MX2008000600A - Formulations for enhanced mucosal delivery of pyy - Google Patents

Abstract

Pharmaceutical formulations are described for enhancing mucosal delivery of peptide YY (PYY) to a mammal. A PYY dosage form is described that is suitable for multi-use administration. The PYY dosage form comprises a bottle containing an aqueous pharmaceutical formulation and an actuator effective intranasal administration of the formulation. The formulation comprises a therapeutically effective amount of PYY, a buffer to control pH, a water-miscible polar organic solvent and a chelating agent for cations. The PYY dosage form exhibits at least 90%PYY recovery after storage as used for greater than about five days.

Description

FORMULATIONS FOR THE IMPROVED MUCOUS SUPPLY OF PYY BACKGROUND OF THE INVENTION Obesity and its associated disorders are common and very serious public health problems in the United States and throughout the world. It has been shown that certain peptides that bind to the Y2 receptor when administered peripherally to a mammal induce weight loss. Peptides binding to the Y2 receptor are neuropeptides that bind to the Y2 receptor. These peptides binding to the Y2 receptor belong to a family of peptides that includes peptide YY (PYY), neuropeptide Y (NPY), and pancreatic peptide (PP). These peptides of about 36 amino acids have a compact helical structure that includes a "fold-PP" in the middle part of the peptide. Specific features include a polyproline helix at residues 1 to 8, a return ß at residues 9 to 14, a helix a at residues 15 to 30, a terminal C for outward projection at residues 30 to 36, and a carboxyl terminal amide, which appears to be critical for biological activity. It has been shown that a 36 amino acid peptide called Peptide YY (1-36) [PYY (1-36)] [YPIKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY, (SEQ ID NO: 1)] when peripherally administered by injection to a Individual produces weight loss and can thus be used as a drug to treat obesity and related diseases, Morley, J., Neuropsychobiology 21: 22-30 (1989). It was then found that to produce this effect, PYY binds to a Y2 receptor, and the binding of a Y2 agonist to the Y2 receptor causes a decrease in the carbohydrate, protein and feed size intake, Leibowitz, S.F. et al., Pepíides 12: 1251-1260 (1991). An alternate molecular form of PYY is PYY (3-36) IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY (Residues 3-36 of SEQ ID NO: 1), Eberlein, Eysselein et al., Pepíides 10: 797-803, 1989). Hereinafter the term PYY refers to full-length PYY and any fragment of PYY that binds to a Y2 receptor. It is known that PYY can be administered by infusion or intravenous injection to treat life-threatening hypotension such as apoplexy, especially that caused by endotoxins (US Patent No. 4,839,343), to inhibit the proliferation of pancreatic tumors in mammals by perfusion, parenteral, intravenous, or subcutaneous administration, and by implant (U.S. Patent No. 5,574,010) and to treat obesity (Morley (1989)) and U.S. Patent Application. No. 20020141985). It is also claimed that PYY can be administered by parenteral, oral, nasal, rectal and topical routes to domesticated animals or human in an effective amount to increase the weight gain of said subject by improving the gastrointestinal absorption of a sodium-dependent cotransported nutrient (U.S. Patent No. 5,912,227). However, for the treatment of obesity and related diseases, including diabetes, the mode of administration has been limited to IV intravenous infusion with ineffective formulations optimized for alternative administration of PYY. None of these teachings of the prior art provide formulations containing PYY or PYY (3-36) combined with excipients designed to improve mucosal (ie, nasal, buccal, oral) delivery or teach the value of binding peptide formulations to the endotoxin-free Y2 receptor for administration not infused. Previously, formulations for intranasal administration of PYY are described in patent applications, including Patent Application Publications Nos. O040563142; US2004 / 0115135; US2004 / 0157777; US2004 / 0209807; and US2005 / 0002927, incorporated herein by reference. These applications describe suitable formulations for dosing between 20 and 200 μg in 0.1 ml, ie, with concentrations between 0.2 and 2.0 mg / ml PYY. The stability of the dosage form of 0.3 mg / ml PYY is tested at various pH values for five days at 40 ° C in a formulation comprising 10 mM citrate and 100 mM NaCl. The optimum pH is found in 4.9 where more than 80% of the peptide remained after five days of incubation. However, it was subsequently found that the stability of PYY is substantially influenced by concentration of PYY: at higher concentration, the stability of PYY decreased. In addition, the formulations previously described and tested for intranasal administration included excipients such as methyl-β-cyclodextrin and L-a-phosphatidylcholine didecanoyl, which are not generally considered as safe excipients (GRAS). Accordingly, to provide formulations and dosage forms that have general utility under common conditions for a pharmaceutical drug, there is a compelling need to develop alternative formulation compositions having improved stability. In addition, there is a compelling need to develop formulations and dosage forms comprising GRAS excipients as a distinct alternative to using non-compendial excipients. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1: PYY3-36 permeation of formulations tested in Example 1. Figure 2: PYY3-36 permeation of formulations tested in Example 2.

Figure 3: PYY3-36 permeation of formulations tested in Example 3. Figure 4: PYY3-36 permeation of formulations tested in Example 4. Figure 5. Recovery of peptide vs. time: (A) storage at 25 ° C; (B) storage at 40 ° C; and (C) storage at 50 ° C. Figure 6. Recovery of peptide vs. time, storage at 40 ° C: (A) formulations based on citrate buffer; (B) formulations based on acetate buffer; (C) formulations based on glutamate buffer; and (D) unregulated formulations. Figure 7. Stability of PYY3-36 at high temperature and spray atomization tension three times a day for samples tested in Example 8. Figure 8. Stability of P? Y3-36 at high temperature and spray atomization voltage three times per day for samples tested in Example 9. Figure 9. Stability of PYY3-36 at elevated temperature and spray atomization stress three times a day, tested in Example 10: (A) samples 5-1, 5-2 and 5-3; (B) samples 5-4, 5-5, 5-6 and 5-7; (C) samples 5-8, 5-9, 5-10 and -11; (D) samples 5-12, 5-13, and 5-14. DETAILED DESCRIPTION OF THE INVENTION To provide a better understanding of the - present invention, the following definitions and detailed description are provided. Peptides binding to the Y2 receptor The Y2 receptor binding peptides used in mucosal formulations of the present invention include three naturally occurring bioactive peptide families, PP, NPY, and PYY. Examples of Y2 receptor binding peptides and their uses are described in the U.S. Patent. No. 5,026,685; Patent of E.U. No. 5,574,010; Patent of E.U. No. 5,604,203; Patent of E.U. No. 5,696,093; Patent of E.U. No. 6,046,167; Gehlert et. al, Proc. Soc. Exp. Biol. Med. 218: 1-22 (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); Grundémar et al. , Regula Iory and Peptides 62: 131-136 (1996); Patent of E.U. No. 5,696,093 (examples of PYY agonists), U.S. Patent. No. 6,046,167. According to the present invention a peptide binding to the Y2 receptor includes the free bases, acid addition salts or metal salts, such as sodium or potassium salts or the Y2 receptor binding peptides that have been modified by such processes as amidation, glycosylation, acylation, sulfation, phosphorylation, acetylation and cyclization, (U.S. Patent No. 6,093,692; and U.S. Patent No. 6,225,445 and PEGylation).

Peptide Agonists YY As used in this, Mp ?? "refers to PYY (1-36) (SEQ ID NO: 1) in a native sequence or in a variant form, as well as derivatives, fragments, and analogs of PYY from any source, whether natural, synthetic, or recombinant, PYY is comprised of at least the last 15 amino acid residues or analogs thereof of the PYY Sequence, PYY (22-36). Other PYY Peptides, which may be used are PYY (1-36) (SEQ. NO: 1), PYY (3-36), PYY (4-36), PYY (5-36), PYY (6-36), PYY (7-36), PYY (8-36), PYY (9 -36), PYY (10-36), PYY (ll-36), PYY (12-36), PYY (13-36), PYY (14-36), PYY (15-36), PYY (16-36) 36), PYY (17-36), PYY (18-36), PYY (19-36), PYY (20-36), and PYY (21-36) .These peptides typically bind to the Y receptors in the brain and elsewhere, especially the Y2 and / or Y5 receptors Typically these peptides are synthesized in pyrogen-free or endotoxin-free forms although not always necessary Other PYY peptides include those PYY peptides in which the residue changes Not me acid preservers have been made, for example, site-specific mutation of a PYY peptide including [Asp15] PYY (15-36) (SEQ ID NO: 2), [Thr13] PYY (13-36) (SEQ ID NO: 3), [Val12] PYY (12-36) (SEQ ID NO: 4), [Glu11] PYY (ll-36) (SEQ ID NO: 5), [Asp10] PYY (10-36) (SEQ ID NO. : 6), [Val7] PYY (7-36) (SEQ ID NO: 7), [Asp6] PYY (6-36) (SEQ ID NO: 8), [Gln4] PYY (4-36) (SEQ ID DO NOT: 9), [Arg4] PYY (4-36) (SEQ ID NO: 10), [Asn4] PYY (4-36) (SEQ ID NO: 11), [Val3] PYY (3-36) (SEQ ID NO : 12) and [Leu3] PYY (3-36) (SEQ ID NO: 13). Other PYY peptides include those peptides in which at least two conservative amino acid residue changes have been made that include [Asp10, Asp15] PYY (10-36) (SEQ ID NO: 14), [Asp6, Thr13] PYY (6 -36) (SEQ ID NO: 15), [Asn4, Asp15] PYY (4-36) (SEQ ID NO: 16), and [Leu3, Asp10] PYY (3-36) (SEQ ID NO: 17). Analogs of a PYY are also included, for example, those described in US Patents. Nos. 5,604,203 and 5,574,010. These include the following peptides: Formula IA Ri R3 / R2 _? A22.A23-A24-A2s-A26-A27-A28-A2 »-A30-A31-A3-A33-A34-A35-A3 < '- R4 For Formula IA the following peptide analogs PYY (22-36) can be created where: X is Cys or is deleted; each of Ri and R2 is bonded to the nitrogen atom of the alpha-amino group of the N-terminal amino acid; R 1 is H, C 1 -C 12 alkyl, C 18 aryl, C 1 -C 2 acyl, C -C aralkyl, or C -C 8 alkaryl; R 2 is H, C 1 -C 2 alkyl, C 6 -C 8 aryl, C 1 -C 2 acyl, C 7 -C 8 aralkyl, or C 7 -C 8 alkaryl; A22 is an aromatic amino acid, Ala, Aib, Anb, N-Me-Ala, - or it is eliminated; A23 is Ser, Thr, Ala, Aib, N-Me-Ser, N-Me-Thr, N-Me-Ala, D-Trp, or is deleted; A24 is Leu, Gly, Lie, Val, Trp, Nle, Nva, Aib, Anb, N-Me-Leu, or is deleted; A25 is Arg, Lys, homo-Arg, diethyl-homo-Arg, Lys-epsilon-NH-R (where R is H, a straight or branched chain C1-C10 alkyl group, or an aryl group), Orn, or It is eliminated; A26 is Ala, His, Thr, 3-Me-His, 1-Me-His, beta-pirozolylalanine, N-Me-His, Arg, Lys, homo-Arg, diethyl-homo-Arg, Lys-epsilon-NH- R (wherein R is H, a straight or branched chain C1-C10 alkyl group, or an aryl group), Orn, or is deleted; A27 is Nal, Beep, Pcp, Tic, Trp, Bth, Thi, or Dip; A28 is Leu, Val, Trp, Nle, Nva, Aib, Anb, or N-Me-Leu; A29 is Asn, Ala, Gln, Gly, Trp, or N-Me-Asn; A30 is Leu, Lie, Val, Trp, Nle, Nva, Aib, Anb, or N-Me-Leu; A31 is Val, Leu, Lie, Trp, Nle, Nva, Aib, Anb, or N-Me-Val; A32 is Thr, Ser, N-Me-Ser, N-Me-Thr, or D-Trp; A33 is Cys, Arg, Lys, homo-Arg, diethyl-homo-Arg, Lys-epsilon-NH-R (where R is H, a straight or branched chain Ci-Cio alkyl group, or C6-C aryl group) 8), or Orn; A34 is Cys, Gln, Asn, Ala, Gly, N-Me-Gin, Aib, or Anb; A35 is Cys, Arg, Lys, homo-Arg, diethyl-homo-Arg, Lys-epsilon-NH-R (where R is H, a straight or branched chain Ci-Cio alkyl group, or a Cß-Ciß aryl group ), or Orn; A36 is an aromatic amino acid, or Cys; R 3 is H, C 1 -C 2 alkyl, C 6 -C 8 aryl, C 1 -C 2 acyl, C 7 -C 8 aralkyl, or C 7 -C 8 alkaryl; R 4 is H, C 1 -C 2 alkyl, C 6 -C 8 aryl, C 1 -C 2 acyl, C -C β aralkyl, or C -C 8 alkaryl, or a pharmaceutically salt - in ¬ acceptable of them. Examples of peptide analogs PYY (22-36) of Formula IA include: N-alpha-Ac-Ala-Ser-Leu-Arg-His-Trp-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg- Tyr-NH2 (SEQ ID NO: 18); N-alpha-Ac-Ala-Ser-Leu-Arg-His-Thi-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-NH2 (SEQ ID NO: 19), or pharmaceutically acceptable salts of the same . Additional analogs of Formula IA may be used which include: Where the bond -CO-NH-- between residues A28 and A29, A29 and A30, A30 and A31, A31 and A32, A33 and A34, A34 and A35, or A35 and A36 is replaced with CH2-NH, CH2-S, CH2-CH2, or CH2-0, or where the CO-NH bond between residues A35 and A36 is replaced with CH2-NH. For Formula IB, the following peptide analogs PYY (22-36) can be created where: X is Cys or is deleted; Ri and R2 are linked to the N-terminal amino acid; R x is H, C 1 -C 2 alkyl, C 6 -C 8 aryl, C 1 -C 2 acyl, C 7 -C 8 aralkyl, or C 7 -C 8 alkaryl; R 2 is H, C 1 -C 12 alkyl, C 6 -C 8 aryl, C 1 -C 2 acyl, C 7 -C 8 aralkyl, or C 7 -C 8 alkaryl; A22 is an aromatic amino acid or is deleted; A23 is Ser, Thr, Ala, Aib, N-Me-Ser, N-Me-Thr, Me-Ala, D-Trp, or is deleted; A24 is Leu, Gly, Lie, Val, Trp, Nle, Nva, Aib, Anb, N-Me-Leu, or is deleted; A25 is Arg, Lys, homo-Arg, diethyl-homo-Arg, Lys-epsilon-NH-R (where R is H, a straight or branched chain C1-C10 alkyl group, or an aryl group), Orn, or is deleted; A26 is Ala, His, Thr, 3-Me-His, 1-Me-His, beta-pirozolylalanine, N-Me-His, Arg, Lys, homo-Arg, diethyl-homo-Arg, Lys-epsilon-NH- -R (where R is H, a straight or branched chain Ci-Cio alkyl group, or an aryl group), or Orn; A27 is an aromatic amino acid other than Tyr; A28 is Leu, Val, Trp, Nle, Nva, Aib, Anb, or N-Me-Leu; A29 is Asn, Ala, Gln, Gly, Trp, or N-Me-Asn; A30 is Leu, Lie, Val, Trp, Nle, Nva, Aib, Anb, or N-Me-Leu; A31 is Val, Leu, He, Trp, Nle, Nva, Aib, Anb, or N-Me-Val; A32 is Thr, Ser, N-Me-Ser, N-Me-Thr, or D-Trp; A33 is Cys, Arg, Lys, homo-Arg, diethyl-homo-Arg, Lys-epsilon-NH-R (where R is H, a Ci-Cio alkyl group, straight or branched chain or C6-Cis aryl group) ), or Orn; A54 is Cys, Gln, Asn, Ala, Gly, N-Me-Gin, Aib, or Anb; A35 is Cys, Arg, Lys, homo-Arg, diethyl-homo-Arg, Lys-epsilon-NH-R (where R is H, a straight or branched chain C? -C? Alkyl group, or aryl group) Cß-Ciß), or Orn; A36 is an aromatic amino acid, or Cys; R 3 is H, C 1 -C 2 alkyl, C 1 -C 18 aryl, C 1 -C 2 acyl, C 7 -C 8 aralkyl, or C 7 -C 8 alkaryl; R 4 is H, C 1 -C 2 alkyl, C 6 -C 8 aryl, C 1 -C 2 acyl, C 7 -C 8 aralkyl, or C -C 8 alkaryl, or a pharmaceutically acceptable salt thereof . Examples of peptide analogue aPYY (22-36) of Formula IB include: N-alpha-Ac-Tyr-Ser-Leu-Arg-His-Phe-Leu-Asn-Leu-Val- Thr-Arg-Gln-Arg-Tyr-NH2 (SEQ ID NO: 20), or a pharmaceutically acceptable salt thereof. Additional analogs of Formula IB can be used, wherein A27 is Phe, Nal, Beep, Pcp, Tic, Trp, Bth, Thi, or Dip. Another example of the peptide analogue aPYY (22-36) of Formula B includes: N-alpha-Ac-Phe-Ser-Leu-Arg-His-Phe-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg -Tyr-NH2 (SEQ ID NO: 21). Formula 2 R, R, For Formula 2 the following PYY peptide analogs (25-36) can be created where: Ri and R2 is bonded to the nitrogen atom of the alpha-amino group of the N-terminal amino acid; R 1 is H, C 1 -C 2 alkyl, C 6 -C 8 aryl, C 1 -C 2 acyl, C 7 -C 8 aralkyl, or C 7 -C 8 alkaryl; R 2 is H, C 1 -C 2 alkyl, C 6 -C 8 aryl, C 1 -C 2 acyl, C 7 -C 8 aralkyl, or C 7 -C 8 alkaryl; A25 is Arg, Lys, homo-Arg, diethyl-homo-Arg, Lys-epsilon-NH-R (wherein R is H, a straight or branched chain C? -C? 0 alkyl group, or an aryl group), Orn, or is deleted; A26 is Ala, His, Thr, 3-Me-His, beta-pirozolylalanine, N-Me-His, Arg, Lys, homo-Arg, diethyl-homo-Arg, Lys-epsilon-NH-R (where R is H, a C? -C? Or straight or branched chain alkyl group, or an aryl group), or Orn; A27 is an aromatic amino acid; A28 is Leu, Val, Trp, Nle, Nva, Aib, Aib, Anb, or N-Me-Leu; A29 is Asn, Ala, Gln, Gly, Trp, or N-Me- Asn; A30 is Leu, He, Val, Trp, Nle, Nva, Aib, Anb, or N-Me-Leu; A31 is Val, Leu, He, Trp, Nle, Nva, Aib, Anb, or N-Me-Val; A32 is Thr, Ser, N-Me-Ser, N-Me-Thr, or D-Trp; A33 is Arg, Lys, homo-Arg, diethyl-homo-Arg, Lys-epsilon-NH-R (where R is H, a straight or branched C? -C? Alkyl group, or aryl group Cd-Ci? ), Cys, or Orn; A is Cys, Gln, Asn, Ala, Gly, N-Me-Gin, Aib, or Anb; A35 is Arg, Lys, homo-Arg, diethyl-homo-Arg, Lys-epsilon-NH-R (where R is H, a straight or branched chain C? -C? Alkyl group, or C6 aryl group) C? 8), Cys, or Orn; A36 is an aromatic amino acid, or Cys; R 3 is H, C 1 -C 2 alkyl, C 6 -C 8 aryl, C x C 2 acyl, C 7 -C aralkyl 8, or C 7 -C 8 alkaryl; and R4 is H, C? -C? 2 alkyl, C? -Ci aryl, C? -C? acyl, C7-C? aralkyl, or C7-C? 8 alkaryl, or a pharmaceutically acceptable salt thereof. Additional analogs may be used where A27 of Formula 2 is Phe, Nal, Beep, Pcp, Tic, Trp, Bth, Thi, or Dip. An example of a PYY analog (22-36) of Formula 2 includes: N-alpha-Ac-Arg-His-Phe-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-TYr-NH2 (SEQ. ID NO: 22), or a pharmaceutically salt - acceptable of them. The additional analogues of the Formula 2 including: Where the bond --CO - NH-- between residues A28 and A29, A29 and A30, A30 and A31, A31 and A32, A32 and A33, A33 and A34, A34 and A35, or A35 and A36 is replaced with CH2-NH, CH2-S, CH2-CH2, or CH2-0. In addition, analogs can include dimeric compounds comprising either two peptides of Formula IA, Formula IB, or Formula 2, or a peptide of the Formula IA and a peptide of the Formula IB, or a peptide of the Formula IA and a peptide of the Formula 2, or a peptide of the Formula IB and a peptide of the Formula 2; wherein said dimer is formed by either an amide bond or a disulfide bridge between said two peptides. Abbreviations include Asp = D = Aspartic Acid; Ala = A = Alanine; Arg = R = Arginine; Asn = N = Aspargin; Cys = C = Cysteine; Gly = G = Glycine; Glu = E = Glutamic Acid; Gln = Q = Glutamine; His = H = Histidine; Ile = I = Isoleucine; Leu = L = Leucine; Lys = K = Lysine; Met = M = Methionine; Phe = F = phenylalanine; Pro = P = Proline; Ser = S = Serine; Thr = T = Threonine; Trp = = Triptofan; Tyr = Y = Tyrosine; Val = V = Valine; 0rn = 0rnitine; Nal = 2-naphthylalanine; Nva = Norvaline; Nle = Norleucine; Thi = 2-thienylalanine; Pcp = 4-chlorophenylalanine; Bth = 3-benzothienialanine; Beep = 4,4'- biphenylalanine; Tic = tetrahydroisoquinoline-3-carboxylic acid; Aib = aminoisobutyric acid; Anb = alpha-aminonormalbutyric acid; Dip = 2, 2-diphenylalanine; and Thz = 4-thiazolylalanine (U.S. Patent No. 5, 604,203). The analogs described in the U.S. Patent. No. 5,574,010 include the following: Formula 3 R? Analogs of Formula 3 where X is a chain of 0-5 amino acids, inclusive, the one of terminal N of which is linked to Ri and R2; And it is a chain of 0-4 amino acids, inclusive, the one of terminal C of which is linked to R3 and R4; R 1 is H, C 1 -C 2 alkyl (eg, methyl), C 6 -C 8 aryl (eg, phenyl, naphthalene acetyl), C 1 -C 2 acyl (eg, formyl, acetyl, and myristoyl), C 7 aralkyl C? 8 (eg, benzyl), or alkaryl CC? 8 (eg, p-methylphenyl); R 2 is H, C 1 -C 2 alkyl (eg, methyl), C 1 -Cis aryl (eg, phenyl, naphthalene acetyl), C 1 -C 2 acyl (eg, formyl, acetyl, and myristoyl), C 7 aralkyl C? S (eg, benzyl), or C7-C? Alkaryl (eg, p-methylphenyl); A22 is an aromatic amino acid, Ala, Aib, Anb, N-Me-Ala, or is deleted; A23 is Ser, Thr, Ala, N-Me-Ser, N-Me-Thr, N-Me-Ala, or is deleted; A24 is Leu, He, Vat, Trp, Gly, - Aib, Anb, N-Me-Leu, or is deleted; A25 is Arg, Lys, homo-Arg, diethyl-homo-Arg, Lys-epsilon-NH-R (where R is H, a straight or branched chain C1-C10 alkyl group, or an aryl group), Orn, or It is eliminated; A26 is His, Thr, 3-Me-His, 1-Me-His, beta-pirozolylalanine, N-Me-His, Arg, Lys, homo-Arg, diethyl-homo-Arg, Lys-epsilon-NH-R ( where R is H, a straight or branched chain C1-C10 alkyl group, or an aryl group), Orn, or is deleted; A27 is an aromatic amino acid other than Tyr; A is Leu, He, Vat, Trp, Aib, Aib, Anb, or N-Me-Leu; A29 is Asn, Ala, Gln, Gly, Trp, or N-Me-Asn; A30 is Leu, He, Val, Trp, Aib, Anb, or N-Me-Leu; A31 is Vat, He, Trp, Aib, Anb, or N-Me-Val; A32 is Thr, Ser, N-Me-Set, or N-Me-Thr; R3 is H, C? -C? 2 alkyl (eg, methyl), C6-C? Aryl (eg, phenyl, naphthalene acetyl), C? -C12 acyl (eg, formyl, acetyl, and myristoyl), C7- aralkyl C 8 (eg, benzyl), or C 7 -C 8 alkaryl (eg, p-methylphenyl); R 4 is H, C 1 -C 2 alkyl (eg, methyl), Cg-C 8 aryl (eg, phenyl, naphthalene acetyl), C 1 -C 12 acyl (eg, formyl, acetyl, and myristoyl), C 7 aralkyl C 8 (eg, benzyl), or C 7 -C 8 alkaryl (eg, p-methylphenyl), or a pharmaceutically acceptable salt thereof. Particularly preferred analogs of Formula 3 include: N-. alpha. -Ala-Ser-Leu-Arg-His-Trp-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-NH2 (SEQ.ID.NO:23). Formula 4 R2 R * Another peptide analog YY is Formula 4 where the N-terminal amino acid is linked to Ri and R2; And it is a chain of 0-4 amino acids, including the one of terminal C from which it binds to R3 and R4; Ri is H, C? -C? 2 alkyl, C6-C? 8 aryl, C-C12 acyl, C7-C? Aralkyl, or C7-C? R 2 is H, C 1 -C 12 alkyl, C 6 -C 8 aryl, C 1 -C 2 acyl, C 7 -C 18 aralkyl, or C 7 -C 8 alkaryl; A25 is Arg, Lys, homo-Arg, diethyl-homo-Arg, Lys-epsilon-NH-R (where R is H, a straight or branched chain C? -C? Alkyl group, or an aryl group), Orn, or is deleted; A26 is Ala, His, Thr, 3-Me-His, 1-Me-His, beta-pirozolylalanine, N-Me-His, Arg, Lys, homo-Arg, diethyl-homo-Arg, Lys-epsilon-NH- R (where R is H, a straight or branched chain C? -C? Alkyl group, or an aryl group), Orn or is deleted; A27 is an aromatic amino acid; A28 is Leu, He, Val, Trp, Aib, Anb, or N-Me-Leu; A29 is Asn, Ala, Gln, Gly, Trp, or N-Me-Asn; A30 is Leu, He, Val, Trp, Aib, Anb, or N-Me-Leu; A31 is Val, He, Trp, Aib, Anb, or N-Me-Val; A32 is Thr, Set, N-Me-Set, or N-Me-Thr or D-Trp; R 3 is H, C -C 2 alkyl, C 6 -C 8 aryl, C 1 -C 2 acyl, C 7 -C 18 aralkyl, or C 7 -C 18 alkaryl; and R 4 is H, C 1 -C 2 alkyl, C 6 -C 8 aryl, C 1 -C 12 acyl, C 7 -C 18 aralkyl, or C 7 -C 8 alkaryl, or a pharmaceutically acceptable salt thereof. - - same. Note that, unless otherwise indicated, for all YY peptide agonists described herein, each amino acid residue, eg, Leu and A1, represents the structure of NH-C (R) H-CO- -, in which R is the side chain. The lines between the amino acid residues represent peptide bonds that bind the amino acids. Also, where the amino acid residue is optically active, it is the proposed L-shape configuration unless the D-shape is expressly designated. Abbreviations: Aib = aminoisobutyric acid; Anb =. alpha - aminonormalbutyric acid; Bip = 4,4'-biphenylalanine; Bth = 3-benzothienialanine; Dip = 2,2-diphenylalanine; Nat = 2-naphthylalanine; Orn = Ornithine; Pcp = 4-chlorophenylalanine; Thi = 2-thienylalanine; Tic = tetrahydroisoquinoline-S-carboxylic acid. (U.S. Patent No. 5, 574, 010). Examples of additional PYY synthesized analogs include: [im-DNP-His26] PYY: YPAKPEAPGEDASPEELSRYYASLR [im-DNP-His26] YLNLVTRQRY-NH2 (SEQ ID No. 24); [Ala32] PYY: ASLRHYLNLV [Ala] RQRY-NH2 (SEQ ID No. 25); [Ala23.32] PYY: A [Ala] LRHYLNLV [Ala] RQRY-NN2 (SEQ ID No. 26); [Glu28] PYY (22-36): ASLRHY [Glu] NLVTRQR and -NH2 (SEQ ID No. 27); N-alpha-Ac-PYY (22-36): N-alpha-Ac-ASLRHYLNLVTRQRY-NH2 (SEQ ID No. 28); N-alpha-Ac [p. CL. Phe. sup.26] PYY: N-alpha-Ac-A SLR [p. Cl. Phe26] YLNLVTRQR (SEQ ID No.29); N-alpha- - Ac [Glu28] PYY: N-alf a-Ac-ASLRHY [Glu] NLVTRQRY-NH2 (SEQ.

No.30); N-alpha-Ac [Phe27] PYY: N-alpha a-Ac-ASLRH [Phe] ENLVTRQR [N-Me-Tyr] -NH2 (SEQ ID No. 31); N-alpha-Ac] 8N-Me-Tyr] TYY N-alpha-Ac-ASLRHYENLVTRQR [N-Me-Tyr] -NH2 (SEQ ID No. 32); N-alpha-myristoyl-PYY (22-36): N-alpha-myristoyl-ASLRHYLNLVTRQRY-NH2 (SEQ ID No. 33); N-alpha a-naphthateacetyl-PYY (22-36): N-alpha-naphthalene acetal-ASLRHYLNLVTRQR '(SEQ ID No. 34); N-alpha-Ac [Phe27] PYY: N-alf a-Ac-ASLRH [Phe] ENLVTRQR [N-Me- TyT] --NH2 (SEQ ID No. 35); N-alpha a-Ac-PYY (22-36): N-alpha-Ac-ASLRHYLNLVTRQRY-NH2 (SEQ ID No. 36); N-alpha a-Ac- [Bth27] PYY (22-36): N-alpha-Ac-ASLRH [Bth] LNLVTRQRY-NH2 (SEQ ID No. 37); N-alpha-Ac- [Bip27] PYY (22-36): N-alf a-Ac-ASLRH [Bip] LNLVTRQRY - NH2 (SEQ ID No. 38); N-alpha a-Ac- [Nal27] PYY (22-36): N-alpha-Ac-ASLRH [NaL] LNLVTRQRY-NH2 (SEQ ID No. 39); N-alpha-Ac- [Trp27] PYY (22-36): N-alpha-Ac-ASLRH [Trp] LNLVTRQRY-NH2 (SEQ.

ID No.40); N-alf a-Ac- [Thi27] PYY (22-36): N-alf a-Ac-ASLRN [Thi] LNLVTRQRY-NH2 (SEQ ID No. 41); N-alf a-Ac- [Tic27] PYY (22-36): N-alpha-Ac-ASLRH [Tic] LNLVTRQRY-NH2 (SEQ ID No. 42); N-alpha-Ac- [Phe27] PYY (25-36): N-alpha-Ac-H [Phe] LNLVTRQRY-H2 (SEQ ID No. 43); N-alpha-Ac- [Phe27, Thi36] PYY (22-36): N-alpha-Ac-ASLRH (Phel LNLVTRQR [Thi] -NH2 (SEQ ID No. 4); N-alpha-Ac- [Thz26, Phe27] PYY (22-36): N-alf a-Ac-ASLR [Thz] [Phe] LNLVTRqRY- NH2 (SEQ ID No. 5); N-alf a-Ac. [Pcp27] PYY (22-36): N-alpha-Ac-ASLRH [Pcp] LNLVTRQRY-NH2 (SEQ ED No.46); N-alpha-Ac- [Ph22 * 27] PYY (22-36): N-alpha-Ac- [Phe] SLRN [Phe] LNLVTRQRY- NH2 (SEQ ID No. 47); N-alpha-Ac- [Tyr22, Phe27] PYY (22-36): N-alpha-Ac- [Tyr] SLRH [Phe] LNLVTRQRY-NH2 (SEQ ID No. 8); N-alpha-Ac- [Trp28] PYY (22-36): N-alpha-Ac-ASLRHY [Trp] NLVTRQRY-NH2 (SEQ ID No. 49); N-alpha-Ac- [Trp28] PYY (22-36): N-alpha-Ac-ASLRHYLN [Trp] VTRQRY-NH2 (SEQ ID No.50); N-alpha-Ac- [Ala.sup.26, Phe27] PYY (22-36): N-alpha-Ac-ASLR [Ala] [Phe] LNLVTRQRY-NH2 (SEQ ID No. 51); N-alpha-Ac- [Bth27] P? Y (22-36): N-alpha-Ac-ASLRH [Bth] LNLVTRQRY-NH2 (SEQ ID No. 52); N-alpha-Ac- [Phe27] PYY (22-36): N-alpha-Ac-ASLRH [Phe] LNLVTRQRY-NH2 (SEQ ID No. 53); N-alpha-Ac- [Phe27'36] PYY (22-36): N-alpha-Ac-ASLRH [Phe] LNLVTRQR [Phe] -NH2 (SEQ ID No. 54); N-alpha-Ac- [Phe27, D-Trp32] PYY (22-36): N-alpha-Ac-ASLRH [Phe] LNLV [D-Trp] RQRY-NH2 (SEQ ID No. 55). Additional analogs are described in Balasubramaniam, et al. , Peptide Research 1: 32 (1988); Japanese Patent Application No.2, 225, 497 (1990); Balasubramaniam, et al. , Peptides 14: 1011, 1993; Grandt, et al. , Reg. Peptides 51: 151 (1994); PCT International Application No.94 / 03380. Balasubramaniam, et al. describes analogs PYY (1- 28); PYY (1-22); PYY (22-28); PYY (22-36); and PYY (27-36). Grandt, et al. treats PYY (1-36) (SEQ ID NO: 1) and PYY (3-36). The peptides described above typically bind to the Y receptors in the brain and elsewhere, especially the Y2 and / or Y5 receptors. Typically these peptides are They synthesize in pyrogen-free or endotoxin-free forms although this is not always necessary. PYY agonists include rat PYY: Tyr Pro Ala Lys Pro Glu Pro Wing Gly Glu Asp Ala Ser Pro Glu Glu Leu Ser Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr (SEQ ID NO: 56 ) and the truncated forms of amino terminal corresponding to the human; Pig PYY: Tyr Pro Wing Lys Pro Glu Wing Pro Gly Glu Asp Wing Pro Pro Glu Glu Leu Ser Arg Tyr Tyr Wing Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr (SEQ ID NO: 57) and forms truncated amino terminal corresponding to human; and Guinea Pig PYY: Tyr Pro Ser Lys Pro Glu Pro Wing Gly Ser Asp Wing Pro Pro Glu Glu Leu Wing Arg Tyr Tyr Wing Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr (SEQ ID NO: 58) and truncated amino terminal forms corresponding to the human. According to the present invention a PYY peptide also includes the free bases, acid addition salts or metal salts, such as sodium or potassium salts of the peptides, and PYY peptides that have been modified by such processes as amidation, 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 the Y2 and / or Y5 receptors.

Typically these peptides are synthesized in pyrogen-free or endotoxin-free forms although this is not always necessary. Neuropeptide agonists AND NPY is another peptide binding to the Y2 receptor. NPY peptides include NPY (1-36) full length human: Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr He Asn Leu He Thr Arg Gln Arg Tyr (SEQ ID NO: 59) as well as fragments of NPY (1-36), which have been truncated at the amino terminal. To be effective at the binding of the Y2 receptor, the NPY agonist should have at least the last 11 amino acid residues at the carboxyl terminus, ie, be compressed from NPY (26-36). Other examples of NPY agonists that bind to the Y2 receptor are NPY (3-36), NPY (4-36), NPY (5-36), -NPY (6-36), NPY (7-36), NPY (8-36), NPY (9-36), NPY (10-36), NPY (ll-36), NPY (12-36), NPY (13-36), NPY (14-36), NPY ( 15-36), NPY (16-36), NPY (17-36), NPY (18-36), NPY (19-36), NPY (20-36), NPY (21-36), NPY (22) -36), NPY (23-36), NPY (24-36), and NPY (25-36). Other NPY agonists include rat NPY: Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr He Asn Leu He Thr Arg Gln Arg Tyr (SEQ ID NO: 60) and the truncated amino terminal forms of NPY (3-36) for NPY (26 ~ 36) as in the form human Rabbit NPY: Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr He Asn Leu He Thr Arg Gln Arg Tyr (SEQ ID NO: 61) and forms truncated from amino terminal of NPY (3-36) to NPY (26-36) as in the human form; NPY of dog: Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp Wing Pro Wing Glu Asp Met Wing Arg Tyr Tyr Wing Wing Leu Arg His Tyr He Asn Leu He Thr Arg Gln Arg Tyr (SEQ ID NO: 62) and forms truncated from amino terminal NPY (3-36) to NPY (26-36) as in the human form; Pork NPY: Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp Wing Pro Wing Glu Asp Leu Wing Arg Tyr Tyr Wing Wing Leu Arg His Tyr He Asn Leu He Thr Arg Gln Arg Tyr (SEQ ID NO: 63) and forms truncated amino terminal from NPY (3-36) to NPY (26-36) as in the human form; Cow NPY: Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp Wing Pro Wing Glu Asp Leu Wing Arg Tyr Tyr Being Wing Leu Arg His Tyr He Asn Leu He Thr Arg Gln Arg Tyr (SEQ ID NO: 64) and forms truncated from amino terminal of NPY (3-36) to NPY (26-36) as in the human form; Sheep NPY: Tyr Pro Ser Lys Pro Asp Asn Pro Gly Asp Asp Ala Pro Ala Glu Asp Leu Ala Arg Tyr Tyr Be Ala Leu Arg His Tyr He Asn Leu He Thr Arg Gln Arg Tyr (SEQ ID NO: 65) and forms truncated from amino terminal of NPY (3-36) to NPY (26-36) as in the human form; and Guinea Pig NPY: Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro Wing Glu Asp Met Wing Arg Tyr Tyr Ser Wing Leu Arg His Tyr He Asn Leu He Thr Arg Gln Arg Tyr (SEQ ID NO: 66) and the truncated forms of amino terminal of NPY (3-36) to NPY (26-36) as in the human form. According to 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 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 the Y2 and / or Y5 receptors. Typically these peptides are synthesized in pyrogen-free or endotoxin-free forms although this is not always necessary. Pancreatic Peptide Pancreatic Peptide (PP) and PP agonist also bind to the Y2 receptor. Examples of PP agonists are full-length PP (l-36): Ala Ser Leu Glu Pro Glu Tyr Pro Gly Asp Asn Wing Thr Pro Glu Gln Met Wing Gln Tyr Wing Wing Glu Leu Arg Arg Tyr He Asn Met Leu Thr Arg Pro Arg Tyr (SEQ HD NO: 67) and a number of PP fragments, which are truncated at the amino terminal. To bind the Y2 receptor the PP agonist must have the last 11 amino acid residues at the carboxyl terminus, PP (26-36). Examples of other PP, which bind to the Y2 receptor, are PP (3-36), PP (4-36), PP (5- 36), PP (6-36), PP (7-36), PP (8-36), PP (9-36), PP (10-36), PP (H-36), PP (12-36) ), PP (13-36), PP (? 4-36), PP (15-36), PP (16-36), PP (17-36), PP (18-36), PP (19-36) ), PP (20-36), PP (21-36), PP (22-36), PP (23-36), PP (24-36), and PP (25-36). Other PP agonists include 00 of sheep: Ala Pro Leu Glu Pro Val Tyr Pro Gly Asp Asn Wing Thr Pro Glu Gln Met Wing Gln Tyr Wing Wing Asp Leu Arg Arg Tyr He Asn Met Leu Thr Arg Pro Arg Tyr (SEQ ID NO: 68) and the truncated forms of amino terminal of PP (3-36) to PP (26-36) as in the human form; Porcupine PP: Ala Pro Leu Glu Pro Val Tyr Pro Gly Asp Asp Wing Thr Pro Glu Met Wing Gln Tyr Wing Wing Glu Leu Arg Arg Tyr He Asn Met Leu Thr Arg Pro Arg Tyr (SEQ ID NO: 69) and truncated forms amino terminal of PP (3-36) to PP (26-36) as in the human form; Dog PP: Ala Pro Leu Glu Pro Val Tyr Pro Gly Asp Asp Ala Thr Pro Glu Gln Met Ala Gln Tyr Ala Wing Glu Leu Arg Arg Tyr He Asn Met Leu Thr Arg Pro Arg Tyr (SEQ ID NO: 70) and the truncated forms of amino terminal PP (3-36) to PP (26-36) as in the human form; Cat PP: Ala Pro Leu Glu Pro Val Tyr Pro Gly Asp Asn Wing Thr Pro Glu Gln Met Wing Gln Tyr Wing Wing Glu Leu Arg Arg Tyr He Asn Met Leu Thr Arg Pro Arg Tyr (SEQ ID NO: 71) and truncated forms of amino terminal PP (3-36) to PP (26-36) as in the human form; Cow PP: Ala Pro Leu Glu Pro Glu Tyr Pro Gly Asp Asp Wing Thr Pro Glu Gln Met Wing Gln Tyr Wing Wing Glu Leu Arg Arg Tyr He Asn Met Leu Thr Arg Pro Arg Tyr (SEQ ID NO: 72) and the truncated amino terminal forms of PP (3-36) to PP (26-36) as in the human form; Rat PP: Wing Pro Leu Glu Pro Met Tyr Pro Gly Asp Tyr Wing Thr His Glu Gln Arg Wing Gln Tyr Glu Thr Gln Leu Arg Arg Tyr He Asn Thr Leu Thr Arg Pro Arg Tyr (SEQ ID NO: 73) and forms truncated amino terminal of PP (3-36) to PP (26-36) as in the human form; Mouse PP: Wing Pro Leu Glu Pro Met Tyr Pro Gly Asp Tyr Wing Thr His Glu Gln Arg Wing Gln Tyr Glu Thr Gln Leu Arg Arg Tyr He Asn Thr Leu Thr Arg Pro Arg Tyr (SEQ ID NO: 74) and forms truncated amino terminal of PP (3-36) to PP (26-36) as in the human form; and guinea pig PP: Wing Pro Leu Glu Pro Met Tyr Pro Gly Asp Tyr Wing Thr Pro Glu Gln Met Wing Gln Tyr Glu Thr Gln Leu Arg Arg Tyr He Asn Thr Leu Thr Arg Pro Arg Tyr (SEQ ID NO: 75) and the truncated amino terminal forms of PP (3-36) to PP (26-36) as in the human form. According to 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 methods of covalent modification. These peptides typically bind to the Y receptors in the brain and elsewhere, especially the Y2 and / or Y5 receptors. Typically these peptides are synthesized in pyrogen-free or free forms of endotoxin although this is not always necessary. Analogies and Imitations of Protein and Peptide Included within the definition of biologically active proteins and peptides for use within the invention are peptides (comprised of two or more covalently linked amino acids), proteins, protein or peptide fragments, peptide analogs or protein, natural or synthetic, therapeutically or prophylactically active, and chemically modified derivatives or salts of active proteins or peptides. A wide variety of useful analogs and mimics of the Y2 receptor binding peptide are contemplated for use within the invention and can be produced and tested for biological activity according to known methods. Frequently, the peptides or proteins of the Y2 receptor binding peptide or other biologically active proteins or peptides for use within the invention, are luteins that are easily obtained by partial substitution, addition, or elimination of amino acids within a protein sequence. or peptide that occurs naturally or natively (e.g., wild type, mutant that occurs naturally, or allelic variant). Additionally, biologically active fragments of native proteins or peptides are included. Such fragments and mutant derivatives substantially retain the desired biological activity of the native proteins or peptide. At - - In case of proteins or peptides 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 exchange capacity of amino acid residues having 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 threonine; A group of amino acids having side chains containing amide is asparagine and glutamine; A group of amino acids that has aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids that has basic side chains is lysine, arginine, and histidine; and a group of amino acids that has side chains containing sulfur is cysteine and methionine. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for others. In the same way, the present invention contemplates the substitution of a polar (hydrophilic) residue such as between arginine and lysine, between glutamine and asparagine, and between threonine and serine.

Additionally, replacement of a basic residue such as lysine, arginine or histidine by another or substitution of an acidic residue such as aspartic acid or glutamic acid by another is also contemplated. Conservative amino acid substitution groups, exemplifying are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. By optimally aligning a protein or peptide analogue with a corresponding native protein or peptide, and by using appropriate assays, eg, receptor binding or adhesion protein assays, protein analogs can be readily identified to determine a selected biological activity. operable peptides for use within the methods and compositions of the invention. The operable protein and peptide analogs are typically specifically immunoreactive with antibodies raised to the corresponding native protein or peptide. Pharmacokinetic Parameters (PK) As used herein, "peak concentration (Cmax) of peptide binding to the Y2 receptor in a blood plasma", "area under concentration vs. time curve (AUC) of peptide binding to the Y2 receptor in a blood plasma "," time for maximum plasma concentration (tmax) of peptide binding to the Y2 receptor in a blood plasma "are pharmacokinetic parameters known to a person skilled in the art.

The matter. Laursen et al., Eur. J. Endocrinology 135: 309-315, 1996. The "concentration vs time curve" measures the concentration of peptide binding to the Y2 receptor in a blood serum of a subject vs. time after administration of a binding peptide dosage to the Y2 receptor to the subject either by intranasal, intramuscular, subcutaneous, or other parenteral administration route. "Cmax" is the maximum concentration of peptide binding to the Y2 receptor in the blood serum of a subject after a single dose of peptide binding to the Y2 receptor to the subject. "tmax" is the time to reach the maximum concentration of peptide binding to the Y2 receptor in a blood serum of a subject after the administration of a single dose of peptide binding to the Y2 receptor to the subject. As used herein, "area under concentration vs. time curve (AUC) of peptide binding to the Y2 receptor in 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 dosages would be detected with a probability of 90% (error ß type II = 10%). The "supply speed" or "absorption speed" is estimated by time comparison (tma) to reach the maximum concentration (Cmax). Both Cmax and tmax are analyzed using non-parametric methods. The Comparisons of the pharmacokinetics of intramuscular, subcutaneous, intravenous and intranasal administrations of peptide binding to the Y2 receptor are performed by the analysis of variance (ANOVA). For par mode comparisons a Bonferroni-Holmes sequential procedure is used to evaluate the meaning. The dose-response relationship between the three nasal doses is estimated by regression analysis. P < 0.05 is considered significant. The results are given as average values +/- SEM. Although the mechanism of absorption promotion can vary with different agents that improve the mucosal delivery 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. particular Y2 receptor or other agent that improves the supply or active. 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 barrier of the mucosa. For such agents that improve delivery to be of value within the invention, it is generally desired that any significant change in mucosal permeability be reversible within an appropriate time frame for the desired duration of drug delivery. Also, you should not there is cumulative, substantial toxicity, or any permanent damaging change induced in the mucosal barrier properties with long-term use. Stability An approach to stabilize the solid protein formulations of the invention is to increase the physical stability of purified protein, e. g. , lyophilized. This will inhibit aggregation through hydrophobic interactions as well as through covalent pathways that can increase as the protein unfolds. Stabilization 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 protein structure, function and stability is well known. Typically, proteins are relatively stable in the solid state with water in large volume removed. However, solid therapeutic protein formulations can be hydrated in storage at high humidity or during delivery of a sustained release device or composition. Protein stability generally falls with increasing hydration. Water can also play a significant role in aggregation of solid protein, for example, by increasing protein flexibility resulting in improved accessibility of reactive groups, by providing a mobile phase for reagents, and by serving as a reagent in various 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 bound water and internal protein movements are low. Above this level, water mobility and protein movements raise those of complete hydration. At one point, increased susceptibility to solid phase aggregation with increasing hydration has been observed in several systems. However, at high water content, 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. This level depends on the nature of the protein, but in general, the proteins held below its "monolayer" water cover will show stability in the higher solid state. A variety of additives, diluents, bases and delivery vehicles are provided within the invention, which effectively control the content of water to improve protein stability. These reagents and carrier materials effective 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 of solid phase of peptides and proteins mixed with them or linked thereto. Certain additives also impart significant physical stability to dry, e.g., lyophilized proteins. These additives can also be used within the invention to protect the proteins against aggregation not only during lyophilization but also during storage in the dry state. Various additional preparation methods and components, as well as specific formulation additives, are provided herein that produce formulations for mucosal delivery of proteins and peptides prone to aggregation, in which the peptide or protein is stabilized in a substantially pure form, not added using a solubilization agent. A range of components and additives are contemplated to be used within these methods and formulations. Examples of these solubilization agents are cyclodextrins (CDs), which bind Selectively 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 CD and the included protein. Such selective inhibition of protein aggregation provides additional advantages within the intranasal delivery methods and compositions of the invention. Additional agents to be used in this context include CD dimers, trimers and tetramers with variable geometries controlled by linkers that specifically block aggregation of peptides and protein. Still the solubilization agents and methods of incorporation within the invention include the use of peptides and peptide mimics to selectively block protein-protein interactions. In one aspect, the specific binding of hydrophobic side chains reported for CD multimers is extended to the proteins through the use of peptides and peptide mimics that similarly block protein aggregation. A wide range of suitable methods and anti-aggregation agents are available for incorporation into the compositions and methods of the invention. Proteinase inhibitors Another excipient that can be included in a Trans-mucosal preparation is a degrading enzyme inhibitor. The mucoadhesive-enzyme polymer inhibitor complexes that are useful within the mucosal delivery formulations and methods of the invention include, but are not limited to: Carboxymethylcellulose-pepstatin (with anti-pepsin activity); Poly (Acrylic acid) -Bo man-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 described in more detail below, certain embodiments of the invention will optionally incorporate a novel chitosan derivative or chemically modified form of chitosan. Such a novel derivative for use within the invention is denoted as a β- [1- »4] -2-guanidino-2-deoxy-D-glucose (poly-GuD) polymer. 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. The enzyme inhibitors useful for the protection of peptides and proteins biologically - - active compounds include, for example, soybean trypsin inhibitor, pancreatic trypsin inhibitor, chymotrypsin inhibitor and trypsin inhibitor, and chymotrypsin isolated from potato tuber (Solanum tuberosum L.). A combination or mixtures of inhibitors may be employed. Additional inhibitors of proteolytic enzymes for use within the invention include ovomucoid enzyme, gabaxate mesylate, alfal-antitrypsin, aprotinin, amastatin, bestatin, puromycin, bacitracin, leupepsin, alpha2-macroglobulin, pepstatin and soybean trypsin inhibitor and egg white These and other inhibitors can be used alone or in combination. The inhibitor (s) can (are) incorporated in or attached to a vehicle, eg, a hydrophilic polymer, coated on the surface of the dosage form that is to contact the nasal mucosa, or incorporated into the surface phase of the surface, 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 react to introduce ethylenic unsaturation necessary to copolymerization with hydrogel forming monomers), and (c) the number of lectin groups, such as glycosides, which are present in the inhibitory molecule. It may also depend on the specific therapeutic agent that it is proposed to administer. 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 to 5 mg / ml of the formulation (ie, a separate protease inhibitor formulation or formulation combined with the inhibitor or biologically active agent). In the case of trypsin inhibition, suitable inhibitors can be selected from, eg, aprotinin, BBI, soybean trypsin inhibitor, chicken ovomucoid, chicken ovoinhibitor, human pancreatic trypsin inhibitor, camostate mesylate, inhibitors of flavonoid, antipain, leupeptin, p-aminobenzamidine, AEBSF, TLCK (tosyllysine chloromethylacetone), APMSF, DFP, PMSF, and poly (acrylate) derivatives. In the case of chymotrypsin inhibition, suitable inhibitors can be selected from, eg, aprotinin, BBI, soybean trypsin inhibitor, chymostatin, benzyloxycarbonyl-Pro-Phe-CHO, FK-448, chicken ovoinhibitor, acid complexes biphenylboronic of sugar, DFP, PMSF, β-phenylpropionate, and poly (acrylate) derivatives. In the case of elastase inhibition, suitable inhibitors can be selected from, eg, elastatinal, methoxy succinyl, Ala, Ala-Pro-Val-chloromethylketone (SQE ID NO: 76) (MI / CMK), BBI, trypsin inhibitor of soybean seed, 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 more detail below, the immobilization of these adjunct agents to matrices or other delivery vehicles, or development of chemically modified analogs, can be easily implemented to reduce or even eliminate the toxic effects, when found. Among this broad group of candidate enzyme inhibitors to be used within the invention are organophosphorus inhibitors, such as diisopropylfluorosphate (DFP) and phenylmethylsulfonyl fluoride (PMSF), which are potent, irreversible inhibitors of serine proteases (eg, trypsin and chymotrypsin). The additional inhibition of acetylcholine esterase by these compounds makes them highly toxic in uncontrolled supply facilities. Another candidate inhibitor, 4- (2-Aminoethyl) fluoride - Benzenesulfonyl (AEBSF), has an inhibitory activity comparable to DFP and PMSF, but is markedly less toxic. (4-Aminophenyl) -methanesulfonyl fluoride hydrochloride (APMSF) is another potent trypsin inhibitor, but it is toxic in uncontrolled facilities. In contrast to these inhibitors, 4- (4-isopropylpiperadinocarbonyl) phenyl 1,2,3,4-tetrahydro-l-naphthoate methanesulfonate (FK-448) is a low toxic substance, which represents a potent and specific inhibitor of chymotrypsin. Additional representatives of this nonprotein group of inhibitor candidates, and also showing 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 amino acids and modified amino acids that interfere with enzymatic degradation of specific therapeutic compounds. For use in this context, amino acids and modified amino acids are substantially non-toxic and can be produced at a low cost. However, due to their low molecular weight and good solubility, they are easily diluted and absorbed in mucosal environments. However, under appropriate conditions, amino acids can act as reversible inhibitors, competitive protease enzymes. Certain modified amino acids may display 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, while they are generally selected to have a much higher affinity for the active site of an enzyme than the substrate itself. The transition state inhibitors are competitive, reversible inhibitors. Examples of this type of inhibitor are 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 that is believed to resemble the transition state of peptides during their hydrolysis by aminopeptidases. These amino acid derivatives are potent and reversible inhibitors of aminopeptidase 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 strong protease inhibitory activity has been reported is N-acetylcysteine, which inhibits the enzymatic activity of aminopeptidase N. This adjunct agent also displays mucolytic properties that can be employed within the methods and compositions of the invention. invention to reduce the effects of the mucosal 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 peptides and modified peptide enzyme inhibitors. An important representative of this class of inhibitors is the cyclic dodecapeptide, bacitracin, obtained from Bacillus licheniformis. In addition to these types of peptides, certain dipeptides and tripeptides display non-specific, weak inhibitory activity towards some protease. By analogy with amino acids, its inhibitory activity can be improved by chemical modifications. For example, dipeptide analogues of phosphonic acid are also "transition state" inhibitors with strong inhibitory activity towards aminopeptidases. They have been used in a reported manner to stabilize leucine enkephalin nasally administered. Another example of a transition state analogue is the modified pentapeptide pepstatin, which is a very potent inhibitor of pepsin. The structural analysis of pepstatin, by testing the inhibitory activity of several synthetic analogues, demonstrated the function characteristics of the main structure of the molecule responsible for the inhibitory activity. Another special type of modified peptide includes inhibitors with a - - aldehyde function located terminally in its structure. For example, the benzyloxycarbonyl-Pro-Phe-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. Chemical structures of additional inhibitors with a terminally located aldehyde function, eg, antipain, leupeptin, chymostatin and elastatinal, are also known in the art, since they are the structures of other known, reversible, modified peptide inhibitors, such as phosphorus starch. , bestatin, puromycin and amastatin. Due to their comparably high molecular mass, polypeptide protease inhibitors are more docile than small compounds to the concentrated supply in a drug-carrier matrix. Additional agents for protease inhibition within the formulations and methods of the invention include the use of composition agents. These agents mediate inhibition of enzyme by depriving the intranasal environment (or therapeutic or preparative composition) of divalent cations, which are co-factors for many proteases. For example, composition agents EDTA and DTPA as adjunct agents administered in a coordinated manner or formulated in a combinatorial manner, in adequate concentration, will be sufficient to inhibit selected proteases to thereby improve 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, due to their propensity to divalent chelate cations, these and other compositional agents are useful within the invention as direct, absorption-promoting agents. As seen in more detail elsewhere herein, it is also contemplated to use various polymers, particularly mucoadhesive polymers, as enzyme inhibitors within coordinated administration, multi-processing and / or combination formulation methods and compositions of the invention. invention. For example, poly (acrylate) derivatives, such as poly (acrylic acid) and polycarbophil, can affect the activity of several proteases, including trypsin, chymotrypsin. The inhibitory effect of these polymers can also be based on the composition of divalent cations such as Ca2 + and Zn2 +. It is further contemplated that these polymers can serve as vehicles or conjugated partners for additional enzyme inhibiting agents, as described above. For example, a chitosan-EDTA conjugate has been developed and is useful within the invention which shows a strong inhibitory effect towards the enzymatic activity of zinc-dependent proteases. The mucoadhesive properties of polymers following the covalent binding of other enzyme inhibitors in this context is not expected to be substantially compromised, nor is the general utility of such polymers as a delivery vehicle for biologically active agents within the invention that are expected to decrease. In contrast, the reduced distance between the delivery vehicle and mucosal surface provided by the mucoadhesive mechanism will minimize the presystemic metabolism of the active agent, while the covalently bound enzyme inhibitors remain concentrated at the drug delivery site, minimizing the effects of dilution unwanted inhibitors as well as toxic and other secondary effects caused by them. In this way, the effective amount of a coordinately administered enzyme inhibitor can be reduced due to the exclusion of dilution effects. Exemplary mucoadhesive polymer-enzyme inhibitor complexes that are useful within the mucosal 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 (anti-trypsin, anti-chymotrypsin, anti-elastase). Mucosal Supply The mucosal delivery formulations of the present invention comprise Y2 receptor binding peptide, analogs and mimics, 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 the other ingredients of the formulation and not producing an unacceptable harmful effect on the subject. Such vehicles are described herein or are otherwise well known to those skilled in the art of pharmacology. Desirably, the formulation should not include substances such as enzymes or oxidizing agents with which the biologically active agent to be administered is known to be incompatible. The formulations can be prepared by any of the methods well known in the art of pharmacy. Within the compositions and methods of the invention, the Y2 receptor binding peptide proteins, analogs and mimics, and other biologically active agents described herein may be administered to subject by a variety of modes of mucosal administration, including oral, rectal, vaginal, intranasal, intrapulmonary, or transdermal, or by topical delivery to eyes, ears, skin, or other mucosal surfaces. Optionally, Y2 receptor binding peptide proteins, analogs and mimics, and other biologically active agents described herein may be administered in a coordinated or adjunct manner by non-mucosal pathways, including intramuscular, subcutaneous, intravenous, intramuscular arterial, intra-articular, intraperitoneal, or parenteral. In other alternative embodiments, the biologically active agent (s) can be administered ex vivo by direct exposure to cells, tissues and organs originating from a mammalian subject, for example, as a component of a treatment formulation of the ex vivo organ or tissue containing the biologically active agent in a suitable vehicle, liquid or solid. The compositions according to the present invention are often administered in an aqueous solution such as a pulmonary or nasal spray and can be distributed in the form of a spray by a variety of methods known to those skilled in the art. Preferred systems for distributing liquids such as a nasal spray are described in the U.S. Patent. No. 4,511,069. The formulations can - 41 presented in multi-dose containers, for example, in the sealed distribution system described in the U.S. Patent. No. 4,511,069. Additional aerosol delivery forms may include, eg, piezoelectric, ultrasonic, jet, and compressed air nebulizers, which deliver the biologically active agent dissolved or suspended in a pharmaceutical solvent, eg, water, ethanol, or a mixture thereof. . The pulmonary or nasal spray solutions of the present invention typically comprise the drug or drug to be delivered, optionally formulated with a surface active agent, such as a nonionic surfactant (e.g., polysorbate-80), and one or more regulators. In some embodiments of the present invention, the nasal spray solution further comprises an impeller. The pH of the nasal spray solution is optionally between about pH 3.0 and 6.0, preferably 4.5 ± 0.5. Regulators suitable for use as described above or as otherwise known in the art. Other components may be added to improve or maintain chemical stability, including preservatives, surfactants, dispersants, or gases. Suitable preservatives include, but are not limited to, phenol, methyl paraben, paraben, m-cresol, thiomersal, chlorobutanol, benzylalconium chloride, and the like. Suitable surfactants include, but are not limited to, oleic acid, sorbitan trioleate, polysorbates, lecithin, phosphatidyl hills, 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, usually lyophilized, of an appropriate particle size, or within a range of appropriate particle size, for delivery intranasal Minimal particle size appropriate for deposition within the nasal or pulmonary passages is frequently approximately 0.5 μ of average mass mean aerodynamic diameter (MMEAD), commonly about 1 μMMEAD, and more typically about 2 μMMEAD. The maximum particle size suitable for deposition within the nasal passages is often about 10 μMMEAD, commonly about 8 μMMEAD, and more typically about 4 μMMEAD. Intranasally 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 dry powders of appropriate MMEAD can be administered to a patient through a conventional dry powder inhaler (DPI), which are based on the breath of the patient, on nasal or pulmonary inhalation, to disperse the powder in a aerosilized amount. Alternatively, the dry powder may be administered through air-assisted devices that use an external energy source to disperse the powder in an aerosolized amount, e.g., a piston pump. To formulate compositions for mucosal delivery within the present invention, the biologically active agent can be combined with various pharmaceutically acceptable additives, as well as a base or vehicle for 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), adsorption inhibitors (eg, Tween 80), agents that improve solubility (eg, cyclodextrins and derivatives of the same), stabilizers (eg, serum albumin) and reducing agents (eg, glutathione) may be included. When the composition for mucosal delivery is a liquid, the tonicity of the formulation, as measured with reference to the tonicity of 0.9% (w / v) of physiological saline taken as a unit, is typically adjusted to a value at which no damage of Irreversible, substantial tissue will be induced in the nasal mucosa at the site of administration. Generally, the tonicity of the solution is adjusted to a value of about 1/3 to 3, 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 capacity to disperse the active agent and any desired additive. The base can also be selected from a wide range of suitable vehicles, including but not limited to, polycarboxylic acid copolymers or salts thereof, carboxylic anhydrides (e.g., 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 non-toxic metal salts thereof. Frequently, a biodegradable polymer is selected as a - base or vehicle, 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, can be employed as carriers. The hydrophilic polymers and other vehicles can be used alone or in combination, and the improved structural integrity can be imparted to the vehicle by partial crystallization, ionic bonding, degradation and the like. The vehicle can be provided in a variety of ways, including, viscous or fluid 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 promotion of absorption of the biologically active agent. The compositions of the invention may alternatively contain as pharmaceutically acceptable carriers, substances as required to approximate physiological conditions, such as pH adjustment and regulating agents, tonicity adjusting agents, wetting agents and the like, for example, sodium, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. For solid compositions, conventional non-toxic pharmaceutically acceptable carriers can be used, which include for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, magnesium carbonate, and the like. The therapeutic compositions for administering the biologically active agent can also be formulated as a solution, microemulsion, or other ordered structure suitable for high concentration of active ingredients. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity for solutions can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of a desired particle size in the case of dispersible formulations, and by the use of surfactants. In certain embodiments of the invention, the biologically active agent is administered in a time release formulation, 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 prolonged supply of the active agent, in various compositions of the invention may originate by including in the composition agents delaying absorption, for example, gelatin and aluminum monostearate hydrogels. When controlled release formulations of the biologically active agent are desired, the controlled release binders for use in accordance with the invention include any biocompatible controlled release material which is inert to the active agent and which is capable of incorporating the biologically active agent. Numerous such materials are known in the art. Useful controlled release binders are materials that are metabolized slowly under physiological conditions after their intranasal delivery (e.g., at the nasal mucosal surface, or in the presence of bodily fluids after transmucosal delivery). Suitable binders include but are not limited to biocompatible polymers and copolymers previously used in the art in sustained release formulations. Such biocompatible compounds are non-toxic and inert to surrounding tissues, and do not activate significant adverse side effects such as nasal irritation, immune response, inflammation, or the like. They are metabolized into metabolic products that are also compatible and eliminated - easily from the body. 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 in 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 methods of preparation include vacuum drying and lyophilization which produces a powder of the active ingredient plus any additional desired ingredient of a previously sterile filtered solution thereof. The prevention of the action of microorganisms can be carried out by various antibacterial and antifungal agents, for example, parabens, chlorbutanol, phenol, sorbic acid, thimerosal, and the like. The mucosal administration according to the invention allows the effective self-administration of the treatment by patients, stipulating that sufficient precautions be placed to control or monitor the dosage and side effects. Mucosal administration also overcomes certain disadvantages of other forms of administration, such as injections, which are painful and expose the patient to possible infections and may present problems of drug bioavailability. For pulmonary and nasal delivery, systems for controlled aerosol distribution of therapeutic liquids such as a spray are well known. In one embodiment, the measured doses of active agent are delivered by means of a specially constructed mechanical pump valve, US Pat. No. 4,511,069. Dosage For treatment and prophylactic purposes, the biologically active agent (s) described herein may be administered to the subject in a single bolus delivery, or in a repeated administration procedure. (e.g., for one hour, daily or weekly, repeated administration procedure). In this context, a therapeutically effective dosage of PYY may include repeated doses within a prolonged prophylaxis or treatment regimen that will produce clinically meaningful results to alleviate one or more symptoms or detectable conditions associated with an objective condition or disease as set forth above. The determination of effective dosages in this context is typically based on animal model studies followed by clinical trials in humans and is guided by determining effective administration and dosing procedures that significantly reduce the occurrence or severity of disease conditions or symptoms in the subject. The right models in - - this aspect includes, for example, murine, rat, porcine, feline, non-human primate, and other accepted animal model subjects known in the art. Alternatively, effective dosages can be determined using in vitro models (e.g., histopathological and immunological assays). Using such methods, only ordinary adjustments and calculations are typically required to determine an appropriate dose and concentration to deliver 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 current dosage of biologically active agents will of course vary according to factors such as the indication of the disease and the particular condition of the subject (eg, factors of susceptibility, degree of symptoms, complexion, size, age, etc. of the subject), time and route of administration, other drugs or treatments that are administered concurrently, as well as the specific pharmacology of the biologically active agent (s) to produce the biological response or activity desired in the subject. Dosage regimens can be adjusted to provide an optimal therapeutic or prophylactic response. A therapeutically effective amount it is also one in which any harmful or toxic side effect of the biologically active agent is excessively in clinical terms by therapeutically beneficial effects. A non-limiting range for a therapeutically effective amount 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 is administered at a sufficiently high dose to promote satiety but low enough so as not to induce any undesired side effects such as nausea. A preferred intranasal dose of PYY3.36 is about 1 μg -10 μg / kg by weight of the patient, more preferably from about 1.5 μg / kg to about 6 μg / kg by weight of the patient. In a standard dose a patient will receive 40 μg to 2000 μg, more preferably approximately 50 μg to 600 μg, more preferably 100 μg to 400 μg. Alternatively, a non-limiting 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 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. The Dosages within this range can be achieved by single or multiple administrations, including, e.g., multiple administrations per day, daily or weekly administrations. 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 sustained, normalized therapeutic levels of peptide binding to the Y2 receptor to maximize the clinical benefits while minimizing the risks of side effects and excessive exposure. This dose can be administered several times a day to promote satiety, preferably a half hour before a meal or when hunger occurs. The goal is to mucosally deliver an amount of the Y2 receptor binding peptide sufficient to cause the concentration of the Y2 receptor binding peptide in the plasma of an individual to mimic the concentration that would normally occur postpandrially, i.e. after the individual has finished eating. The dosage of Y2 agonists such as PYY can be varied by the physician or patient, if self-administered an excess of the counter dosage form, to maintain a desired concentration at the target site. In an alternative embodiment, the invention provides compositions and methods for delivery intranasal peptide binding to the Y2 receptor, in which the Y2 receptor binding peptide compound (s) is administered (n) repeatedly through an effective intranasal dosing regimen that includes multiple administrations of the binding peptide to the Y2 receptor to the subject during a daily or weekly schedule to maintain a high or decreased therapeutically effective pulsatile level of the Y2 receptor binding peptide during an extended dosing period. The compositions and method provide 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 and low peptide effective therapeutically pulsatile level. of binding to the Y2 receptor during an extended dosing period of 8 hours to 24 hours. The present invention also includes equipment, packages and multiple container units containing the pharmaceutical compositions described above, active ingredients, and / or means for administering the same for use in the prevention and treatment of diseases and other conditions in mammalian subjects. Briefly, these kits include a container or formulation containing one or more Y2 receptor binding peptide proteins, analogs or mimics, and / or other biologically active agents in combination with the agents - Improving the mucosal delivery described herein, formulated in a pharmaceutical preparation for mucosal delivery. The intranasal formulations of the present invention can be administered using any syringe or spray bottle. An example of a rolling bottle is the "Nasal Spray Puma w / Safety Clip, Pfeiffer SAP # 60548, which provides a dose of 0.1 ml per square and has a length of 36.05 mm depth tube. It can be purchased from Pfeiffer of America of Princeton, NJ Intranasal doses of a Y2 receptor binding peptide such as PYY may vary from 0.1 μg / kg to about 1500 μg / kg When administered as an intranasal spray, it is preferable that the particle size of the spray be 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 at a sufficiently high dose to promote satiety but low enough not to induce any undesired side effects such as nausea.A preferred intranasal dose of a Y2 receptor binding peptide such as PYY (3-36) is approximately 3 μg-10 μg / kg patient weight, more preferably about 6 μg / kg patient weight. In a standard dose a patient would receive 50 μg to 800 μg, more preferably approximately - - between 100 μg to 400 μg, more preferably 150 μg to about 200 μg. The Y2 receptor binding peptide such as PYY (3-36) is preferably administered at least ten minutes to one hour before eating to prevent nausea but no more than about twelve hours 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. 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, it is found that PYY (3-36) reduces appetite. The examples also show that the first-time post-prandial physiological levels of a PYY peptide could be achieved via an intranasal route of administration using the Y2 receptor binding peptide formulations of the present invention in which PYY (3- 36) was the peptide binding to the Y2 receptor. PYY Aerosol Nasal Administration We have discovered that PYY in the formulations described above can be administered intranasally using an aerosol or nasal spray. This is amazing - - because many proteins and peptides have been shown to be shared or denatured due to the mechanical forces generated by the actuator to produce the spray or aerosol. In this area the following definitions are useful. Aerosol - A product that is packaged under pressure and contains therapeutically active ingredients that are released in the activation of an appropriate valve system. Measured aerosol - A pressurized dosage comprised of metered dose valves, which allow the supply of a uniform amount of spray at each activation. Powdered aerosol - A product that is packaged under pressure and contains therapeutically active ingredients in the form of a powder, which are released upon activation of an appropriate valve system. Spray Aerosol - An aerosol product that uses a compressed gas as the impeller to provide the force necessary to expel the product as a wet spray; generally applicable to solutions of medicinal agents in aqueous solvents. Spray - A liquid divided minutely as by a jet of air or steam. Nasal spray drug products contain therapeutically active ingredients dissolved or suspended in solutions or mixtures of excipients in non-pressurized dispensers.

Measured Dew - A non-pressurized dosage form consisting of valves that allow the distribution of a specified amount of dew on each activation. Suspension spray - A liquid preparation containing solid particles distributed in a liquid vehicle and in the form of, of course, droplets or as finally divided solids. The dynamic characterization of aerosol spray fluid emitted by nasal spray pumps measured as a drug delivery device ("DDD"). Spray characterization is an integral part of the regulatory filings required for approval by the Food and Drug Administration ("FDA") for research and development, quality assurance and stability testing procedures for new and existing nasal spray pumps . Through the characterization of the spray geometry it has been found that it is the best indicator of the total performance of 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, uniformity and particle / droplet distribution (spray pattern); and the evolution of dew time in development has been found to be the most representative performance quantities in the characterization of a nasal spray pump. During quality assurance and stability testing, column geometry and spray pattern measurements are key identifiers to verify consistency and compliance with approved data criteria for nasal spray pumps. The following definitions apply to spray properties. Column height - the measurement from the tip of the actuator to the tip at which the angle of the pen becomes non-linear due to the interruption of the linear flow. Based on the visual examination of digital images, and to establish a measurement point for width that is consistent with the measurement point farthest from the spray pattern, a height of 30 mm is defined for this study. Main Shaft - the longest cord that can be removed within the adjusted spray pattern that crosses COMw in base units (mm). Minor Axis - the smallest bead that can be extracted within the adjusted dew pattern that crosses COMw in base units (mm). Proportion of ellipticity - the ratio of the principal axis to the minor axis. Dio _ the droplet diameter for which 10% of the total liquid sample volume consists of droplets of one smaller diameter (μm) D50 - the droplet diameter for which 50% of the total liquid sample volume consists of droplets of a smaller diameter (μm), also known as the average mass diameter. D90 - the droplet diameter for which 90% of the total liquid sample volume consists of droplets of a smaller diameter (μm). Lapse - measurement of the width of the distribution. The smaller the value, the narrower the distribution. The span is calculated as: (D90-D10). D50% RSD - percent relative standard deviation, the standard deviation divided by the series average and multiplied by 100, also known as% CV. A nasal spray device is comprised of a bottle in which the PYY formulation is placed, and an actuator, which when actuated or engaged, forces a spray column, of PYY out of the spray bottle through the actuator. The bottles may be plain glass bottles comprised of borosilicate glass Type I. The bottles may have a threaded upper part and a concave upper part. The lids can be lined with three layers of polypropylene. Three-layer WP consists of a 0.0005"transparent polyester that is bonded by 0.00067" of LDPE white to a 0.0035"aluminum foil which is bonded to a film / foam / LDPE film co-extrusion All components of this liner are GRAS The lids may be comprised of polypropylene and are appropriately threaded for use with the proposed bottles EXAMPLES EXAMPLE 1 Evaluation of In Vitro Tissue Model Model Various Formulations of PYY3-36 In vitro im- perion of PYY3_36 in the presence of several excipients (EDTA, polysorbate 80 (Tween 80), oleic acid, sorbitol and ethanol) is evaluated. The formulation is adjusted to pH 4 with 10 mM citrate buffer (citric acid / sodium citrate). Several tested formulations are presented in Table 1. All samples are clear and colorless.

Table 1 Description of the Formulations Tested in Example 1 Samples are evaluated in a tissue model in vi uro. The cell line used were normal, human-derived tracheal / bronchial epithelial cells (EpiAirway ™ Fabric Model, MatTek Corporation). The cells are provided as inserts that grow for confluence in Millipore Millicell-CM filters comprised of transparent hydrophilic Teflon (PTFE). At the reception, the membranes are cultured in 1 ml of base medium (Dulbecco's Modified Tagle Medium (DMEM) free of hydrocortisone and free - - of red phenol) at 37 ° C / 5% C02 for 24-48 hours before use. Inserts are fed for each day of recovery. Each tissue insert is placed in an individual cavity containing 1 ml of MatTek basal medium. At the apex surface of the inserts, 50 μl of test formulation are applied according to the study design in Table 1, and the samples are placed on a shaker (~ 100 rpm) for 1 h at 37 ° C. Samples of the underlying culture medium are stored at 4 ° C for up to 48 hours for lactate dehydrogenase (LDH, cytotoxicity) and sample permeation evaluations (enzyme immunoassay (EIA)). The transepithelial electrical resistance (TER) is measured before and after a 1-h incubation. After incubation, the cellular inserts are analyzed for cell viability through the mitochondrial dehydrogenase (MDH) assay. 1. Electrical Resistance Through a Single Cell Layer TER measurements are made using the Endohm-12 Tissue Resistance Measurement Chamber connected to the EVOM Epithelial Voltmeter (World Precision Instruments, Sarasota, FL) with the electrode leads. The electrodes and a blank insert of tissue culture are equilibrated for at least 20 minutes in MatTek medium with the power turned off before the calibration calibration. The above resistance is measured with 1.5 ml Medium in the chamber - Endohm tissue and 300 μl Medium in the insert in white. The upper electrode is adjusted so that it is close to, but does not contact, the upper surface of the insertion membrane. The previous resistance of the blank insert was approximately 5-20 ohms. For TER determination, 300 μl of MatTek medium is added to the insert followed by placement in the Endohm chamber. The resistance is expressed as (measured resistance - blank space) X 0.6 cm2. The results show that the negative control and medium control show no significant change in TER after one hour exposure. The Triton control shows essentially a complete reduction in TER. The samples with EDTA and ethanol showed a decrease in TER, consistent with the opening of hermetic joints. 2. Cell Viability Cell viability is assessed using the MTT assay (MTT-100, MatTek equipment). Concentrated MTT diluted and thawed is intubated (300 μl) in a 24-well plate. The tissue inserts are gently dried, placed in the plate cavities, and incubated at 37 ° C for 3 hours. After incubation, each insert is removed from the plate, grouped gently, and placed in 24-well extraction plate. The cell culture inserts are then submerged in 2.0 ml of the solution extracted by cavity (to completely cover the sample). The extraction plate is covered and sealed to reduce the evaporation of extract. After an overnight incubation at room temperature in the dark, the liquid inside each insert is decanted again in the cavity from which it is taken and the inserts are discarded. The extractor solution (200 μl in at least duplicate) is intubated in a 96-well microconcentration plate, together with extracted blanks. The optical density of the samples is measured at 550 nm in a plate reader. All the samples and the negative and medium controls showed an acceptable cellular viability, that is, at least 80% compared to the average control. The Triton X control substantially decreased cell viability, as expected. 3. Cytotoxicity The amount of cell death (cytotoxicity) is assayed by measuring the loss of lactate dehydrogenase (LDH) from the cells using a CytoTox 96 Cytotoxicity Test Kit (Promega Corp., Madison, Wl). LDH analysis of the apex apex is evaluated. The appropriate amount of medium is added to the apex surface for the total of 300 uL, taking into consideration the loading volume of the initial sample. The inserts are shaken for 5 minutes, and then 150 uL of the half apex is removed and Distribute in eppendorf tubes and centrifuge at 10,000 rpm for 3 minutes. A volume of 2 uL of the supernatant is removed and added to a 96-well plate. A volume of 48 uL of medium is used to dilute the supernatant to make a 25x dilution. For LDH analysis of the basolateral medium, 50 uL of sample is loaded in a 96-well assay plate. Cell-free, fresh culture medium is used as a blank space. Fifty microliters of substrate solution is added to each well and the plates are incubated for 30 minutes at room temperature in the dark. After incubation, 50 ul of stopping solution is added to each well and the plates are read in a plate reader of optical density at 490 nm. All samples and medium and negative controls had relatively low cytotoxicity by basolateral LDH assay, ie, no more than 20% compared to the mean control. The Triton X control had relatively high cytotoxicity, as expected. 4. Cell Permeation Equipments EIA PYY3_36 are purchased from Phoenix Pharmaceuticals, Inc. (Belmont, CA), and the assay is conducted following the instructions provided. The results of the permeation (Figure 1) showed that 10 mM EDTA or 1-2% ethanol provided substantial permeation%, ie, approximately 1.5 to 2% Perfected drug in one hour. In contrast,% very low permeation, e.g., < 0.3% is observed for the negative control (breeders without permeation, isotonic). The highest permeation% is observed for samples 11 (1 mg / ml EDTA, 1% ethanol) and 12 (10 mg / ml EDTA, 2% ethanol). The combination of EDTA and ethanol provides the reduction in TER, consistent with opening of tight junctions and increases the permeation of PYY3-36, with low acceptable cytotoxicity and high cell viability. EXAMPLE 2 In Vitro Evaluation of Various Formulations of PYY3-36 The objective was to further examine the effect of ethanol, EDTA, and Tween 80 as permeation enhancers for PYY3_36. The formulations are adjusted to pH 4 with 10 mM citrate buffer (citric acid / sodium citrate). The various formulations tested in Example 2 are presented in Table 2. In addition to these samples, a negative isotonic control, a cell culture medium control, and a Triton X control are also included.

Table 2 Description of Formulations Tested in Example 2 The various samples in Table 2 are examined for permeation of TER, MTT, LDH and PYY3-36; the methodologies for the various assays are performed as described in Example 1. The negative control and medium control did not show any significant change in TER after one hour exposure. The Triton control showed essentially a complete reduction in TER. All samples revealed a fall in TER. These data illustrate that EDTA, ethanol, Tween 80, and combinations of these excipients decreased TER, which is consistent with the opening of hermetic joints. All samples and medium and negative controls had relatively low cytotoxicity by the basolateral LDH assay, ie, only 20% toxicity compared to the mean control. The Triton X control had high relative cytotoxicity.

Data for% permeation are shown in Figure 2. The negative control had very low permeation, consistent with Example 1. The three GRAS samples showed substantially high permeation under the conditions tested. These data further confirm that EDTA, ethanol and Tween 80, and combinations of these excipients reduce TER and improve the PYY3-36 permeation, with low acceptable cytotoxicity. EXAMPLE 3 Evaluation of the In Vitro Tissue Model of Various Formulations of PYY3-36 The objective of this study was to further examine the effect of ethanol, EDTA, and Tween 80 as potential permeation enhancers for PYY3-36. The in vitro permeation of PYY3-36 in the presence of several excipients (EDTA, ethanol, Tween 80, DDPC, and methyl-beta-cyclodextrin) is evaluated. The formulations are adjusted to pH 4.2-4.3 with 10 mM citrate buffer (citric acid / sodium citrate). The various formulations tested are shown in Table 3. In addition to these samples, a negative isotonic control, a cell culture medium control, and a Triton X control are also included. Sample 3-1 contained a combination of methyl-beta-cyclodextrin (M-β-CD), DDPC, and EDTA, in a combination shown previously to provide permeation enhancement - of PYY3-36 (U.S. Patent Application No. 10 / 768,288 [Publication No. 20040209807]). The various samples in Table 3 are examined for permeation of TER, MTT, LDH and PYY3-36; the methodologies for the various tests are carried out as described in Example 1. Table 3 Description of Formulations Tested in Example 3 All samples are clear and without color. Negative and average controls show no significant change in TER after a one-hour exposure. Samples 3-1 to 3-13 all showed a substantial decrease in TER in contrast to the mean control, showing EDTA, ethanol, Tween 80 and combinations of these TER excipients decreased. The data also shows that a formulation containing methyl-beta-cyclodextrin, EDTA and DDPC decrease TER. The data for% permeation (Figure 3) show that the relatively high permeation% is shown for all samples 3-1 to 3-13. The negative control had very low permeation%, consistent with Example 1. These data further confirm that EDTA, ethanol, Tween 80, and combinations thereof provide a reduction in TER, consistent with the opening of tight junctions and PYY3 permeation. -36 increased. These formulations achieve comparable or better permeation than for previously described formulations containing EDTA, DDPC and methyl-beta-cyclodextrin (U.S. Patent Application No. 10 / 768,288). EXAMPLE 4 Evaluation of the In Vitro Tissue Model of Various Formulations of PYY3-36 The objective of this study was to further examine the effect of ethanol, EDTA, and Tween 80 as potential permeation enhancers for PYY3-36. In this example, different regulators are tested (citrate buffer, acetate buffer, and glutamate buffer), as well as different preservatives (chlorobutanol and benzalkonium chloride). Data for all formulations are compared to a formulation with methyl-beta-cyclodextrin, DDPC and EDTA.

The in vitro permeation of PYY3-36 in the presence of several excipients (EDTA, ethanol, Tween 80, DDPC, and methyl-beta-cyclodextrin) is evaluated. The formulations are adjusted to pH 4 with 10 mM citrate buffer (citric acid / sodium citrate). The various formulations tested are presented in Table 4. In addition to these samples, a negative isotonic control, a cell culture medium control, and a Triton X control are also included. Sample 4-1 contained a formulation with methyl-beta-cyclodextrin, DDPC, and EDTA, previously shown to provide PYY3_36 permeation enhancement (US patent application 10 / 768,288). Table 4 Description of Formulations Tested in Example 4 The various samples in Table 4 are examined for permeation of TER, MTT, LDH and PYY3-36; The methodologies for the various tests are carried out as described in the Example 1. In Table 4, "BZK" = 0.2 mg / ml benzalkonium chloride and "CB" = 5 mg / ml chlorobutanol. The Triton X control showed essentially a complete reduction in TER. The mean and negative control showed no change in TER. All the test samples showed a substantial decrease in TER in contrast to the negative control. The data show that the acetate and glutamate regulators can be replaced by citrate buffer in the formulation YY3-36. The data further support that preservatives such as benzalkonium chloride and chlorobutanol can be added to the formulation PYY3-36. The permeation data (Figure 4) show relatively high permeation% for all samples, and the highest permeation% was shown for 4-4, 4-6 and 4-9. These data further confirm the effect that improves the permeation of EDTA, ethanol, Tween 80, and combinations thereof. Acetate and glutamate regulators are substituted for citrate buffer without loss of permeation. In addition, benzalkonium chloride and chlorobutanol are successfully added as preservatives. These data confirm that the EDTA, ethanol, and Tween 80 formulations can achieve comparable permeation% or better than that for the formulation containing EDTA, DDPC and methyl-beta-cyclodextrin; acetate and glutamate regulators can be substituted for buffer - of citrate in the formulation of PYY3-36; and preservatives such as benzalkonium chloride and chlorobutanol can be added to the formulation of PYY3-36. EXAMPLE 5 Pharmacokinetics Test of PYY3-36 Formulations Intranasal Containers of Acetate, Ethanol and EDTA Absorber The pharmacokinetic test of PYY3-36 in various intranasal formulations is tested in mammals. The formulations included EDTA and ethanol as permeation enhancers (acetate buffer, pH 4.0). For comparison, a formulation is also dosed intranasally containing methyl-beta-cyclodextrin, DDPC, and EDTA as permeation enhancers (it is previously shown that this combination of excipients provides improved permeation of PYY3-36 (U.S. Patent Application No. / 768,288). In addition, a formulation provided with improvers is dosed to produce the potency of the permeation enhancers to improve drug delivery. The various formulations tested in Example 5 are described in Table 5. Sample 5-1 contained methyl-beta-cyclodextrin, DDPC, and EDTA as enhancers and CB as a preservative, 5-1 is dosed IN for comparison. Samples 5-2, 5-3 and 5-4 contained EDTA in either 1 or 10 - mg / ml and ethanol in either 0, 10 or 20 mg / mL, and BZK as a preservative. Sample 5-5 is formulated in buffer and exceeds permeation enhancers. Table 5 Description of Formulations Tested in Example 5 Pharmacokinetic evaluation (PK) in white rabbits from New Zealand adhered to the Principles of Care of Laboratory Animals (NIH publication 86-23, revised in 1985). Blood samples are taken from the marginal ear vein in the pre-dose and 2.5, 5, 10, 15, 30, 45, 60 and 120 min after dosing IN. The concentration of PYY3-36 in plasma is determined by EIA (Foerder C, et al., Quan ti ta íive Determination of Peptide YY3-36 in Plasma by Radioimmunoassay, AAPS 2004 National Biotechnology Conference, Boston, MA, May 2004). The PK calculations are performed using WinNonlin software (Pharsight Corporation, Version 4.0, Mountain View, CA) using a Model approach without compartment. Data are presented as average + standard error. The PK results are summarized in Table 6. Tmax by IN route was between approximately 26-43 for all samples. Sample 5-1 had higher Cmax and AUC, the latter was 27 times better compared to the case without breeders (sample 5-5). Samples 5-2 and 5-3 showed a lower standard deviation in their Cmax compared to sample 5-1. Also, sample 5-2 showed a lower standard deviation in AUCúitima compared to sample 5-1. Sample 5-2 showed almost the same AUCuitima as sample 5-1. Table 6 PK Summary of Formulations Tested in Example 5 n / d = not determined; * compared without enhancers (8-5) All groups containing combinations of EDTA and / or ethanol (samples 5-2, 5-3 and 5-4) showed substantially improved permeation compared without enhancers, up to about 20 to 25 times plus. EXAMPLE 6 - Effect of Various Regulators on Thermal Stress Stability for PYY3-36 in the Absence of Any Additional Excipient The formulations are manufactured as indicated in Table 7. Regulators tested included citrate, tartrate, acetate and glutamate. In all cases, PYY3-36 was present at 1 mg / ml and the pH was 5.0. Non-salinized amber bottles of 1c are filled with the test formulations, 1 ml of filling per bottle, and the bottles are fitted with a cover lined with three sheets. The jars are purchased from SGD Glass Inc. (New York, NY). These bottles had a threaded upper part and a concave lower part (U-shaped configuration). The bottles are comprised of Type I borosilicate glass. The caps are purchased from O 'Berk Company (Union, NJ) and are comprised of polypropylene and are appropriately threaded for use with the proposed bottles. The lids are lined with Three Sheets. Three WP Sheets consisted of a 0.0005 # clear polyester that is bonded by 0.0067"white LDPE to an 0.0035" aluminum foil that is bonded to a co-extrusion film LDPE / foam / film. All the components of the lining were GRAS (Generally Recognized as Insurance). The bottles are stored at 25, 40 and 50 ° C, and at various time points are tested by HPLC to examine the chemical stability, as reflected in the recovery 1 - . 1 - of the peptide (% peptide measured relative to the data at t = 0). The HPLC method uses a C18 5-micron column (Supelco, BIO Wide-pore, 250 x 4.6 mm) at 45 ° C with mobile phase components of 0.1% trifluoroacetic acid (A) and 0.08% trifluoroacetic acid in acetonitrile (B) supplied isocratically at 27% A / 73% B. The detection was by UV at 210 nm. The quantification is carried out by external standard method. Table 7 Formulations Evaluated in Example 6 The HPLC data showed that the best stability (highest recovery after storage under the various conditions) is achieved using the glutamate and acetate regulators. The results of the peptide recovery are shown in Figures 5A, 5B and 5C for 25, 40 and 50 ° C, respectively. The results of this experiment highlight that those regulators that best preserve the stability of PYY3-36 were monovalent regulators. , while those that do not improve the stability of PYY3-3e were regulators polyvalent Monovalent regulators probably increase the stability of PYY3-36 under thermal stress. EXAMPLE 7 Effect of Shock Type and pH on Thermal Stress Stability for PYY3-36 in the Presence of Sorbitol as Toner Formulations are manufactured as indicated in Table 8. Regulators tested were citrate, acetate, and glutamate. In all cases, PYY3-36 was present at 2 mg / ml and sorbitol was present to provide osmolarity of 225 mOsm. The pH is varied from 3.5 to 5.0. These formulations are then filled in amber non-silanized bottles of 1 cup, 1 ml of filling per bottle, and fitted with a polypropylene lid with a three-leaf liner. The samples are stored and tested for recovery as described in Example 6. 16 -. 16 - Table 8 Formulations Evaluated in Example 7 The HPLC data shows that the best performance formulations for thermal stability over the temperatures evaluated are the glutamate and acetate regulators as well as the unregulated formulations. The results of the recovery of the peptide are represented in Figures 6A, 6B, 6C and 6D for cases where the buffer was citrate, acetate, glutamate or without regulator, respectively. As in Example 6, the best performing formulations are those that contain either a monovalent buffer (i.e., acetate or glutamate) or that do not contain a buffer over the range of pH and temperatures - evaluated. Those formulations that contain a polyvalent buffer (ie, citrate) do not achieve optimal performance. In addition, it seems that the pH that maintains the optimum stability for PYY3-36 seems to be pH 3.5-pH 4.5 without considering the buffer used. EXAMPLE 8 Thermal Stability and Atomization Stress Stability for Various Formulations of PYY3-36 The objective of this study was to examine the stability against atomization and thermal stresses for PYY3-36 formulations containing ethanol, EDTA, and Tween 80 as enhancers of Potential permeation for PYY3-36. In this example, different regulators are added (acetate buffer and glutamate buffer), as well as a preservative to allow multi-use formulations ("benzalkonium chloride") The solutions are filled to ~ 3.9 ml in non-amber glass jars 3 mL silanized bottles are fixed with 100 μL actuators The actuators are purchased from Pfeiffer of America (Princeton, NJ) The bottles are primed by actuation until the first complete spray is achieved. First full spray, an additional two drives is performed to confirm full priming After the priming is complete, the next spray is considered to be the first spray from the study.

All drives are driven by hand. The bottles are stored at 30 ° C / 65% relative humidity between all sprays (during the day as well as during the night). After the removal of the camera, the bottles are sprayed (within 5 min) and then returned to the chamber. There is a minimum of 1 hour between all sprays. The bottles are sprayed three times a day (TID) for 10 days. The HPLC method is conducted as described in Example 1. Data for peptide stability are presented as recovery% (native peptide concentration relative to that initially at = 0) and% purity (native peptide peak area divided by area of all peaks associated with peptide). The HPLC data for peptide content is measured by day = 0, day = 5 and day = 10 of the exposure at high temperature and spray atomization voltage three times a day. The various formulations tested in Example 8 are described in Table 9. All samples contained 6 mg / ml PYY3-36. Samples contained 10 mg / ml EDTA, 20 mg / ml ethanol, 0.02% benzalkonium chloride (BZK) (as a representative preservative to allow multiple use), and either 0 or 1 mg / ml Tween 80. Samples 3- 1 and 3-2 contained 10 mM acetate buffer (acetic acid / sodium acetate buffer system) at pH 4.3. The sample 3-3 - contained 10 mM glutamate buffer (glutamic acid buffer system / sodium glutamate) at pH 4.3. Table 9 Description of Formulations Tested in Example 8 The HPLC data for peptide content on day = 0, day = 5 and day = 10 of the exposure at high temperature and spray atomization voltage three times a day are shown in Figure 7. The data show that in the formulations with 10 mg / ml EDTA and 20 mg / ml ethanol, the presence of 1 mg / ml Tween-80 (3-1, filled triangles) had a stabilizing effect on the same formulation - without Tween-80 (3-2, open squares ). Glutamate cushion (3-3, open diamonds) provided more stability compared to the acetate buffer (3-2, open squares). The data for purity of PYY3_36 show that the predominant species that remain in solution have the same retention time by HPLC as native PYY3-36, consistent with the loss of peptide due to aggregation. The precipitate consisted predominantly of PYY3 monomer. 36, which shows that the loss in PYY3-36 in the submission to - - atomization and thermal stresses are due to a hydrophobic aggregation (e.g., non-covalent). The PYY3-36 formulations are subjected to hydrophobic aggregation, e.g., non-covalent, on exposure to elevated temperatures combined with the spray voltage three times a day. Under certain conditions, the presence of Tween-80 improves this. Also, the data shows that glutamate may be a preferred buffer system with respect to stability compared to acetate. EXAMPLE 9 Thermal Stability and Atomization Stress Stability for Various Formulations of PYY3-36 The objective of this study was to examine the stability against atomization and thermal stresses for PYY3-36 formulations containing ethanol, EDTA, and Tween 80 as buffers. potential permeation for PYY3-.36. In this example, the acetate buffer is tested from a pH range of pH 3.8 to 4.4, and chlorobutanol is used as a preservative to allow multiple use. The methodologies employed in this example were the same as described in Example 8 above. As in Example 8, the bottles are stored at 30 ° C / 65% relative humidity between all sprays (during the day as well as during the night), the bottles are sprayed three times a day (TID) for 10 days, and HPLC is - used to determine content of PYY3-36 in the spray (last dew of the day) on day = 0, 5 and 10 of spray. The various formulations tested in this example are described in Table 10. All samples contained 6 mg / ml PYY3-36, 10 mM acetate buffer (acetic acid / sodium acetate buffer system) and 5 mg / ml chlorobutanol (CB). Samples 4-1 to 4-8 contained 10 mg / ml EDTA, 20 mg / ml ethanol, and either 0 or 1 mg / ml Tween 80, and the pH was varied from 3.8 to 4.4. For comparison, the last sample, 4-9, contained 45 mg / ml of methyl-beta-cyclodextrin, 1 mg / ml DDPC, and 1 mg / ml EDTA, pH 4.0. The latter formulation is previously described (patent application of E.U. 10/768288, Quay et al., "Compositions and methods for enhanced mucosal delivery of Y2 receptor-binding peptides and methods for treating and preventing obesity").

Table 10: Formulation Test Description in Example 9 Abbreviations: Me-ß-CD = methyl-beta-cyclodextrin EDTA = disodium edentate DDPC = didecanoil La-phosphatidylcholine HPLC data for peptide content on day = 0, day = 5 and day = 10 of exposure at high temperature and stress Spray atomization three times a day are depicted in Figure 8. The data show that the presence of 1 mg / ml Tween-80 improves the stability of PYY3-36 after combining atomization and thermal stresses (compare sample 4-1). and 4- 2 (full and open diamonds, respectively), compare sample 4-3 and 4-4 (full and open squares, respectively), compare sample 4-5 and 4-6 (full and open circles, respectively) and compare sample 4-7 and 4-8 (full and open triangles, respectively)).

There is also an improved stability tendency as the pH decreases from 4.4 to 3.8. The sample at pH 3.8 containing 1 mg / ml Tween-80 (4-1) showed almost the same stability as that for sample 4.9. The presence of 1 mg / ml Tween-80 provided stabilization towards spray and thermal stresses for PYY3-3d formulations containing 10 mg / ml EDTA and 20 mg / ml ethanol. The stability is improved as the pH decreases from 4.4 to 3.8.

EXAMPLE 10 Thermal Stability and Atomization Stress Stability for PYY3-36 Formulations The objective of this study was to examine stability against atomization and thermal stresses for PYY3-36 formulations containing ethanol, EDTA, and Tween 80 as potential permeation enhancers. for PYY3-36. In this example, the buffer was either acetate or glutamate, the pH was 4.0, and the Tween-80 level was varied from 0 to 50 mg / mL. Chlorobutanol is added as a preservative. The methods employed in this example are described in Example 8 The bottles are stored at 30 ° C / 65% relative humidity between all sprays, the bottles are sprayed TID for 10 days, and HPLC is used to determine the content of PYY3_36 (last spray of the day) the day = 0, 5 and 10 of spray. The various formulations tested in this example are described in Table 11. All samples contained 6 mg / ml PYY3-36 and 5 mg / ml chlorobutanol (CB). Samples 5-1 to 5-13 contained 1-10 mg / ml EDTA, 10-20 mg / ml ethanol, 0-50 mg / ml Tween-80, either 10 mM acetate buffer / pH 5 or 10 mM buffer of glutamate / pH 4. For comparison, the last sample, 5-14 contained 45 mg / ml methyl-beta-cyclodextrin, 1 mg / ml DDPC, and 1 mg / ml EDTA, pH 4.0.

- Table 11 Description of the Formulations Tested in Example 10 Figure 9A shows the stability data for samples 5-1, 5-2 and 5-3. The comparison of 5-1 and 5-2 reveals that addition of 1 mg / ml Tween 80 did not have an improved effect since excellent stability is achieved for both samples under the conditions tested (eg, 1 mg / ml EDTA and 10 mg / ml ethanol, pH 4). Comparison of sample 5-2 to 5-3 revealed a slightly lower stability observed for glutamate buffer v. acetate buffer under the conditions tested. Figure 9B represents the stability data for -4, 5-5, 5-6 and 5-7. All these samples contained 10 mg / ml EDTA, 20 mg / ml ethanol, and 10 mM buffer acetate / pH 4.0. These series of samples had variable levels of Tween 80, mainly from 0 to 50 mg / mL. In general, the stability observed under the conditions in Figure 9B was slightly lower compared to the samples in Figure 9A. The highest stability is observed for sample 5-7 that contained the highest level of Tween 80 (50 mg / ml) in these series. Figure 9C presents the effect of atomization and thermal stress for samples 5-4, 5-5, 5-6 and 5-7. All these samples contained 10 mg / ml EDTA, 20 mg / ml ethanol, and 10 mM glutamate buffer / pH 4.0. This series of samples had variable levels of Tween 80, mainly from 0 to 50 mg / mL. In general, the stability observed under the conditions in Figure 9C was slightly lower compared to the samples in Figure 9A and Figure 9B. The highest stability is observed for sample 5-11 which contained the highest level of Tween 80 (50 mg / mL) in this series. Finally, the stability data for samples 5-12, 5-13 and 5-14 are illustrated in Figure 9D. All these three samples show excellent stability, mainly, there is no substantial change in peptide recovery during the 10 days of exposure to atomization and thermal stress.

Claims (93)

1. - CLAIMS 1. A pharmaceutical formulation for improving the mucosal delivery of PYY to a mammal, wherein the formulation comprises a therapeutically effective amount of PYY, a polar organic solvent miscible in water and a chelating agent for cations.
2. 2. The formulation of claim 1, wherein PYY is PYY (3-36), the polar organic solvent miscible in water is ethanol and the chelating agent for cations is EDTA.
3. 3. The formulation of claim 2, wherein the ethanol is at a formula concentration of about 1% (v / v) or greater. The formulation of claim 2, wherein the ethanol is at a formula concentration of about 2% (v / v) or greater. The formulation of claim 2, wherein the ethanol is at a formula concentration of about 10% (v / v) or greater. 6. The formulation of claim 2, wherein the EDTA is at a concentration of at least about 1 mg / ml in the formulation. The formulation of claim 2, wherein the EDTA is at a concentration of at least about 2 mg / ml in the formulation. 8. The formulation of claim 2, in - - where the EDTA is at a concentration of at least about 10 mg / ml in the formulation. 9. The formulation of claim 2, further comprising a surface active agent. The formulation of claim 9, wherein the surface active agent is Tween-80. The formulation of claim 10, wherein Tween-80 is present at 50 mg / ml or less in the formulation. The formulation of claim 10, wherein Tween-80 is present at 10 mg / ml or less in the formulation. The formulation of claim 10, wherein Tween-80 is present at 1 mg / ml or less in the formulation. The formulation of claim 2, further comprising a buffer salt. 15. The formulation of claim 9, further comprising a buffer salt. 16. The formulation of claim 14, wherein the buffer salt is acetate or glutamate. The formulation of claim 15, wherein the buffer salt is acetate or glutamate. The formulation of claim 16, wherein the buffer salt is glutamate. 19. The formulation of claim 17, wherein the buffer salt is glutamate. The formulation of claim 2, wherein the pH is about 5.0 or less. The formulation of claim 2, wherein the pH is about 4.4 or less. 22. The formulation of claim 2, wherein the pH is about 4.0 or less. The formulation of claim 2, wherein the pH is about 3.8 or less. The formulation of claim 2, further comprising a preservative. The formulation of claim 24, wherein the preservative is chlorobutanol or benzalkonium chloride. The formulation of claim 2, wherein administration of the formulation by contacting a monolayer of mucosal cells results in a Papp measured of approximately 2-fold or greater compared to the Papp measured in an isotonic solution exceta permeation The formulation of claim 2, wherein administration of the formulation by contacting a monolayer of mucosal cells results in a Papp measured of approximately 5 times or greater compared to the Papp. - - measured in an isotonic solution exceeds permeation enhancers. The formulation of claim 2, wherein administration of the formulation by contacting a monolayer of mucosal cells results in a Papp measured of approximately 10-fold or greater compared to the Papp measured in an isotonic solution excenta of buffers. permeation 29. The formulation of claim 2, wherein administration of the formulation by contacting a monolayer of mucous cells results in a Papp measured of approximately 10-fold or greater compared to the Papp measured in an isotonic solution excetan of buffers. permeation 30. The formulation of claim 26, 27, 28, or 29 where the mucosal cells are bronchial epithelial cells. The formulation of claim 2, wherein the administration of the formulation intranasally in a mammal results in an AUCuitimate measurement of approximately 2-fold or greater as compared to the AUCuitimate measured for intranasal administration of an isotonic saline solution exceeding the breeders of permeation 32. The formulation of claim 2, wherein the administration of the formulation intranasally in - - a mammal results in an AUCuitima measure of about 5 times or greater compared to the AUCuitimate measured for intranasal administration of an isotonic saline solution exceeding permeation enhancers. The formulation of claim 2, wherein the administration of the formulation intranasally in a mammal results in a UCu? T measured as approximately 10-fold or greater compared to the AUCuitimate measured for intranasal administration of an isotonic saline solution. excenta of permeation enhancers. 34. The formulation of claim 2, wherein administering the formulation intranasally in a mammal results in an AUCuitimate measurement of approximately 20-fold or greater compared to the AUCuitime measured for intranasal administration of an isotonic saline solution exceeding BMP enhancers. permeation 35. A pharmaceutical formulation for improving the mucosal delivery of PYY to a mammal, wherein the formulation comprises a therapeutically effective amount of PYY, about 2% (v / v) ethanol, about 10 mM EDTA, about 1% Tween-80, and a pH of about 4.0. 36. The formulation of claim 35, further comprising a preservative, wherein the preservative is chlorobutanol or benzalkonium chloride. - - 37. The formulation of claim 36, further comprising a buffer salt, wherein the buffer salt is acetate or glutamate. 38. The formulation of claim 37, further comprising a buffer salt, wherein the buffer salt is glutamate. 39. A PYY dosage form suitable for multi-use administration comprising a sealed bottle containing an aqueous pharmaceutical formulation, wherein the formulation comprises a therapeutically effective amount of PYY, a water-miscible polar organic solvent and a chelating agent for cations, and wherein such a dosage form of PYY exhibits al. minus 90% recovery of PYY after storage for at least 10 days at 5 ° C 40. The PYY dosage form of claim 39, which has more than about 90% recovery of PYY after at least six months in Storage at 5 ° C. 41. The PYY dosage form of claim 39, which has more than about 90% recovery of PYY after one year in storage at 5 ° C. 42. The PYY dosage form of claim 39, which has more than about 90% recovery of PYY after two years in storage at 5 ° C. 43. A suitable PYY dosage form for multi-use administration comprising a bottle containing an aqueous pharmaceutical formulation and an effective driver for intranasal administration of the formulation, wherein the formulation comprises a therapeutically effective amount of PYY, a solvent polar organic miscible in water and a chelating agent for cations, and wherein such a dosage form exhibits at least 90% recovery of PYY after storage as it is used for more than about five days. 44. The PYY dosage form of claim 43, wherein the administration is three times daily. 45. The PYY dosage form of claim 44, which has more than about 90% recovery of PYY at 30 ° C / 65% relative humidity between all sprays. 46. The PYY dosage form of claims 39 and 43, further comprising a pH buffer having a single ionogenic residue net with a pKa within two pH units of the formulation pH. 47. The PYY dosage form of claim 46, wherein said shock absorber has a 1 net single ionogenic residue with a pKa within a pH unit of the formulation pH. 48. The PYY dosage form of claim 47, wherein said buffer is selected from the list consisting of glutamate, acetate, glycine, histidine, arginine, lysine, methionine, lactate, formate, and glycoate. 49. The PYY dosage form of claim 48, further comprising a glutamate or acetate buffer. 50. The PYY dosage form of claim 48, wherein the pH is about 5.0 or less. 51. The PYY dosage form of claim 48, wherein the pH is about 4.4 or less. 52. The PYY dosage form of claim 48, wherein the pH is about 4.0 or less. 53. The PYY dosage form of claim 48, wherein the pH is about 3.8 or less. 54. The PYY dosage form of claim 40 and 45, wherein PYY is PYY (3-36). 55. The dosage form of PYY of the - claim 54, wherein the concentration of PYY is at least about 20 μg / ml. 56. The PYY dosage form of claim 54, wherein the concentration of PYY is at least about 100 μg / ml. 57. The PYY dosage form of claim 54, wherein the concentration of PYY is at least about 200 μg / ml. 58. The PYY dosage form of claim 54, wherein the concentration of PYY is at least about 1 mg / ml or greater. 59. The PYY dosage form of claim 54, wherein the concentration of PYY is at least about 2 mg / ml or greater. 60. The PYY dosage form of claim 54, wherein the concentration of PYY is at least about 6 mg / ml or greater. 61. The PYY dosage form of claim 54, wherein the concentration of PYY is at least about 10 mg / ml or greater. 62. The PYY dosage form of claim 54, wherein said dosage form is suitable for intranasal administration to achieve a dose of from about 2 μg to about 1000 μg of said PYY. 63. The PYY dosage form of claim 54, wherein said dosage form is suitable for intranasal administration to achieve a dose of from about 100 μg to about 600 μg of said PYY. 64. The PYY dosage form of claim 54, wherein the polar organic solvent miscible in water is ethanol and the chelating agent for cations is EDTA. 65. The PYY dosage form of claim 64, wherein the ethanol is at a formula concentration of at least about 0.1% (v / v). 66. The PYY dosage form of claim 64, wherein the ethanol is at a formula concentration of at least about 1% (v / v). 67. The PYY dosage form of claim 64, wherein the ethanol is at a formula concentration of at least about 10% (v / v). 68. The PYY dosage form of claim 64, wherein the EDTA is at a concentration of at least about 1 mg / ml in the formulation. 69. The PYY dosage form of claim 64, wherein the EDTA is at a concentration of at least about 10 mg / ml in the formulation. 70. The PYY dosage form of claim 64, wherein the EDTA is at a concentration of at least about 50 mg / ml in the formulation. 71. The PYY dosage form of claim 64, further comprising a surface active agent. 72. The PYY dosage form of claim 71, wherein the surface active agent is Tween-80. 73. The PYY dosage form of claim 72, wherein Tween-80 is present at least about 1 mg / ml in the formulation. 74. The PYY dosage form of claim 72, wherein Tween-80 is present at least about 10 mg / ml in the formulation. 75. The PYY dosage form of claim 72, wherein Tween-80 is present at least about 50 mg / ml in the formulation. 76. The PYY dosage form of claim 64, further comprising a conservative. 77. The PYY dosage form of claim 76, wherein the preservative is chlorobutanol or benzalkonium chloride. 78. A pharmaceutical formulation for improving the mucosal delivery of a peptide binding to the Y2 receptor to a mammal, wherein the formulation comprises a therapeutically effective amount of the peptide, a water-miscible polar organic solvent and a chelating agent for cations. 79. A pharmaceutical formulation for improving the mucosal delivery of a functional analogue of PYY to a mammal, wherein the formulation comprises a therapeutically effective amount of the analog, a water-miscible polar organic solvent and a chelating agent for cations. 80. The formulation of claim 78 or 79, wherein the polar organic solvent miscible in water is ethanol and the chelating agent for cations is EDTA. 81. The formulation of claim 80, wherein the ethanol is at a formula concentration of about 1% (v / v) or greater. 82. The formulation of claim 80, wherein the EDTA is at a concentration of at least about 1 mg / ml in the formulation. 83. The formulation of claim 80, further comprising a surface active agent. 84. The formulation of claim 83, in where the surface active agent is Tween-80. 85. The formulation of claim 84, wherein Tween-80 is present at 50 mg / ml or less in the formulation. 86. The formulation of claim 80, further comprises a buffer salt. 87. The formulation of claim 80, wherein the pH is about 5.0 or less. 88. The formulation of claim 80, further comprising a preservative. 89. The formulation of claim 88, wherein the preservative is chlorobutanol or benzalkonium chloride. 90. A pharmaceutical formulation for improving the mucosal delivery of a Y2 receptor binding peptide to a mammal, wherein the formulation comprises a therapeutically effective amount of the peptide, about 2% (v / v) ethanol, about 10 mM EDTA, about 1% Tween-80, and a pH of about 4.0. 91. A pharmaceutical formulation for improving the mucosal delivery of a functional PYY analog to a mammal, wherein the formulation comprises a therapeutically effective amount of the analog, approximately 2% (v / v) ethanol, approximately 10 mM EDTA, approximately 1% Tween-80, and a pH of approximately 4.0. 92. The formulation of claim 90 or 91, further comprising a preservative, wherein the preservative is chlorobutanol or benzalkonium chloride. 93. The formulation of claim 92, further comprising a buffer salt, wherein the buffer salt is acetate or glutamate.