3.8.2 20 μl - 200 μl (Fisher, Cat. No. 21-371-10) (or equivalent) 3.8.3 100 μl - 1000 μl (Fisher, Cat. No. 21-371-13) (or equivalent) 3.9 Eppendorf Repetition Pipettor (Fisher , Cat. No. 21-380-9) (or equivalent) 3.10 Eppendorf repetitive repeating pipette tipper 3.10.1 2.5 ml (Fisher, Cat. No. 21-381-331) (or equivalent) 3.10.2 25 ml (Fisher, Cat. No. 21-381-115) (or equivalent) 3.11 Positive displacement pipette (Fisher, Cat. No. 21-169-10A) (or equivalent) 4. 0 Procedure DAY 1 4.1 Reagents and samples required for freezing for the test. Prepare RIA damper to concentration IX (RIAB) if sufficient quantity is not available. 4.2 Prepare samples of standard curves in agglomerated human naked plasma. Prepare as follows if a standard concentration of 12.8 μg / ml is used. 4.2.1 Add 990 μl RIAB to tube O. 4.2.2 Add 990 μl of agglomerated plasma to tube A. 4.2.3 Add 500 μl of agglomerated plasma to B-H tubes. 4.2.4 Add 10 μl 12.8 μg / ml Standard to tube O. Shake. 4.2.5 Add 10 μl of solution from tube O to tube A. Shake. 4.2.6 Add 500 μl of solution from tube A to tube B. Shake. 4.2.7 Add 500 μl of solution from tube B to tube C. Shake. 4.2.8 Repeat dilutions as in 4.2.7 through tube H. (See Diagram # 1) 4.3 Dilute samples of known human plasma to be tested if necessary. Samples should be diluted in agglomerated human naked plasma. 4.4 Add 1.2 ml of cold alcohol to the empty tubes for NSB, TB, all Standards, QC samples, and human plasma samples to be tested. - 4.5 Add 400 μl of naked human plasma agglomerated to NSB and TB tubes. Cap, Vortex. 4.6 Add 400 μl of prepared standard sample from 4.2.5 to 4.2.8 to the standard H-A repurposing curve tubes (See Diagram # 1). Cap, Shake 4. 7 Add 400 μl of QC samples to the respective tubes. Cap, Shake 4.8 Add 400 μl of each sample to be tested to its respective tube. Cap, Shake 4.9 Incubate all samples on ice for 30-60 minutes. 4.10 Flip the cold separator switch on the Concentrator. 4.11 Centrifuge all tubes at 3000 rpm, 4 ° C for 15 minutes. 4.12 Transfer 1.3 ml of supernatant from each sample to a new set of empty tubes. Store in an ice bath or at 2-8 ° C if not turned immediately. 4. 13 Place the samples in the Concentrator. 4.14 The samples are rotated for two hours at 40 ° C, then at room temperature for a total of 5 hours or until dry. 4.15 Remove the samples to dry, cover and store overnight at 2-8 ° C.

Day 2 4. 16 Remove the dry tubes from the cooler at 2-8 ° C. 4. 17 Add 100 μl of 4 × RIA absorber concentrate to each tube. 4.18 Add 100 μl of TX100 0.6% to each tube. (Annex # 1) Shake for a minimum of 30 seconds to ensure that all extracts are completely reconstituted. 4.19 Incubate all samples on ice for 30-60 minutes. 4.20 Add 200 μl of distilled water to each tube. Shake. 4.21 Transfer 100 μl of each sample extract to the respective tube. Note: The NSB Standard Curve samples, TB, TC, and QCs were typically run in triplicate, requiring three tubes per sample. Many samples of human plasma were treated in any variation (up to three replicates) depending on the availability of the sample. 4.22 Prepare rabbit anti-PYY as described in the insert of the Peninsula Laboratories team. 4.23 Add 100 μl RIAB to each NSB tube. 4.24 Add 200 μl RIAB to each TC tube. 4.25 Add 100 μl rabbit anti-PYY to all remaining tubes. Shake. 4.26 Cover with foil and store overnight at 2-8 ° C.

Day 3 4. 27 Remove the cooler tubes at 2-8 ° C. 4.28 Prepare traces 125I-Peptide YY (Appendix # 2). 4.29 Add 100 μl of prepared traces to all tubes. Cover and shake. 4.30 Store overnight at 2-8 ° C.

Day 4 4. 31 Remove the cooler tubes 2-8 ° C. 4. 32 Prepare goat anti-Rabbit IgG serum (GARGG) and normal Rabbit serum (NRS) as described in the insert of the Peninsula Laboratories team. 4.33 Add 100 μl of GARGG to each tube (except TC tubes). 4.34 Add 100 μl of NRS to each tube (except TC tubes). Shake. 4.35 Incubate 90-120 minutes at room temperature. 4. 36 Add 500 μl of RIAB to the tubes to centrifuge immediately (except TC tubes). Shake Note: 500 μl of RIAB should be added to the tubes just before centrifugation. Add only RIAB to the number of tubes that is ready to be centrifuged. 500 μl of RIAB should be added to the additional tubes when they are ready to be centrifuged. 4.37 Centrifuge tubes (containing 500 μl of RIAB) at 3000 rpm at 4 ° C, for 15 minutes. The TC tubes are not centrifuged. 4.38 Aspirate the supernatant from the centrifuged tubes. 4.39 Place the tubes in the black shelves designated for counting in the Gamma counter. The first shelf must have the appropriate Program number attached. All shelves that follow should not contain a program number. Samples should be added in the following order: 4.39.1 NSB tubes 4.39.2 TB tubes 4.39.3 TC tubes 4.39.4 Standard tubes (increased concentration) 4.39.5 QC samples (3 concentrations) 4.39.6 Human samples unknown 4.39.7 QC Samples (3 concentrations) 4.40 Place an empty black shelf with the label Alto attached after having counted all the samples 4.41 Press Start 'on the Gamma Counter keypad to start counting. 4.42 Press E 'to enter the keyboard of the Gamma Counter to display the CPM results. . 0 Evaluation of results . 1 The following guidelines apply to the identification and rejection profiles in the trial.

In order for a result to qualify as a profile and not be included in the final calculation of results, the following conditions must be met: 5.1.1 QCs and unknown samples: 5.1.1.1 The% CV of all replicates must be greater than twenty%. 5.1.1.2 There must be at least three results to evaluate. 5.1.1.3 The difference between the expected profile and the result following the closest value must be greater than 20%. 5.1.1.4 The difference between the remaining major and minor results must be less than 20%. 5.1.2 Samples of the Standard Curve: 5.1.2.1 The% CV of all the replicas must be greater than 15%. 5.1.2.2 There must be at least three results to evaluate 5.1.2.3 The difference between the expected profile and the result following the closest value must be greater than 15%. 5.1.2.4 The difference between the results - remaining major and minor must be less than 15%. 6.0 Test Specifications 6. 1 QC samples are prepared at the following concentrations. Two QC samples are tested in each concentration in one assay. Four of the six QC samples tested must be within the following ranges (+ 30% of the nominal concentration). At least one of the two QCs tested at any concentration should be within the test range for the data to be acceptable 6.1.1 QC1 (100 pg / ml) 70-130 pg / ml 6.1.2 QC2 (200 pg / ml) 140-260 pg / ml 6.1.3 QC3 (500 pg / ml) 350-650 pg / ml 6.2 TBD requirements of the standard curve parameter.

RY Standard of PYY: Annex # 1 0.6% TX-100 Reagent: 0.6% TX-100 Materials: Distilled water Milli-Q TX-100 Preparation: 1) Measure 50 ml of Distilled Water Milli- Q 2) Add 300 μl of TX-100 using positive displacement pipette 3) Mixing well. Appendix 2 Traces of 1l25O-PYY peptide Reagent: Traces of 12A501 - PYY peptide Materials: Damper lx RIA 125I - PYY peptide Preparation: 1) Reconstitute traces with 1 ml of Ix RIA buffer. 2) Measure the amount of the traces in the Gamma Counter. Transfer 10 μl of reconstituted traces to a tube. Place them on a black shelf by the Gamma Counter with Program # 30 attached. 3) Place the shelf on the Gamma Counter with the High shelf behind it. 4) Press Start "to start counting, then E 'to see the CPM results 5) Determine the amount of traces (X μl) to prepare and RIAB (Y ml) necessary as follows: X μl = (5 μl) (cpm value) (# tubes + 10) (cpm of the shelf solution) And ml = (0.1) (# tubes + 10) - 6) Combine X μl of 125 I-Peptide YY with Y ml of RIAB. Mixing well.

EXAMPLE 28 Preparation of a NPY Formulation Free of a Stabilizer that is a Protein. A PYY formulation suitable for intranasal administration of NPY, which is substantially free of a stabilizer which is a protein is prepared having the formulation listed below. 1. Approximately add water to a beaker and stir with a stir bar on a stir plate and add sodium citrate until it is completely dissolved. 2. EDTA is then added and stirred until completely dissolved. 3. The citric acid is then added and stirred until it completely dissolves. 4. Methyl-β-cyclodextrin is added and stirred until it completely dissolves. 5. DDPC is then added and stirred until it is completely dissolved. 6. Lactose is then added and stirred until it is completely dissolved. 7. Sorbitol is then added and stirred until it dissolves completely. 8. Then add chlorobutanol and stir until completely dissolved. 9. Add NPY (3-36) and stir gently until it dissolves. 10. Check the pH to make it more secure at 5.0 ± 0.25. Dilute HCl or diluted NaOH is added to adjust the pH. 11. - Water is added to the final volume.

Table 12 - - Formulation pH 5 +/- 0.25 Osmolarity ~ 250 EXAMPLE 29 A second formulation was prepared as above, except that the concentration of NPY (3-36) is 15 mg / ml as shown below in Table 13.

Table 13 Formulation pH 5 +/- 0.25 EXAMPLE 30 Preparation of the Pancreatic Peptide (PP) Formulation Free of Stabilizer which is a Protein. A formulation of PYY suitable for intranasal administration of PP, which is substantially free of a stabilizer which is a protein is prepared having the formulation listed below.

Approximately H of water is added to a beaker and stirred with a stir bar on a stir plate and sodium citrate is added until it is completely dissolved. EDTA is then added and stirred until completely dissolved. The citric acid is then added and stirred until it completely dissolves. Methyl-β-cyclodextrin is added and stirred until completely dissolved.

- . DDPC is then added and stirred until it is completely dissolved. 6. Lactose is then added and stirred until it is completely dissolved. 7. Sorbitol is then added and stirred until it dissolves completely. 8. Then add chlorobutanol and stir until completely dissolved. 9. Add NPY (3-36) and stir gently until it dissolves. 10. Check the pH to make it more secure at 5.0 ± 0.25. Dilute HCl or diluted NaOH is added to adjust the pH. 11. Water is added to the final volume.

Table 14 Formulation pH 5 +/- 0.25 Osmolarity -250 EXAMPLE 31 A second formulation was prepared as above, except that the concentration of PP (3-36) is 15 mg / ml as shown below in Table 15.

Table 15 - - Formulation pH 5 +/- 0.25 EXAMPLE 32 This example describes a pharmaceutical composition product comprising an aqueous solution formulation of a compound that binds to the Y2 receptor at a concentration sufficient to produce therapeutically effective plasma concentrations and an actuator to produce an aerosol of said solution, where the electicity ratio of the spray pattern of said aerosol is between 1.00 and 1.40 when measured at a height between 0.5 cm and 10 cm from the tip of the actuator. Surprisingly a PYY (3-36) formulation of the present specification can be made in aerosol and still be therapeutically effective (as shown in Example 8a). The volume of the aerosol can be between approximately 5 microliters and 1.0 ml, preferably between 20 and 200 microliters. This test method describes the procedure for characterizing the pen geometry of the nasal solution formulations of the compound that binds to the Y2 receptor using the SprayView NSP system. The boom geometry is characterized using a SprayView High Speed Optical Spray System Characterization (SprayView NSP) High Speed Optical Spray Characterization System with the Integrated SprayView NSx actuation station (Image Ther Engineering, Inc ., Sudbury, MA) according to the methods described in the US Patent. No. 6,665,421 and Patent Application Publication No. 20030018416 published on January 23, 2003. Using the formulation of Table 14 or placebo the characterization of the spray and the size of the drops of the formulation in both a bottle of 1 ml and one of 3 ml having both a Nasal Spray Pump for Safety Clip, Pfeiffer SAP # 60548, which supplies a dose of 0.1 ml per jet and has a tube depth length of 36.05 mm.

The droplet size data is shown in the - next table.

Drop Size for Nasal Spray Bottle and Pfeiffer SAP # 60548% < 10 microm Compound that binds 3ml to the Y2 receptor (PYY) 23.26 92.31 610.46 6.60 0.59 The results of the sprinkling pattern and the pen geometry are listed below Although the above invention has been described in detail by way of example for the purpose of clarity of understanding it will be apparent to a technician that certain changes and modifications are comprehensive by the disclosure and can be practiced without due experimentation within the scope of the appended claims. , which are presented as a non-limiting illustration.

Claims (45)

1. -
2. CLAIMS 1. A pharmaceutical composition product comprising: a. an aqueous composition formulation comprising a PYY compound and a protein free stabilizer at a concentration sufficient to produce therapeutically effective plasma concentrations of the PYY compound; and • b. an actuator capable of producing an aerosol of said solution, wherein the electicity ratio of the spray pattern of said aerosol is between 1.00 and 1.40 when measured at a height of 0.5 cm and 10 cm distance from the tip of the actuator . 2. The product of the pharmaceutical composition of claim 1 wherein the PYY compound is a variant, derivative, analog or fragment of PYY.
3. 3. The product of the pharmaceutical composition of claim 1 wherein the compound PYY is selected from the group consisting of SEQ ID Nos: 1-21, 72-74 and 90-105.
4. 4. The product of the pharmaceutical composition of claim 1 wherein the PYY compound is PYY (3-36).
5. 5. The product of the pharmaceutical composition of claim 1 wherein the amount of the PYY compound in the aerosol is at least 50 μg per actuation.
6. 6. The product of the pharmaceutical composition of claim 1 wherein the amount of the PYY compound in the aerosol is at least 200 μg per actuation.
7. 7. The product of the pharmaceutical composition of claim 1 wherein the actuator produces an ellipse of between 1.00 and 1.30.
8. 8. The product of the pharmaceutical composition of claim 1 wherein the actuator produces an ellipse of between 1.15 and 1.25.
9. 9. A product of the pharmaceutical composition comprising: a. an aqueous solution formulation comprising a PYY compound and a protein free stabilizer at a concentration sufficient to produce therapeutically effective plasma concentrations of the PYY compound; and b. an actuator to produce an aerosol of said solution, wherein the main spray pattern and the major axis of said spray are between 10 and 50 mm when measured at a height of 0.5 cm and 10 cm from the tip of the actuator
10. 10. The product of the pharmaceutical composition of claim 9 wherein the PYY compound is a variant, derivative, analog or fragment of PYY.
11. 11. The product of the pharmaceutical composition of claim 9 wherein the compound PYY is selected from the group consisting of SEQ ID Nos: 1-21, 72-74 and 90-105.
12. 12. The product of the pharmaceutical composition of the claim 9 wherein the PYY compound is PYY (3-36).
13. 13. The product of the pharmaceutical composition of claim 7 wherein the amount of said PYY compound in the aerosol is at least 50 μg per actuation. The product of the pharmaceutical composition of claim 7 wherein the amount of said PYY compound in the aerosol is at least 200 μg per actuation. 15. A product of the pharmaceutical composition comprising: a. an aqueous solution formulation comprising a PYY compound and a protein free stabilizer at a concentration sufficient to produce therapeutically effective plasma concentrations of the PYY compound; and b. an actuator for producing an aerosol of said solution, wherein less than 10% of the droplets are smaller than 10 microns of size 16. The product of the pharmaceutical composition of claim 15 wherein the PYY compound is a variant, derivative , analog or fragment of PYY. 17. The product of the pharmaceutical composition of claim 15 wherein the compound PYY is selected from the group consisting of SEQ ID Nos: 1-21, 72-74 and 90-105. 18. The product of the pharmaceutical composition of Claim 15 wherein the PYY compound is PYY (3-36). 19. The product of the pharmaceutical composition of claim 15 wherein the amount of said PYY compound in the aerosol is at least 50 μg per actuation. 20. The product of the pharmaceutical composition of claim 15 wherein the amount of said PYY compound in the aerosol is at least 200 μg per actuation. The product of the pharmaceutical composition of claim 15 wherein less than 5% of the droplets are smaller than 10 microns in size 22. The product of the pharmaceutical composition of claim 15 wherein less than 1% of the droplets They are smaller than 10 microns in size. 23. A pharmaceutical composition product comprising: a. an aqueous solution formulation comprising a PYY compound and a protein free stabilizer at a concentration sufficient to produce therapeutically effective plasma concentrations of the PYY compound; and b. an actuator selected to produce an aerosol of said solution, wherein droplets of between 25 and 700 microns are produced. 24. The product of the pharmaceutical composition of claim 23 wherein the PYY compound is a variant, derivative, analog or fragment of PYY. 25. The product of the pharmaceutical composition of claim 23 wherein the compound PYY is selected from the group consisting of SEQ ID Nos: 1-21, 72-74 and 90-105. 26. The product of the pharmaceutical composition of claim 23 wherein the PYY compound is PYY (3-36). 27. The product of the pharmaceutical composition of claim 23 wherein the amount of said PYY compound in the aerosol is at least 50 μg per actuation. 28. The product of the pharmaceutical composition of claim 23 wherein the amount of the compound of PYY in the aerosol is at least 200 μg per act 29. The product of the pharmaceutical composition of claim 1 wherein the protein free stabilizer is selected from the group consisting of polyols, surface active agents, solubilizing agents and chelating agents. . 30. The product of the pharmaceutical composition of claim 29 wherein the surfactant is didecanoyl phosphatidylcholine. 31. The product of the pharmaceutical composition of claim 29 wherein the solubilizing agent is cyclodextrin. 32. The product of the pharmaceutical composition of claim 29 wherein the protein free stabilizer is selected from the group consisting of cyclodextrins, chitosans, DDPC, Tween 80, disodium edetate, citric acid, lactose and sorbitol. 33. The product of the pharmaceutical composition of claim 9 wherein the protein free stabilizer is selected from the group consisting of polyols, surface active agents, solubilizing agents and chelating agents. 34. The product of the pharmaceutical composition of claim 33 wherein the surfactant is didecanoyl phosphatidylcholine. 35. The product of the pharmaceutical composition of claim 33 wherein the solubilizing agent is cyclodextrin. 36. The product of the pharmaceutical composition of claim 33 wherein the protein free stabilizer is selected from the group consisting of cyclodextrins, chitosans, DDPC, Tween 80, disodium edetate, citric acid, lactose and sorbitol. 37. The product of the pharmaceutical composition of claim 15 wherein the protein free stabilizer is selected from the group consisting of polyols, surface active agents, solubilizing agents and chelating agents. 38.- The product of the pharmaceutical composition of claim 37 wherein the surfactant is didecanoyl phosphatidylcholine. 39. The product of the pharmaceutical composition of claim 37 wherein the solubilizing agent is cyclodextrin. 40. The product of the pharmaceutical composition of claim 37 wherein the protein free stabilizer is selected from the group consisting of cyclodextrins, chitosans, DDPC, Tween 80, disodium edetate, citric acid, lactose and sorbitol. 41. The product of the pharmaceutical composition of claim 23 wherein the protein free stabilizer is selected from the group consisting of polyols, surface active agents, solubilizing agents and chelating agents. 42. The product of the pharmaceutical composition of claim 41 wherein the surfactant is didecanoyl phosphatidylcholine. 43. The product of the pharmaceutical composition of claim 41 wherein the solubilizing agent is cyclodextrin. 44. The product of the pharmaceutical composition of claim 41 wherein the protein free stabilizer is selected from the group consisting of cyclodextrins, chitosans, DDPC, Tween 80, disodium edetate, citric acid, lactose and sorbitol. 45. A product of pharmaceutical composition comprising: a. an aqueous composition formulation comprising a protein free stabilizer and a Pancreatic Peptide at a concentration sufficient to produce therapeutically effective plasma concentrations of the Pancreatic Peptide; and b. an actuator capable of producing an aerosol of said solution, wherein the ellipticity ratio of the spray pattern of said aerosol is between 1.00 and 1.40 when measured at a height of 0.5 cm and 10 cm from the tip of the actuator .