KR100878137B1 - Mixtures and methods for enhanced mucosal delivery of Ｙ2 receptor-binding peptides and methods for the prevention and treatment of obesity - Google Patents

Abstract

The present invention relates to pharmaceutical compositions and methods relating to at least one YY peptide mixture and one or more intranasal delivery enhancing agents for enhancing intranasal delivery of YY peptides for the treatment of various diseases and symptoms in mammalian subjects, including obesity. It is about the field. In one aspect of the invention, the intranasal delivery formulations and methods allow for enhanced delivery of the YY peptide to plasma or central nervous system (CNS) tissue or body fluids. For example, the highest concentration of YY peptide (C _max ) in the subject's plasma or CNS tissue or body fluid is determined by the subject's plasma or CNS following administration of the same concentration of PYY peptide or subcutaneous injection of PYY peptide. 20% or more when compared to the highest YY peptide concentration in tissue or body fluid.

Description

     COMPOSITIONS AND METHODS FOR ENHANCED MUCOSAL DELIVERY OF Y2 RECEPTOR-BINDING PEPTIDES AND METHODS FOR TREATING AND PREVENTING OBESITY}

[Background of invention]

Obesity and related diseases are generally very serious public health threats in the United States and around the world. Overweight is a serious risk factor for the disease known as type 2 diabetes and is an important risk factor for heart disease. Obesity is a recognized risk factor for increased incidence of genital diseases such as hypertension, arteriosclerosis, cerebral hemorrhage, seizures, gallbladder disease, osteoarthritis, heart attack, polycystic ovary syndrome, breast cancer, prostate cancer and general paralysis. It shortens life expectancy and acts as a major factor in the severe co-pathology mentioned above. It also leads to abnormalities such as infections, varicose veins, melanoma cells, eczema, maladaptation, insulin resistance, hypertension hypercholesterolemia, gallstones, orthopedic damage, and thromboembolic diseases. Obesity is also a major risk factor for a situation called "X syndrome" or insulin resistance syndrome.

Certain types of peptides that bind to Y2 receptors have resulted in weight loss when administered peripherally to mammals. The Y2 receptor-binding peptides are neuropeptides that bind to the Y2 receptor. Neuropeptides are small peptides derived from large precursor proteins synthesized by peptidic neurons and endocrine / paracrine cells. Often the precursors include peptides with various biological activities. There are a wide variety of neuropeptides in the brain, due to the selective splicing of the first gene transcription and various precursor processes. The neuropeptide receptors are provided to activate the appropriate signal and to distinguish between the ligands. These Y2 receptor-binding peptides belong to a group of peptides including YY peptide (PYY), neuropeptide Y (NPY) and pancreatic peptide (PP).

NPY is a 36-amino acid peptide and is the most abundant neuropeptide identified in the mammalian brain. NPY is an important controller in both the central and peripheral nervous systems and affects a variety of physiological variables that affect activity in psychomotor activity, food intake, central endocrine secretion, and cardiovascular system. High concentrations of NPY are found in the sympathetic nervous system supplied to coronary, cerebral, and renal vessels and cause vasoconstriction. NPY binding sites have been identified in a variety of tissues including the spleen, tabernacle, brain, aortic smooth muscle, kidney, testes, and placenta.

Neuropeptide Y (NPY) receptor pharmacology is currently defined by structural activity relationships within the pancreatic polypeptide group. This group includes NPY, which is first synthesized in neurons; PYY, which is first synthesized by endocrine cells in the digestive tract; PP, which is first synthesized by endocrine cells in the pancreas. These about 36 amino acid peptides consist of a complex helicar structure with a "PP-fold" in the central portion of the peptide. Features include polyprolineline helix at residues 1-8, beta-turn at residues 9-14, alpha-helix at residues 15-30, and C-terminal protruding out at residues 30-36. And carboxy terminal amides that appear to be important for biological activity. The peptides are defined as at least five receptor subtypes known as Y1, Y2, Y3, Y4 and Y5. Y1 receptor recognition by NPY acts on both the N-terminus and C-terminus of the peptide; In the 34th, the conversion of glutamine to proline is properly done. Y2 receptor recognition by NPY is initially dependent on the four C-terminal residues of the peptide (arginine ³³ -glutamine ³⁴ -arginine ³⁵ -tryptophan ³⁶ -NH ₂ ) and is advanced by alpha-helix; In the 34th, the conversion of glutamine to proline is not well done. One of the pharmacological properties that distinguishes Y1 from Y2 is that the Y2 receptor (not the Y1 receptor) has a high affinity for the NPY peptide carboxy-terminal fragment NPY (13-36 and the PYY fragment PYY 22-36). to be.

36 amino acids known as peptide YY (1-36) [PYY (1-36)] [YPIKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY, SEQ ID NO. : 1] is known to produce weight loss results when individually administered peripherally by injection. Therefore, it can be used as a medicament for treating obesity and related diseases {Morley, J. Neuropsychobiology 21: 22-30 (1989)}. It was later found to produce an effect of binding PYY to the Y2 receptor, the binding of the Y2 antagonist to the Y2 receptor resulted in a reduction in the intake of carbohydrates, proteins and meals {Leibowitz, SF et al.Peptides, 12. 1251-1260 (1991)}. An alternative molecular form of PYY is PYY (3-36) IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY [SEQ ID NO .: 2] {Eberlein, Eysselein et al. Peptides 10: 797-803, (1989)}. These fragments consist of about 40% of total PYY and cross-immune reactions in human and intestinal extracts and about 36% of total serum PYY cross-immunity at about 50% faster after a meal. It seems to be the product of PYY by dipeptidyl peptidase-IV (DPP4) action. PYY3-36 is known ligands selected from the Y2 and Y5 receptors, which exhibit pharmacological characteristics in selecting truncated N-terminal (eg, C-terminal fragment) NPY analogs. It also indicates that PYY fragments having only 22-36 residues can still bind to the Y2 receptor. However, if any carboxy terminus of the peptide is degraded, it loses its ability to bind the Y2 receptor. Thus, PYY antagonists are peptides with some sequence of PYY full-length, capable of binding to Y2 receptors in the correct nucleus of the hypothalamus. In view of this fact, the term PYY is meant to refer to both the PYY full-length and any PYY fragment that binds to the Y2 receptor. PYY and PYY3-36 can be administered in the form of intravenous and subcutaneous injections specifically for the treatment of life-threatening hypotension caused by endotoxins and causing shock (US Pat. No. 4,839,343). Pancreatic cancer may be inhibited by parenteral methods, intravenous injection or subcutaneous injection (US Pat. No. 5,574,010), and obesity may be treated (Morley, J. Neuropsychobiology 21: 22-30) 1989) and US Patent Application No. 20020141985). PYY also provides parenteral, oral, intranasal, rectal, and topical dosages to animals or humans in effective dosages to reduce the weight of the aforementioned patients by enhancing the gastrointestinal absorption of sodium-dependent cotransmitted nutrients. It is disclosed that it can be administered by route (US Pat. No. 5,912, 227). However, for the treatment of related diseases including obesity and diabetes, the method of administration has been limited to intravenous IV forms because no other forms of administration have been adequately effective for the selective administration of PYY3-36. None of the above prior arts provided a form comprising PYY or PYY (3-36) in combination with modified additives to enhance mucosal (eg, intranasal, oral, oral) delivery and further they It also did not describe the value of the endotoxin-free Y2-receptor in combination with the peptide form for the method of administration. Therefore, there was a need for developing formulations and methods for PYY3-36 administration.

[Summary of invention]

The present invention is effective for mucosal, especially intranasal delivery of Y2 receptor-binding peptides such as PYY, pancreatic peptide (PP) and NPY to treat obesity, enhance weight-loss in one individual, and prevent or treat diabetes. New methods and compositions are provided. In addition, the present invention relates to the Y2 antagonist formulation in order to be able to increase the concentration of the Y2 receptor-binding peptide of at least 5 pmol, preferably at least 10 pmol, in mammalian serum when the Y2 antagonist is administered intranasally. Receptor-binding peptides are delivered. Furthermore, the preferred formulation was that when the Y2 receptor-binding peptide was administered intranasally, the concentration of the Y2 receptor-binding peptide in mammalian serum could be increased to at least 10 pmol, more preferably at least 20 pmol. When 150 μg was administered intranasally, the concentration of the Y2 antagonist in mammalian serum could be increased to over 40 pmol per liter of serum. When the Y2 receptor-binding peptide is administered at 200 μg intranasally, the formulation of the present invention leads to an increase of at least 80 pmol per liter of serum of the Y2 receptor-binding peptide.

The Y2 receptor-binding peptide is preferably a PP, PYY or NPY peptide and the mammal is preferably a human. The most preferred Y2 receptor-binding peptide is PYY (3-36), which is a PYY peptide, and the mammal is a human.

In addition, the present invention provides a Y2 receptor-binding that can increase the concentration of the Y2 receptor-binding peptide in mammalian serum to 5 pM or more when a drug dose containing 50 µg or more of the Y2 receptor-binding peptide is administered to the mammal. It relates to peptide formulations.

In addition, the present invention provides a Y2 receptor-binding that can increase the concentration of the Y2 receptor-binding peptide in mammalian serum up to 20 pM or more when a drug dose including 100 µg or more of the Y2 receptor-binding peptide is administered to the mammal. It relates to peptide formulations.

The present invention also provides a Y2 receptor- that can increase the concentration of the Y2 receptor-binding peptide to 30 pM or more in mammalian serum when a drug dose containing 150 µg or more of the Y2 receptor-binding peptide is administered to the mammal. Binding peptide formulations.

In addition, the present invention provides a Y2 receptor-binding that can increase the concentration of the Y2 receptor-binding peptide to 60 pM or more in mammalian serum when a drug dose containing 200 μg or more of the Y2 receptor-binding peptide is administered to the mammal. It relates to peptide formulations.

The invention also relates directly to the intranasal formulation of the Y2 receptor-antagonist, which is substantially a protein or polypeptide that stabilizes the formulation. More specifically, the formulation is preferably a protein such as albumin and a collagen-derived protein such as gelatin.

The mucosal Y2 receptor-binding peptide formulation according to the invention consists of a Y2 receptor-binding peptide, water and a solubilizer at pH 3-6.5. The solubilizing agent is preferably cyclodextrin.

In an embodiment according to the invention the mucosal Y2 receptor-binding peptide formulation consists of a Y2 receptor-binding peptide, water, a solubilizer (preferably cyclodextrin) and at least one polyol (preferably two polyols). . In an alternative embodiment the formulation comprises one or all of the reagents mentioned below: chelating agent, surface-agent (surfactant) and buffer.

In another embodiment according to the invention said formulation consists of a Y2 receptor-binding peptide, water, a chelating agent and a solubilizing agent.

In another embodiment according to the present invention the formulation consists of a Y2 receptor-binding peptide, water and a chelating agent of pH 3-6.5.

In another embodiment according to the invention said formulation consists of a Y2 receptor-binding peptide, water, a chelating agent and at least one polyol (preferably two polyols). Still other embodiments include one or more of the following reagents: surface-agents, solubilizers and buffers.

In another embodiment according to the invention the formulation consists of a Y2 receptor-binding peptide, water and at least two polyols (such as lactose and sorbitol). Additives added to the formulations include, but are not limited to, solubilizers, chelating agents, one or more buffers and surface-agents.

Enhancing intranasal delivery of Y2 receptor-binding peptide antagonists according to the methods and compositions of the present invention applies to the effective pharmaceutical use of such reagents to treat a variety of diseases and conditions in mammalian diseases.

The present invention fulfills this need by providing a liquid or dehydrated Y2 receptor-binding peptide formulation. The formulation is substantially free of stabilizers that are polypeptides or proteins. The liquid PYY formulation consists of at least one additive selected from the group consisting of water, PYY and polyols, surface-agents, solubilizers and chelating agents. The pH of the formulation is preferably 3-7, more preferably 4.5-6.0, most preferably 5.0 ± 0.3.

Another embodiment of the invention is the water soluble Y2 receptor-binding formulation of the invention, which consists of water, Y2 receptor-binding peptides, polyols and surface-agents, wherein the pH of the formulation is about 3-6.5, The formulation is substantially free of stabilizers that are proteins or polypeptides.

Another embodiment of the invention is a water soluble Y2 receptor-binding peptide formulation, which consists of water, Y2 receptor-binding peptide, polyols and solubilizers, wherein the pH of the formulation is about 3-6.5, and the formulation is substantially There is no stabilizer that is a protein or polypeptide.

Another embodiment of the invention is a water soluble Y2 receptor-binding peptide formulation, which consists of water, Y2 receptor-binding peptide, solubilizer and surface-agent, wherein the pH of the formulation is about 3-6.5 and the formulation is substantially There is no stabilizer that is a protein or polypeptide.

Another embodiment of the invention is a water soluble Y2 receptor-binding peptide formulation, which consists of water, Y2 receptor-binding peptides, solubilizers, polyols and surface-agents, wherein the pH of the formulation is about 3-6.5. Emulation is substantially free of stabilizers that are proteins or polypeptides.

In another aspect of the invention, the stable water soluble formulation is a dehydrated Y2 receptor-binding consisting of a Y2 receptor-binding peptide and at least one additive selected from the group consisting of polyols, surface-agents, solubilizers and chelating agents. It is dehydrated to produce a peptide formulation. The dehydrated Y2 receptor-binding peptide formulation lacks stabilizers that are proteins such as albumin, collagen or polypeptides or collagen-derived proteins such as collagen. The dehydration reaction is carried out by various methods such as lyophilization, spray drying, salt-induced precipitation and drying, vacuum drying, rotary evaporation or supercritical CO ₂ precipitation.

In one embodiment, the dehydrated Y2 receptor-binding peptide consists of a Y2 receptor-binding peptide, a polyol and a solubilizer, wherein the formulation is substantially free of stabilizers that are proteins.

In another embodiment, the dehydrated Y2 receptor-binding peptide formulation consists of a Y2 receptor-binding peptide, a polyol and a surface-agent, wherein the Y2 receptor-binding peptide formulation is substantially free of stabilizers that are proteins or polypeptides.

In another embodiment, the dehydrated Y2 receptor-binding peptide formulation consists of a Y2 receptor-binding peptide, a surface-agent and a solubilizer, wherein the Y2 receptor-binding peptide formulation is free of stabilizers that are proteins or polypeptides.

In another embodiment of the invention the dehydrated Y2 receptor-binding peptide formulation consists of a Y2 receptor-binding peptide, a polyol, a surface-agent and a solubilizer, wherein the Y2 receptor-binding peptide formulation is substantially a protein or polypeptide. There is no stabilizer.

Any solubilizer may be used, but is preferably selected from the group consisting of hydroxypropyl-β-cyclodextran, sulfobutylether-β-cyclodextran, methyl-β-cyclodextrin and chitosan.

In general, the polyol is selected from the group consisting of lactose, sorbitol, trihalose, sucrose, mannose and the derivatives and homologues thereof.

Surface-agents include L-α-phosphatidylcholine didecanoyl (DDPC), polysorbate 20 (Tween 20), polysorbate 80 (Tween 80), polyethylene glycol (PEG), cetyl alcohol, polyvinylpyrrolidone (PVP ), Polyvinyl alcohol (PVA), lanolin alcohol and sorbitan monooleate.

In a preferred formulation the Y2 receptor-binding peptide formulation also consists of a chelating agent such as ethylene diamine tetraacetic acid (EDTA) or ethylene glycol tetraacetic acid (EGTA). Preservatives such as chlorobutanol or benzylconium chloride may also be added to the formulation to inhibit the growth of microorganisms.

The pH is generally adjusted using buffers such as sodium citrate, citric acid, sodium acetate and acetic acid. Optional buffers are acetic acid and sodium acetate or succinic acid and sodium hydroxide.

Preferred Y2 receptor-binding peptides are PYY, PP or NPY peptides, with PYY (3-36) peptides being more preferred.

The present invention also includes formulations wherein the concentration of the Y2 receptor-binding peptide is 0.1-15.0 mg / mL, preferably 1.0-2 mg / mL, and the pH of the aqueous solution is 3.0-6.5, preferably pH is 5.0 ± 0.3.

The present invention further encompasses Y2 receptor-binding peptide formulations wherein the concentration of the polyol is about 0.1-10% (w / v) and in particular cases about 0.1-3% (w / v).

The invention also includes formulations wherein the concentration of the surface-agent is about 0.00001-5% (w / v), preferably about 0.0002-0.1% (w / v).

The present invention also includes formulations wherein the concentration of the solubilizer is about 1-10% (w / v), preferably about 2-5% (w / v).

The finished solution may be filtered and lyophilized using the method according to the manufacturer's instructions of the method and the method well known to those skilled in the art and lyophilization apparatus.

In another embodiment of the present invention, the Y2 receptor-binding peptide formulation is composed of a Y2 receptor-binding peptide and a pharmacologically acceptable medium, wherein the Y2 receptor-binding peptide formulation is used for in vivo tissue penetration analysis. And a higher penetration rate of at least 1%, preferably 3%, more preferably 6%, compared to a regulatory formulation consisting of a buffer and a Y2 receptor-binding peptide, which is determined by transepithelial electrical resistance analysis. It is shown in Experimental Examples 2 and 7. In a preferred embodiment, the Y2 receptor-binding formulation further consists of at least one additive selected from the group consisting of surface-agents, solubilizers, polyols and chelating agents. The Y2 receptor-binding peptide is preferably a PYY peptide, an NPY peptide or a PP peptide.

In another embodiment according to the invention the Y2 receptor-binding peptide formulation is characterized in that at least 5 pmoles per liter of plasma in mammalian plasma, at least 5 pmole, preferably 10, 20, when 100 μg of said formulation is administered in mammalian nasal passages. It is possible to increase the Y2 receptor-binding peptide concentration above 40, 60, 80 pmole.

In an embodiment, said enhanced delivery methods and compositions according to the present invention provide effective mucosal delivery treatment of said Y2 receptor-binding peptide antagonist for the prevention and treatment of obesity and eating disorders in mammalian patients. In one aspect of the present invention, pharmaceutical formulations suitable for intranasal administration determine effective dosages for the treatment of Y2 receptor-binding peptides and one or more intranasal delivery enhancers described above, which formulations may be associated with obesity and It is particularly effective in the nasal mucosal delivery method of the present invention for preventing the onset and progression of eating disorders. Intranasal mucosal delivery doses effective in the treatment of Y2 receptor-binding peptide antagonists and one or more intranasal delivery enhancers can increase the concentration of the Y2 receptor-binding peptide antagonist in patients and control food intake in mammalian patients to cause symptoms of obesity or eating disorders. To alleviate

The enhanced delivery methods and compositions according to the present invention are effective in the treatment of mucosal delivery of Y2 receptor-binding peptides for the prevention and treatment of various diseases and conditions in mammalian patients. Y2 receptor-binding peptides can be administered via a variety of mucosal pathways, for example the contact of the Y2 receptor-binding peptide to the nasal mucosal epithelial tissue, bronchial tissue or lung mucosal epithelium, the oral surface or the oral and small intestinal mucosal surfaces. Is done. In specific embodiments, the methods and compositions are delivered either directly or in nasal delivery (eg, nasal mucosal delivery or intranasal mucosal delivery).

In one aspect of the present invention, pharmaceutical formulations suitable for intranasal administration determine effective doses for the treatment of Y2 receptor-binding peptides and one or more intranasal delivery enhancers described above, which formulations may be associated with obesity, Particularly effective in the nasal mucosal delivery method of the present invention for preventing the development and progression of diabetes, cancer or malnutrition or wasting diseases associated with cancer, and also for treating Alzheimer's disease, colon cancer, colon adenocarcinoma, pancreatic cancer, pancreatic epithelial cancer, breast cancer Likewise, it is effective in relieving one or more chronically well known symptoms of obesity.

In another aspect of the invention the pharmaceutical formulations and methods are direct administration of Y2 receptor-binding peptide antagonists in combination with vitamin E succinic acid. Y2 receptor-binding peptide antagonists in combination with vitamin E succinic acid may be administered to reduce the incidence of cancer (eg, colorectal cancer, pancreatic epithelial cancer or breast cancer) or to prevent or alleviate analgesic.

In another aspect of the invention the use of endotoxin-free Y2 receptor binding peptides (eg, PYY (3-36)) increases mucosal delivery compared to peptides without endotoxin removal. Thus, the use of the endotoxin-free Y2 receptor binding peptides in pharmaceutical formulations is suitable for non-injection administration including mucosal delivery such as nasal, oral, lung, vaginal, rectal and the like.

The mucosal Y2 receptor-binding peptide formulations described above and the delivery and preparation methods according to the present invention increase mucosal delivery of Y2 receptor-binding peptides in mammalian patients. The compositions and methods provide a combined formulation and are involved in the administration of one or more Y2 receptor-binding peptides with one or more mucosal delivery enhancers. The mucosal delivery enhancers are selected to achieve the following formulations and methods: (a) solubilizers; (b) charge converting agents; (c) pH adjusting agents; (d) degrading enzyme inhibitors; (e) viscous or mucus removers; (f) ciliostatic agents; (g) cell membrane penetration enhancers (e.g., (i) surfactants, (ii) bile salts, (ii) phospholipids or fatty acid additives, mixed micelles, liposomes, or mediators, (iii) alcohols, (iv) enamels) (V) NO donor compound, (vi) long chain amphiphilic molecule (vii) small hydrophobic penetration enhancer; (viii) sodium or salicylic acid derivative; (ix) glycerol ester of acetoacetic acid (x) cyclodextrin or beta-cyclo Dextrin derivatives, (xi) medium-chain fatty acids, (xii) chelating agents, (xiii) amino acids or salts thereof, (xiv) N-acetylamino acids or salts thereof, (xv) degrading enzymes for selected cell membrane components, ( ix) fatty acid synthesis inhibitors, (x) cholesterol synthesis inhibitors, or (xi) cell membrane penetration enhancing compound reagents of (i)-(x); (h) nitric oxide (NO) stimulants, chitosan, and chitosan derivatives; Modulators on the same epithelial tissue physiology; (i) vasodilators; (j) selective delivery-enhancing agents; and (k) stable delivery media, mediators, supports or complex-shaped traits, and the Y2 receptor-binding peptide (s) may enhance mucosal delivery enhancement. To be effectively structured, bound, included, encapsulated or bound to stabilize the activator.

In various embodiments according to the present invention peptide YY is combined with one, two, three, four or more of the mucosal delivery enhancers mentioned in (a)-(k) above. Such mucosal delivery-enhancing agents may be mixed alone or in combination with peptide YY or other materials or delivery media associated in a pharmaceutically acceptable formulation. The formulation of the peptide YY with one or more mucosal delivery-enhancing agents according to the technique provides an increase in the physiological acceptability of the peptide YY in delivering the substance to the mucosal surface of a mammalian patient.

Accordingly, the present invention relates to a method for inhibiting appetite, promoting weight loss, reducing food intake, or treating obesity and / or diabetes in a mammal by administering a formulation consisting of a Y2 receptor-binding peptide through the mucosa. will be. When 50 μg of the Y2 receptor is administered to the mammalian mucosa, the concentration of the Y2 receptor-binding peptide in the mammalian plasma increases by at least 5 pmol / l and preferably by 10 pmol / l plasma. Examples of such formulations have been described above.

The present invention further provides the use of Y2 receptor-binding peptides for the production of drugs for mucosal absorption, administration of Y2 receptor-binding peptides to suppress appetite, promote weight loss, reduce food intake or treat obesity. to provide. When 50 μg of the Y2 receptor was administered to the mammalian mucosa, the concentration of the Y2 receptor-binding peptide in the mammalian plasma increased to at least 5 pmol per liter of plasma. When 100 μg of the Y2 receptor was administered to the mammalian mucosa, the concentration of the Y2 receptor-binding peptide in the mammalian plasma increased to at least 20 pmol per plasma. When the 150 μg was administered, the concentration

Effective dosages for mucosal absorption of peptide YY in the pharmaceutical formulations according to the invention are, for example, about 0.001-100 pmol per kg of body, or about 0.01-10 pmol per kg of body, or body 1 It is about 0.1-5 pmol per kg. In another embodiment, the dosage of peptide YY is about 0.5-10 pmol per kg of body. In a preferred embodiment the intranasal dose is 50-400 μg, more preferably 100-200 μg, most preferably about 100-150 μg. The pharmaceutical formulations of the present invention may be administered one or more times per day and may also be administered three times a week or once a week between one and at least 96 weeks or regularly throughout the life of the patient. In some embodiments, the pharmaceutical formulations of the present invention are administered at least once a day, twice a day, four times a day, six times a day, or eight times a day.

Intranasal delivery-enhancers function to enhance peptide YY delivery into or across intranasal mucosal surfaces. Passively absorbing the drug, wherein the route around and through the cell for drug delivery is determined by the pKa, partition coefficient, molecular radii and dose of the drug, the drug being the absorbed surface area and the vessel to which the drug is delivered. Depends on the pH of the environment. The intranasal delivery-enhancing agent of the present invention may be a pH adjusting agent. The pH of the pharmaceutical formulation of the present invention is a factor influencing the uptake of peptide YY via pathways around and through cells in drug delivery. In one embodiment the pH of the pharmaceutical formulation of the present invention is applied at about 3-6.5. In another embodiment, the pH of the pharmaceutical formulation of the present invention is applied at about 4.0-5.0. Typically the pH is in the range of 5.0 ± 0.3.

[Brief Description of Drawings]

Figure 1 shows the influence of pH values from 3.0 to 7.4 on the stability of PYY3-36 at high temperatures of 40 ° C.

FIG. 2 shows data for TEER of penetration enhancers.

3 shows the cell viability of test PYY formulations.

4 shows the cytotoxic effects of the test formulations. The abbreviations mentioned in FIG. 2 to FIG. 4 are defined as follows.

EN1 = PBS pH 5. 0

EN2 = L-arginine (10% w / v)

EN3 = poly-L-arginine (0.5% w / v)

EN4 = gamma-cyclodextrin (1% w / v)

EN5 = alpha-cyclodextrin (5% w / v)

EN6 = methyl-beta-cyclodextrin (3% w / v)

EN7 = n-sodium caprate (0.075% w / v)

EN8 = chitosan (0.5% w / v)

EN9 = L-alpha-phosphatidylcholine didecanyl (3.5% w / v)

EN10 = S-nitroso-N-acetylphenicylamine, (0.02% w / v)

EN11 = palmotoyl-DL-carnitine (0.5% w / v)

EN12 = Pluronic-127 (0.3% w / v)

EN13 = sodium nitroprusside (0.3% w / v)

EN14 = sodium glycocholate (1% w / v)

5 shows the energetic effect of the various compositional components on drug penetration. In FIG. 5, EN1 is DDPC, EN2 is methyl-β-cyclodextrin, and EX1 is EDTA.

FIG. 6 shows the concentration of PYY3-36 in rat plasma, where □ is 4.1 μg / kg, △ is 41 μg / kg, and ○ is 205 μg / kg. It is shown.

FIG. 7 shows dose linearity according to the dose μg / kg and blood concentration Cmax-Cbas pg / mL following intranasal administration of PYY3-36 to mice.

FIG. 8 shows dose linearity according to dose μg / kg and AUC as a result of intranasal administration of PYY3-36 to rats.

FIG. 9 shows the mean plasma concentration of PYY over time in three human volunteers, each intranasally administered 20 μg of PYY (3-36).

FIG. 10 shows the mean plasma concentrations of PYY over time in three human volunteers intranasally administered 50 μg of PYY (3-36), respectively.

FIG. 11 shows the mean plasma concentrations of PYY over time in three human volunteers intranasally administered 100 μg of PYY (3-36), respectively.

FIG. 12 shows the mean plasma concentrations of PYY over time in three human volunteers each administered 150 μg of PYY (3-36) intranasally.

FIG. 13 shows the mean plasma concentrations of PYY over time in three human volunteers each administered 200 μg of PYY (3-36) intranasally.

14 shows concentrations of PYY over time for 5 groups of healthy human volunteers who received PYY (3-36) intranasally. The PYY3-36 doses are 200 μg, 150 μg, 100 μg, 50 μg and 20 μg, respectively.

FIG. 15 shows the dose linearity for Cmax pg / mL, which is the maximum PYY concentration in blood according to the PYY3-36 dose administered to human volunteers.

FIG. 16 shows dose linearity for PYY mean AUG according to PYY3-36 dose administered to human volunteers.

Figure 17 shows the visual analog scale (VAS) according to the PYY3-36 dose administered to human volunteers. The question is, "How hungry are you?" Relatively less hungry subjects had lower values than the 100 point scale.

18 shows the visual analog scale (VAS) according to the PYY3-36 dose administered to human volunteers. The question is, how much can you eat? Relatively less hungry subjects had lower values than the 100 point scale.

19 shows the visual analog scale (VAS) according to the PYY3-36 dose administered to human volunteers. The question is, "How full do you feel?" Relatively less satisfied individuals had values lower than the 100 point scale.

FIG. 20 depicts% penetration of PYY (3-36) including endotoxin to endotoxin-free PYY (3-36).

The present invention provides compositions and methods for mucosal delivery of improved Y2 receptor-binding peptides for the prevention and treatment of various diseases and situations in mammalian patients. Prophylactic methods and treatments suitable for mammalian patients according to the present invention include, but are not limited to, human and non-human primates, including poultry species such as dogs, cats, mice, guinea pigs, and rabbits, as well as animal species such as horses, cattle, sheep and goats It doesn't happen.

The invention may be understood in more detail by the definitions provided below.

Y2 receptor-binding peptides

Y2 receptor-binding peptides used in the mucosal formulations of the present invention include a group of pancreatic polypeptides, which are characterized by consisting of three peptides, PP, NPY and PYY, which naturally produce physiological activity. Experimental examples of Y2 receptor-binding peptides and their uses are described in US Pat. Nos. 5,026, 685; US Patent No. 5,574,010; US Patent No. 5,604, 203; US Patent No. 5,696,093; US Patent No. 6,046,167; Gehlertet. al., Proc Soc Exp Biol Med 218: 7-22 (1998); Sheikh et al. Am JP hysiol, 261: 701-15 (1991); Fournier et al., Mol Pharmacol 45: 93-101 (1994); Kirby et al., J Med C / em 38: 4579-4586 (1995); Rist et al., Eur J Biochena 247: 1019-1028 (1997); Kirby et al., J Med Clze Tn 36: 3802-3808 (1993); Grundemar et al., Regulatory peptides 62: 131-136 (1996); US Patent No. 5,696,093 (Experiments of PYY Antagonists), US Patent No. 6,046,167.

The Y2 receptor-binding peptides according to the invention are free bases, acid addition salts or metal salts such as sodium salts or potassium or amidation, glycosylation, acylation, sulfate, phosphorylation, acetylation and Y2 receptor-binding peptides converted by reactions such as cyclization (US Pat. No. 6,093, 692; and US Pat. No. 6,225, 445) and pegylaton.

Peptide YY Antagonists

The above-mentioned "PYY" means PYY (1-36) in native-sequence and also refers to derivatives, fragments and PYY analogs of various traits depending on natural, synthetic or recombination. The PYY should consist of at least the last 15 amino acid residues or a corresponding analog of the PYY sequence consisting of PYY (22-36) (SEQ ID NO: 3). Other PYY peptides may be used with PYY having the following sequence: PYY (1-36) (SEQ ID NO: 1) PYY (3-36) SEQ ID NO: 2) PYY (4-36) (SEQ ID NO: 4) PYY (5-36) (SEQ ID NO: 5), PYY (6-36) (SEQ ID NO: 6), PYY (7-36) (SEQ ID NO: 7) PYY (8- 36) (SEQ ID NO: 8), PYY9-36 (SEQ ID NO: 9) PYY (10-36) (SEQ ID NO: 10), PYY (11-36) (SEQ ID NO: 11), PYY ( 12-36) (SEQ ID NO: 12), PYY (13-36) (SEQ ID NO: 13), PYY (14-36) (SEQ ID NO: 14), PYY (15-36) (SEQ ID NO : 15), PYY (16-36) (SEQ ID NO: 16), PYY (17-36) (SEQ ID NO: 17), PYY (18-36) (SEQ ID NO: 18), PYY (19- 36) (SEQ ID NO: 19), PYY (20-36) (SEQ ID NO: 20) and PYY (21-36) (SEQ ID NO: 21). Such peptides typically bind to Y receptors, in particular Y2 and / or Y5 receptors in the brain and elsewhere. Typically such peptides are synthesized in endotoxin-free or pyrogen-free traits, although not always necessary. Other PYY peptides include, for example, PYY peptides in which existing amino acid residue changes resulted in specific site mutations of the PYY peptide. [Asp ¹⁵ ] PYY (15-36) (SEQ ID NO: 90), [ Thr ¹³ ] PYY (13-36) (SEQ ID NO: 91), [Val ¹² ] PYY (12-36) (SEQ ID NO: 92), [Glu ¹¹ ] PYY (11-36) (SEQ ID NO: 93), [Asp ¹⁰ ] PYY (10-36) (SEQ ID NO: 94), [Val ⁷ ] PYY (7-36) (SEQ ID NO: 95), [Asp ⁶ ] PYY (6-36) ( SEQ ID NO: 96), [Gln ⁴ ] PYY (4-36) (SEQ ID NO: 97), [Arg ⁴ ] PYY (4-36) (SEQ ID NO: 98), [Asn ⁴ ] PYY (4 -36) (SEQ ID NO: 99), [Val ³ ] PYY (3-36) (SEQ ID NO: 100) and [Leu ³ ] PYY (3-36) (SEQ ID NO: 101). Other PYY peptides include those peptides with at least two or more existing amino acid residues changed as follows: [Asp ¹⁰ Asp ¹⁵ ] PYY (10-36) (SEQ ID NO: 102), [Asp ⁶ , Thr ¹³ ] PYY (6-36) (SEQ ID NO: 103), [Asn ⁴ , Asp ¹⁵ ] PYY (4-36) (SEQ ID NO: 104) and [Leu ³ , Asp ¹⁰ ] PYY (3-36) (SEQ ID NO: 105). PYY analogs are also disclosed in the following documents: US Pat. Nos. 5,604, 203 and 5,574,010; 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 at., Reg. Peptides 51: 151, (1994); PCT International Application Nos. 94/03380, US Pat. Nos. 5,604,203 and 5,574,010. Such peptides typically bind to Y receptors, in particular Y2 and / or Y5 receptors, in the brain and elsewhere. Typically such peptides are synthesized in endotoxin-free or pyrogen-free traits, although not always necessary.

PYY antagonists include traits truncated amino terminal ends corresponding to murine PYY (SEQ ID NO: 72) and humans, traits truncated pig terminal PYY (SEQ ID NO: 73) and amino terminal ends corresponding to humans, guinea pig PYY (SEQ ID NO: 74) and traits with truncated amino group ends corresponding to humans.

PYY peptides according to the invention can also be used as free bases, acid addition salts or sodium salts or metal salts such as potassium or amidation, glycosylation, acylation, sulfate, phosphorylation, acetylation, cyclization And PYY peptides converted to other notably known covalent transformation methods. Such peptides typically bind to Y receptors, in particular Y2 and / or Y5 receptors, in the brain and elsewhere. Typically such peptides are synthesized in endotoxin-free or pyrogen-free traits, although not always necessary.

Neuropeptide Y Antagonists

NPY is another Y2 receptor-binding peptide. NPY peptides include full-length NPY (1-36) (SEQ ID NO: 22) as well as fragments of NPY (1-36) cut at the amino end groups. NPY antagonists for the effect of Y2 receptor binding should consist of NPY (26-36) (SEQ ID NO: 23) at least the last 11 amino acid residues at the carboxyl terminus. Other experimental examples of NPY antagonists that bind to the Y2 receptor include NPY (3-36) (SEQ ID NO: 24), NPY (4-36) (SEQ ID NO: 25), NPY (5-36) (SEQ ID NO: 26), NPY (6-36) (SEQ ID NO: 27), NPY (7-36) (SEQ ID NO: 28), NPY (8-36) (SEQ ID NO: 29), NPY (9 -36) (SEQ ID NO: 30), NPY (10-36) (SEQ ID NO: 31), NPY (11-36) (SEQ ID NO: 32), NPY (12-36) (SEQ ID NO: 33), NPY (13-36) (SEQ ID NO: 34), NPY (14-36) (SEQ ID NO: 35), NPY (15-36) (SEQ ID NO: 36), NPY (16-36 ) (SEQ ID NO: 37), NPY (17-36) (SEQ ID NO: 38), NPY (18-36) (SEQ ID NO: 39), NPY (19-36) (SEQ ID NO: 40) , NPY (20-36) (SEQ ID NO: 41), NPY (21-36) (SEQ ID NO: 42), NPY (22-36) (SEQ ID NO: 43), NPY (23-36) ( SEQ ID NO: 44), NPY (24-36) (SEQ ID NO: 45) and NPY (25-36) (SEQ ID NO: 46).

Other NPY antagonists include mouse NPY (SEQ ID NO: 75) and traits truncated amino groups from NPY (3-36) to NPY (26-36) for human traits, mouse NPY (SEQ ID NO: 76) and Amino acid terminal truncated traits from NPY (3-36) to NPY (26-36) for human traits, dog NPY (SEQ ID NO: 77) and NPY (26) from NPY (3-36) for human traits Traits truncated amino group ends up to -36), traits truncated amino group ends from NPY (3-36) to NPY (26-36) for bovine NPY (SEQ ID NO: 78) and human traits, bovine NPY (SEQ ID NO: 79) and amino acid terminal truncated traits from NPY (3-36) to NPY (26-36) in human traits, sheep NPY (SEQ ID NO: 80) and NPY in human traits ( Traits truncated amino groups from 3-36) to NPY (26-36) and amino groups from NPY (3-36) to NPY (26-36) for guinea pig NPY (SEQ ID NO: 81) and human traits Including truncated traits .

NPY peptides according to the invention can also be used as free base, acid addition salts or sodium salts or metal salts such as potassium or amide, glycosylation, acylation, sulfate, phosphorylation, acetylation, cyclization And PYY peptides converted to other notably known covalent transformation methods. Such peptides typically bind to Y receptors, in particular Y2 and / or Y5 receptors, in the brain and elsewhere. Typically such peptides are synthesized in endotoxin-free or pyrogen-free traits, although not always necessary.

Pancreatic peptide

Pancreatic peptide (PP) and PP antagonists are also bound to the Y2 receptor. Examples of such PP antagonists are full-length PP (1-36) (SEQ ID NO: 47) and multiple PP fragments cleaved at the amino group terminus. To bind to the Y2 receptor, the PP antagonist must be PP (26-36), (SEQ ID NO: 48), the last 11 amino acid residues at the carboxyl-terminus. Other PP examples for binding to the Y2 receptor include PP (3-36) (SEQ ID NO: 49), PP (4-36) (SEQ ID NO: 50), PP (5-36) (SEQ ID NO: 51), PP (6-36) (SEQ ID NO: 52), PP (7-36) (SEQ ID NO: 53), PP (8-36) (SEQ ID NO: 54), PP (9-36) ) (SEQ ID NO: 55), PP (10-36) (SEQ ID NO: 56), PP (11-36) (SEQ ID NO: 57), PP (12-36) (SEQ ID NO: 58) , PP (13-36) (SEQ ID NO: 59), PP (14-36) (SEQ ID NO: 60), PP (15-36) (SEQ ID NO: 61), PP (16-36) ( SEQ ID NO: 62), PP (17-36) (SEQ ID NO: 63), PP (18-36) (SEQID NO: 64), PP (19-36) (SEQ ID NO: 65), PP ( 20-36) (SEQ ID NO: 66), PP (21-36) (SEQ ID NO: 67), PP (22-36) (SEQ ID NO: 68), PP (23-36) (SEQ ID NO : 69), PP (24-36) (SEQ ID NO: 70) and PP (25-36) (SEQ ID NO: 71).

Other PP antagonists include sheep PP (SEQ ID NO: 82) and traits with amino terminal truncations from PP (3-36) to PP (26-36) in human traits, pig PP (SEQ ID NO: 83) and Amino acid terminal truncated traits from PP (3-36) to PP (26-36) for human traits, dog PP (SEQ ID NO: 84) and PP (26) from PP (3-36) for human traits Traits truncated amino group ends up to -36), cat PP (SEQ ID NO: 85) and traits truncated amino group ends from PP (3-36) to PP (26-36) in human traits, bovine PP (SEQ ID NO: 86) and traits with amino terminal truncations from PP (3-36) to PP (26-36) in human traits, rat PP (SEQ ID NO: 87) and PP in human traits ( Traits with amino group ends truncated from 3-36) to PP (26-36), traits with amino group ends truncated from PP (3-36) to PP (26-36) in mouse (SEQ 88) and human traits And guinea pig PP (SEQ ID NO: 89).

The PP peptides according to the invention can also be used for free bases, acid addition salts or metal salts such as sodium salts or potassium or amidation, glycosylation, acylation, sulfate, phosphorylation, acetylation, cyclization And PYY peptides converted to other notably known covalent transformation methods. Such peptides typically bind to Y receptors, in particular Y2 and / or Y5 receptors, in the brain and elsewhere. Typically such peptides are synthesized in endotoxin-free or pyrogen-free traits, although not always necessary.

Mucosal Delivery Enhancers

"Muscle delivery enhancers" are defined as chemical agents and other ingredients, which, when added to a formulation consisting of water, salts and / or general buffers and Y2 receptor-binding peptides (regulatory formulations above), The maximum concentration (C _max ) measured in serum, or cerebrospinal fluid, or the concentration gradient over time results in a significant increase in the transport of Y2 receptor-binding peptides across the mucosa as seen in AUC. The mucous membranes include the nasal, oral, intestinal, perioral, intrabronchial, intravaginal, and rectal mucosal surfaces and include virtually all-off cell membranes that connect all of the body's pores or transporters within the human body to the outside. do. Mucosal delivery enhancers are sometimes referred to as mediators.

Endotoxin-Free Formulation

"Endotoxin-free formulation" means a formulation comprising a Y2-receptor-binding peptide and one or more mucosal delivery enhancers, wherein the mucosal delivery enhancers are substantially free of endotoxins and / or associated pyrogenic substances. Endotoxins include toxins confined within a microorganism and toxins that are only released when the microorganism is destroyed or killed. Pyrogeneic materials include exothermic-induced, thermostable materials (glycoproteins) from the outer cell membrane and other microorganisms. All of these substances can cause fever, hypotension and shock when administered to humans. The creation of endotoxin-free formulations requires special equipment, specialists and can be significantly more expensive compared to the production of endotoxin-free formulations.

Intravenous administration of NPY or PYY simultaneously in rodent infusions is known to prevent hypotension and even death associated with endotoxin alone (US Pat. No. 4,839,343), and the production of endotoxin-free formulations of such therapeutic agents is non-injection It was not expected to be necessary for administration.

Non-infusion administration

"Non-injection administration" means not a delivery method that directly injects into an arterial or venous blood vessel, which is a method (typically using liquid) into a body part, in particular by a needle, syringe or other invasive method. ) It is a method of injecting material. Non-injection administration includes non-injection methods for subcutaneous injection, intramuscular injection, intraparatonial infusion and mucosal delivery.

Obesity Treatment and Prevention

As mentioned above, the present invention provides improved and useful methods and compositions for intranasal mucosal delivery of Y2 receptor-binding peptides in the prevention and treatment of obesity in mammalian patients. The prevention and treatment of obesity means reducing food intake during meals and / or maintaining reduced weight before losing weight or gaining weight during administration, thereby reducing the incidence and lowering the severity of chronic obesity.

The present invention provides improved and useful methods and compositions for intranasal mucosal delivery of Y2 receptor-binding peptides in the brain region to prevent and treat obesity in mammalian patients. Bottom, or friomilanocortin (POMC) and NPY real neurons. The Y2 receptor-binding peptide may also be administered in conjunction with a Y1 receptor antagonist such as dihydropyridine.

Delivery Methods and Compositions

Improved methods and compositions for mucosal administration of Y2 receptor-binding peptides to mammalian patients optimize Y2 receptor-binding peptide administration schedules. The present invention provides mucosal delivery of Y2 receptor-binding peptides formulated with one or more mucosal delivery-enhancing agents, wherein the Y2 receptor-binding peptide dose release is generally standardized and / or effective in the range of Y2 receptor-binding peptide release. The delivery period is about 0.1-2.0 hours for mucosal administration; 0.4 ~ 1.5 hours; Maintained for 0.8 ~ 1.0 hours. Sustained release of the finished Y2 receptor-binding peptide is facilitated by repeated administration of the exogenous Y2 receptor-binding peptide using the methods and compositions according to the invention.

Sustained Release Compositions and Methods

Improved compositions and methods for mucosal administration of Y2 receptor-binding peptides in mammalian patients optimize Y2 receptor-binding peptide administration schedules. The present invention provides an improved mucosal (eg, nasal) delivery formulation consisting of one or more mucosal delivery-enhancing agents and an optimized sustained-enhancing or binding agent to a Y2 receptor-binding peptide. The mucosal delivery-enhancing agents of the invention provide an increase in delivery, ie an increase in maximum plasma concentration (C _max ) to enhance the therapeutic activity of the Y2 receptor-binding peptide administered to the mucosa.

The second factor affecting the therapeutic activity of Y2 receptor-binding peptides in the plasma and CNS is retention time (RT). Sustained-potentiators combined with intranasal delivery-enhancers increase C _max and increase the retention time (RT) of the Y2 receptor-binding peptide. Other agents and methods of the present invention that provide polymerization delivery media and sustained-enhancement formulations are disclosed above, for example, polyethylene glycol (PEG). The present invention provides an improved Y2 receptor-binding peptide delivery method and dosage form for the treatment of symptoms associated with obesity, colon cancer, pancreatic cancer, or breast cancer in mammalian patients.

Within the mucosal delivery formulations and methods of the present invention, the Y2 receptor-binding peptide is often administered in concert or in combination with appropriate carriers and media for mucosal delivery. As mentioned above, "medium" means a pharmaceutically acceptable solid or liquid filler, diluent, or encapsulated material. Water containing liquid carriers may contain pharmaceutically acceptable oxidizing agents, alkalizers, antimicrobial preservatives, antioxidants, buffers, chelating agents, synthetics, solubilizers, wetting agents, solvents, suspensions And / or additives such as viscosity-increasing agents, reinforcing agents, wetting agents or other physiologically compatible materials. Tabulation of the components described by these categories can be found in US Pharmacopeia National Formulary, 1857-1859, (1990). Examples of substances that can be used as pharmaceutically acceptable mediators include the following non-toxic compatible substances used in pharmaceutical formulations: sugars such as lactose, glucose and sucrose; Starches such as corn starch and potato starch; Cellulose and its derivatives such as sodium carboxy methyl cellulose, ethyl cellulose, cellulose acetate; Powdered tragacanth rubber; malt; gelatin; talc; Cocoa butter and suppository waxes and contents; Oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; Glycols such as propylene glycol; Polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; Esters such as ethyl oleate and ethyl laurate; Agar; Buffers such as magnesium hydroxide and aluminum hydroxide; Arginine acid; Pyrogen-free water; Isotonic saline; Ringer's solution, ethyl alcohol and phosphate buffers. Depending on the formulator's requirements, wetting agents, suspending agents and lubricants such as sodium lauryl sulfate and magnesium sterate may also be present in the composition, such as colorants, release agents, coatings, sweeteners, flavoring and fragrances, preservatives and antioxidants. Can be. Examples of pharmaceutically acceptable antioxidants include water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite, and the like; Fat-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol and the like; And metal-chelating agents such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like. The amount of active ingredient that can be combined with the delivery agents to provide a single dosage form will vary depending upon the particular mode of administration.

In mucosal delivery compositions and methods of the invention, various delivery-enhancing agents are used to enhance delivery of Y2 receptor-binding peptides into or through mucosal surfaces. In this regard, delivery of Y2 receptor-binding peptides through the mucosal epithelial tissue can result in "transcellular" or "paracellular". The extent to which these pathways affect the total flux and physiological applicability of the Y2 receptor-binding peptide depends on the environment of the mucosa, the physico-chemical activity of the drug, and the properties of the mucosal epithelium. Whereas intracellular migration can be either passively promoted or caused by an active process, only extracellular diffusion is used. In general, hydrophilic, passively shifted, polar solutes use the intercellular migration. In the Y2 receptor-binding peptides selected according to the present invention, the uptake and physiological activity (e.g., reflected by penetration coefficient and physical analysis) of the solutes absorbed, passively and actively, is dependent on the extracellular and intercellular delivery components. It can be easily evaluated. For passively absorbed drugs, drug delivery-related extracellular and intercellular pathways depend on pKa, partition coefficient, molecular radius and drug charge, the spatial environment in which the drug is delivered, and the pH of the absorbent surface.

The extracellular pathway means access to a relatively small portion of the surface of nasal mucosal epithelial tissue. In general, it has been reported that cell membranes occupy mucosal surfaces thousands of times larger than the area occupied by extracellular spaces. The size and charge based differentiation for the smaller accessible regions and polymers thus suggests that extracellular pathways are generally less friendly pathways for drug delivery than intercellular delivery.

Surprisingly, the methods and compositions of the present invention significantly enhanced delivery of physiological treatments into and through mucosal epithelial tissue via extracellular pathways. Therefore, the methods and compositions of the present invention successfully target both extracellular and intercellular pathways or within a single method and composition.

As mentioned above, “mucosal delivery-enhancing agents” refers to the release or solubility (eg, from formulation delivery media), diffusion rate, penetration and penetration time of Y2 receptor-binding peptide or other physiologically activated compound (s). Medicaments fortified with ingestion, residence time, stability, effective half-life, maximum or maintenance concentration levels, elimination and other desirable mucosal delivery properties (measured at the delivery site or selected active target site, such as blood or central nervous system) Include them. Thus, enhanced mucosal delivery can be caused by a variety of mechanisms. Various mechanisms refer to the water solubility of calcium and other ions that regulate intracellular or extracellular penetration, for example, by increasing cell membrane fluidity, such as by increasing the stability, proliferation, delivery, or retention of Y2 receptor-binding peptides. Or changes in activity, solubility of mucosal membrane components (eg lipids), changes in sulfhydryl levels of non-proteins and proteins in mucosal tissue, increased water flow through mucosal surfaces, physical of epithelial tissue Changes in binding, a decrease in the viscosity of the mucosa overlying the mucosal epithelial tissue, a decrease in the rate of mucosal removal, and other mechanisms.

The above-mentioned "effective amount on the mucosa of the Y2 receptor-binding peptide" makes it possible to anticipate effective mucosal delivery of the Y2 receptor-binding peptide at the target site for drug activity in patients providing a variety of delivery or transport pathways. For example, it may be found that a given active agent reaches the vascular wall adjacent to the mucosal cells through removal between the mucosal cells, while agents by other pathways are passive or active mucosa to act in the cells. The cells are discharged or transported out of the cells to enter or reach secondary target sites such as systematic circulation.

The methods and compositions of the present invention may facilitate altering the site of active agents according to one or more such selected routes, or may directly direct the mucosal tissue or basal vessel to facilitate absorption or penetration of activator (s). Can be activated in the tissue. The absorption or penetration promotion mentioned in this paragraph is not limited to the above mechanisms.

As mentioned above, "maximum concentration of Y2 receptor-binding peptide in plasma (C _max )", "concentration region of Y2 receptor-binding peptide in plasma according to time curve (AUC)", "Y2 receptor in plasma The maximum plasma concentration (t _max ) of the binding peptide "is the drug bioreaction parameters known in the art. Laursen et al., Eur. J. Endocrinolo, 135: 309-315,1996. The term "concentration according to the time curve" refers to the concentration of serum in the patient's serum over time after administration of the Y2 receptor-binding peptide administered to the patient by intranasal, intramuscular, subcutaneous, or other parenteral methods of administration, respectively. Means the concentration of Y2 receptor-binding peptide. “(C _max )” is the maximum concentration of Y2 receptor-binding peptide in serum following a single dose of Y2 receptor-binding peptide in a patient. "(t _max )" means the time to reach the maximum concentration of Y2 receptor-binding peptide in serum following a single dose of Y2 receptor-binding peptide in a patient.

The aforementioned "constant concentration region of Y2 receptor-binding peptide in plasma according to time curve (AUC)" is calculated according to the addition of residual regions and the linear trapezoidal law. A 23% decrease and an increase of 30% between the two doses detect a 90% probability (type II error β = 10%). The "delivery rate" or "absorption rate" is measured by comparing the time t _max to reach the maximum concentration C _max . C _max and t _max are analyzed by using invariant-variables. Comparison of drug bioresponse of intramuscular injection, subcutaneous injection, intravenous injection and intranasal administration to Y2 receptor-binding peptides is performed by diversity analysis (ANOVA). Specificity can be measured via the Bonferroni-Holmes continuous process. The drug-response relationship between the three nasal doses is measured by alleviation analysis. P <0.05 is considered to be specific. The results are given as +/- SEM averages.

While absorption promotion mechanisms can be varied with other mucosal delivery-enhancing agents of the present invention, the reagents used in this chapter generally do not partially reverse Y2 receptor-binding peptides or other activation or delivery without adversely affecting the mucosal tissue. It will be chosen according to the physicochemical properties of the enhancer. In this chapter, delivery-enhancing agents that increase the penetration or osmoticity of mucosal tissue often result in deterioration with protective osmotic barriers of the mucosa. In order for such delivery-enhancing agents to be characterized in the present invention, it is generally desirable that certain specific changes in the osmoticity of the mucosa are reversible within a suitable time period during the desired drug delivery. Furthermore, there should be no substantial, chronic toxicity or any deleterious changes induced by the gateway properties of the mucosa due to long term use.

In certain aspects of the invention, absorption-promoting agents for formulations that aid or bind with the Y2 receptor-binding peptides of the invention include dimethyl sulfoxide (DMSO), dimethylformamide, ethanol, propylene glycol, and It is selected from, but not limited to, small hydrophilic molecules containing 2-pyrrolidones. Optionally, long-chain amphiphilic molecules such as diacylmethyl sulfoxide, azone, sodium laurylsulfate, oleic acid, and the bile salts are used for enhanced mucosal penetration of the Y2 receptor-binding peptide. Furthermore, surfactants (eg, polysorbates) are used as formulations to add to intranasal delivery of additive compounds, processing agents, or Y2 receptor-binding peptides. Agents such as DMSO, polyethylene glycol, and ethanol enter the aqueous solution of the mucosa and change their solubility, if the delivery environment is a significant concentration, to enhance the distribution of Y2 receptor-binding peptides from the mediator into the mucosa. .

Additive mucosal delivery-enhancing agents that facilitate formulation and aid administration in combination with the method according to the invention include, but are not limited to, the following materials: mixed micelles; Enamine; Nitrogen monoxide donors (eg, S-nitrozo-N-acetyl-DL-penicillamine, NOR1, NOR4-—the above materials are treated with NO scavengers such as carboxy-PITO or doclofenac sodium) desirable); Sodium salicylate; Glycerol esters of acetoacetic acid (eg, glyceryl-1,3-diacetoacetate or 1,2-isopropylideneglycerin-3-acetoacetate) and other release-diffusion or intra-or physiologically compatible for mucosal delivery Penetration-promoting agent of trans epithelial tissue. Other absorption-promoting agents are selected from mediators, bases and additives that enhance mucosal delivery, stability, activity or trans-epithelial tissue penetration of the Y2 receptor-binding peptide. Such materials include, inter alia, cyclodextrins and (3-cyclodextrin derivatives such as 2-hydroxypropyl- (3-cyclodextrin and heptakis (2,6-di-O-methyl-β-cyclodextrin) These compounds are optionally combined with one or more activated ingredients and further formulated selectively in an oily base to enhance physiological activity in the mucosal formulations of the present invention, but with additional absorption applied to mucosal delivery. Enhancers included medium-chain fatty acids including mono- and diglycerides (eg sodium caprate-coconut oil extract, Capmul), and triglycerides (eg amylodextrin, Estaram 299, Miglyol 810).

The mucosal therapeutic and prophylactic compositions of the invention can be supplemented with a suitable penetration-promoting agent that penetrates the mucosa and promotes absorption, diffusion, and penetration of Y2 receptor-binding peptides. The penetration promoter may be a pharmaceutically acceptable promoter. Therefore, in more detail, the composition of the present invention comprises sodium salicylate and salicylic acid derivatives (acetyl salicylate, choline salicylate, salicylate, etc.); Amino acids and their salts (e.g., monoaminocarboxylic acids such as glycine, alanine, phenylalanine, proline, hydroxyproline, etc .; hydroxyamino acids such as serine; acidic amino acids such as aspartic acid, glutamic acid, and the like; Basic amino acids, including alkali metal or alkaline earth metal salts); And N-acetylamino acids (N-acetylalanine, N-acetylphenylalanine, N-acetylserine, N-acetylglycine, N-acetyllysine, N-acetylglutamic acid, N-acetylproline, N-acetylhydroxyproline, etc. ) And one or more penetration-promoting agents selected from their salts (alkali metal salts and alkaline earth metal salts). Also provided as penetration-promoting agents in the above methods, the compositions of the invention are generally emulsifiers (eg, sodium oleyl phosphate, sodium lauryl phosphate, sodium lauryl sulphite, sodium myristyl sulphite, poly Oxyethylene alkyl ethers, polyoxyylene alkyl esters, etc.), caproic acid, lactic acid, malic acid, citric acid, and salts according to alkali metals, pyrrolidonecarboxylic acids, alkylpyrrolidonecarboxylic acid esters, N-alkylpyrrolidone , Proline acyl esters, and the like.

In various aspects of the invention, improved nasal mucosal delivery formulations and methods are within the scope of the invention when targets are selected across the nasal mucosa when Y2 receptor-binding peptides and other therapeutic agents are administered to the body. To be communicated to. Some formulations are specifically adapted to the selected target cell, tissue, organ, or particular disease state. In other respects, formulations and methods are provided for effective and selective endo- or transcytosis of Y2 receptor-binding peptides that specifically follow distinct intracellular or extracellular pathways. Typically, the Y2 receptor-binding peptide is effectively loaded and delivered at an effective concentration in the mediator or other delivery vehicle. And Y2 receptor-binding peptides travel through intracellular spaces and membranes, for example, away from the nasal mucosa and / or target sites for drug reactions (eg, blood flow, tissue, organs, or extracellular space). It remains stable during the process. Y2 receptor-binding peptides are provided as delivery carriers or otherwise modified (eg, during the activation or release of Y2 receptor-binding peptides induced by physiological stimuli (eg, pH changes, lysosomal enzymes, etc.). In the form of a precursor of the drug). Often Y2 receptor-binding peptides are pharmacologically inactivated until they reach the target site to be activated. In most cases, the Y2 receptor-binding peptides and other formulation compositions are nontoxic and non-immune. Mediators or other formulation compositions in the present invention were generally selected that could quickly lose their properties and were selected under physiological conditions. At the same time the formulations are chemically and physiologically stable under the conditions of administration for effective storage.

Peptide and protein analogs and mimetics

Included in the definition of biologically active peptides and proteins for use in the present invention are natural or synthetic, therapeutic or prophylactically active peptides (consisting of covalent bonds of two or more amino acids), proteins, peptides or proteins. Fragments, peptides or protein analogs, and salts of chemically modified derivatives or active peptides or amino acids. Useful analogs of the Y2 receptor-binding peptides and various variants of mimetics have been studied for use preferably within the invention and have been produced and tested for biological activity according to known methods. Often, the peptides and proteins or other biologically active peptides or proteins of the Y2 receptor-binding peptide suitable for use within the scope of the present invention are naturally occurring or native (eg, wild type, naturally occurring mutations, Genetic mutations) are muteins readily obtainable by partial substitution, addition or deletion of amino acids in the peptide or protein sequence. This includes biologically active fragments of the native peptides or proteins. Such mutant derivatives and substantial fragments retain the desired biological activity from the original peptides or proteins. Carbohydrate chains, peptides or proteins with biologically active mutations indicated by alterations of these carbohydrate types, are also included within the scope of this invention.

As used herein, the term “conservative amino acid substitutions” refers to the general exchangeability of amino acid residues having similar side chains. For example, common exchangeable groups of amino acids with aliphatic side chains are alanine, valine, leucine, and isoleucine; For amino acid groups with aliphatic hydroxy side chains are serine and tyrosine; For amino acid groups having side chains containing amides are asparagine and glutamine; For amino acid groups having aromatic side chains are phenylalanine, tyrosine, and tryptophan; For amino acid groups with base side chains are lysine, arginine, histidine; Amino acid groups with chains between sulfur containing are cysteine and methionine. Examples of conservative substitutions also include substitution of nonpolar (hydrophobic) residues such as isoleucine, valine, leucine, methionine. The present invention also contemplates the substitution of polar (hydrophilic) residues such as between arginine and leucine, between glutamine and asparagine, and between threonine and serine. In addition, substitution of basic residues such as lysine, arginine, or histidine or acidic residues such as aspartic acid, glutamic acid is also contemplated. Typical conservative amino acid substitution groups are; Valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Available peptides suitable for use in the methods and compositions of the invention by an array of peptides or protein analogs similar to the original peptide, or using suitable assays such as fixed protein or receptor binding assays to determine the biological activity selected; Proteins can be easily identified.

Methods for stabilizing solid protein formulations of the invention include increasing the physical stability of purified proteins by methods such as lyophilization. This would not only go through a covalent pathway that could increase protein unwinding but also inhibit aggregation through hydrophobic reactions. Stabilization of formulations in the present invention also often includes formulations based on polymers, such as hydrogel formulation / delivery systems that can degrade naturally. As mentioned earlier, it is well known that water plays an important role in the structure, function and stability of proteins. Typically, they are relatively stable in the solid phase from which large amounts of water have been removed. However, therapeutic protein formulations in the solid state may be hydrated upon storage at high humidity or during delivery from a sustained release composition or device. As hydration increases, protein stability generally decreases. Water, for example, increases the protein fluidity due to the enhanced accessibility of the reaction groups, provides a fluid state of the reactants, or acts as a reactant in many detrimental processes such as beta-removal and hydrolysis, resulting in aggregation of body proteins Plays an important role.

Protein preparations containing between about 6% and 28% water are the most unstable. At lower levels, the combined fluidity of water and protein internal forces is low. At higher levels, the fluidity of the water and the power of the protein approach the state of complete hydration. It has been observed in many systems that up to the end, hydration increases, making solid phase aggregation easier. However, when the amount of water was higher, the aggregation was observed to be reduced due to the dilution effect.

In accordance with these principles, an effective method of stabilizing peptides and proteins to prevent solid phase agglomeration during mucosal delivery is to control the amount of water in solid formulations and maintain water activity at an appropriate level in the formulation. This level of maintenance depends on the protein's properties, and proteins that generally maintain water below the monolayer will show superior solid phase stability.

Various additives, diluents, bases and delivery vehicles are provided within the scope of the present invention that effectively control the amount of water for protein stability. In this sense, the following agents and agents may be used as agents for preventing aggregation. Various functional polymers such as polyethylene glycol, dextran, diethylaminoethyldextran, carboxymethyl cellulose can reduce solid phase aggregation of peptides and proteins added or interconnected and significantly increase stability. In some cases, the activity or physical stability of proteins may be enhanced by various additives added to peptides or aqueous protein drug solutions. For example, polyols (such as sugar), amino acids, proteins such as collagen and gelatin, and various salts can be used as additives.

Some additives—particularly sugars and other polyols—also give significant physical stability to lyophilized (dried) proteins. These additives can be used to prevent agglomeration of proteins not only during lyophilization but also during storage in the present invention. For example, it can be seen that sucrose and Ficoll 70 (a polymer composed of sucrose) prevent agglomeration of peptides or proteins during solid phase incubation under a variety of conditions. These additives can also enhance the stability of the solid proteins inserted into the polymer matrix.

Additional additives, such as sugars, also stabilize the protein against solid phase agglomeration in a humid air at elevated temperatures, which can be seen in the sustained release formulations of the present invention. Proteins such as gelatin and collagen are also used as agents to stabilize unstable proteins or as agents that have an important effect on reducing protein denaturation and aggregation. These additives are included in the polymer dissolution processes and the compositions of the invention. For example, polypeptide microparticles can be prepared simply by lyophilization or by spraying and drying a solution containing the various stabilizing additives described above. Thus slow release of unaggregated peptides and proteins can be obtained over an extended time.

Various additional preliminary compositions and methods are provided herein as well as specific formulation additives, which make formulations for mucosal delivery of peptides and proteins that are easy to aggregate. The peptides and proteins are stabilized into substantially pure and unaggregated form using a solubilizer. The range of compositions and additives has been studied for use in the above methods and formulations. Examples of solubilizers are linked dimers (CDs) of cyclodextrins to which the hydrophobic side chains of the polypeptides are selectively bound. The CD dimers are known to bind to hydrophobic portions of the protein and significantly inhibit protein aggregation. This inhibition is selective for CD dimers and related proteins. Selective inhibition of this protein aggregation provides additional advantages to the above invention regarding intranasal delivery methods and compositions. Additives used in the present invention include CD terpolymers and tetrapolymers having a variety of structures controlled by linkers that reliably prevent the aggregation of peptides and proteins. Solvents and methods for mixing in the invention also include peptide mimetics that prevent the use of peptides and optionally protein-protein reactions. In one aspect, specific binding of hydrophobic side chains known in CD multimers similarly extends to proteins through the use of peptides and peptide mimetics that block protein aggregation. A wide range of suitable methods and anti-agglomerating agents can be used in admixture with the compositions of the invention.

Charge conversion and pH regulators and methods

The invention also describes herein to improve the transport capacity of biologically active agents (including Y2 receptor-binding peptides, other active peptides and proteins, and polymers and small molecule drugs) to enhance delivery forces across hydrophobic mucosa. Reagents and techniques for charge conversion of selected biologically active agents or delivery-enhancing agents are provided. In this regard, the relative permeability of polymers is generally related to their partition coefficient. The degree of ionization of the molecule depends on the pKa of the molecule and the pH of the mucosal cell membrane surface, and also affects the permeability of the molecule. Since mucosal delivery can be facilitated by a change in charge or by charge dispersion of the active agent or penetrant, penetration and distribution of biological agents (including Y2 receptor-binding peptides and analogs of the invention) are achieved. Infiltration and distribution are achieved, for example, by changes in charged functional groups, changes in the pH of the solution containing the delivery vehicle or delivery vehicle.

Consistent with general teaching, mucosal delivery of charged polymers comprising Y2 receptor-binding peptides and other biologically active peptides and proteins may occur when the active agent is delivered to a mucosal surface that is substantially non-ionized or in a neutral or electrically charged state. It is considerably improved within the scope of the invention.

Certain Y2 receptor-binding peptides and other biologically active peptides and the compositions and protein compositions of mucosal formulations used within the scope of the invention are altered in charge, resulting in an increase in the positive charge density of the peptide and protein. This change in charge causes cationization of peptides and protein conjugates, mediators and other delivery agents known herein. Cationization provides a convenient means of altering the transport properties and distribution of proteins and polymers within the scope of the present invention. Cationization begins with a substantial preservation of the biological activity of the active agent and potentially inhibits side effects such as lethal or toxic to tissue.

Enzyme Inhibitors and Methods

Another additive included in mucosal delivery aids is the Degradative Enzyme Inhibitor. Basic mucoadhesive polymer-enzyme complexes used in the mucosal delivery formulations and methods of the invention include, but are not limited to: Carboxymethylcellulose-pepstatin (having anti pepsin activity); Poly (acrylic acid) -baumannschamber inhibitors (anti-chymotrypsin); Poly (acrylic acid) -chymotrypsin (anti-chymotrypsin); Poly (acrylic acid) -elastatinal (anti elastase); Carboxymethylcellulose elaststatin (anti elastase); Polycarbophil-elastinal (anti elastase); Chitosan-antipine (antitrypsin); Poly (acrylic acid) -bacitracin (anti aminopeptidase N); Chitosan--EDTA (anti aminopeptidase N, anti carboxypeptidase A); Chitosan--EDTA-- Antipine (antitrypsin, antichymotrypsin, antielastase). As will be described in detail below, certain embodiments of the invention combine with new chitosan derivatives or chemically modified chitosan structures. Such new derivatives used in the present invention are termed β- [l → 4] -2-guanidino-2-dioxy-D-glucose polymers (poly-GuD).

Inhibitors that inhibit the activity of enzymes to protect biologically active agents may be useful in the compositions and methods of the present invention. Enzyme inhibitors useful for protecting biologically active proteins and peptides are included. For example, soybean trypsin inhibitors, interest trypsin inhibitors, chymotrypsin inhibitors, and crimotrypsin inhibitors isolated from potato (solanum tuberosum L.) tubers. Combinations or mixtures of inhibitors may be used. Additional inhibitors of proteolytic enzymes include ovomucoid-enzyme, gabaxatemesylate, alpha-antitrypsin, aprotinin, amastatin, beta Statins, puromycins, bacitracins, leupepsin, alpha2-macroglobulin, pepstatin and egg white or soybean trypsin inhibitors.

These and other inhibitors may be used alone or in combination. The inhibitor (s) may be combined with, or mixed with, a mediator, such as a hydrophilic polymer, which has a membrane over its surface in dosage form for contact with the nasal mucosa. It is then separated, combined, or mixed in a formulation form (eg, before administration) that is combined or administered with a biologically active agent.

The amount of inhibitor, such as a protease inhibitor that selectively binds with the compositions of the present invention, will vary depending on the following. (a) the properties of the specific inhibitor, (b) the number of functional groups present in the molecule that can be reacted to induce ethylenic unsaturation necessary for copolymerization with the hydrogel forming the monomer, and (c The number of lectin groups, such as glycosides present in the inhibitory molecule, also depends on the particular therapeutic agent intended for administration. In general, a useful amount of enzyme inhibitor is a formulation (i.e. each protease inhibitor formulation or a combination of inhibitor and biologically active agent) at about 0.1 mg / ml to about 50 mg / ml, often at about 0.2 mg / ml About 25 mg / ml, and more generally from about 0.5 mg / ml to 5 mg / ml.

In the case of trypsin inhibition, suitable inhibitors may be selected in the following examples. Aprotinin, BBI, soybean trypsin inhibitor, chicken ovomucoid, chicken ovoinhibitor, human interest trypsin inhibitor, camostat mesilate, flavonoid inhibitors, anti-pine, leupetin , p-aminobenzamidine, AEBSF, TLCK (tosylysine chloromethyl ketone), APMSF, DFP, PMSF, and poly (acrylate) derivatives. In the case of chymotrypsin inhibition, suitable inhibitors may be selected in the following examples. Aprotinin, BBI, soybean trypsin inhibitor, chymostatin, benzyloxycarbonyl-Pro-Phe-CHO, FK-448, chicken blotting agent, sugar biphenylboronic acids complexes, DFP, PMSF , β-phenylpropionate, and poly (acrylate) derivatives. For elastase inhibition, suitable inhibitors are as follows. Elastatin, mesoxyoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone (MPCMK), BBI, soybean trypsin inhibitor, chicken overdosers, DFP, and PMSF.

Additive enzyme inhibitors used within the scope of the present invention are selected from a wide range of nonprotein inhibitors that vary in their toxicity and potency. As described in detail below, the generation of non-flowing or chemically modified analogs of the matrix or other delivery vehicle accessory makes it easy to reduce or eliminate toxicity when they meet. Among the broad group of future enzyme inhibitors used within the scope of the invention, such as diisopropylflurophosphate (DFP) and phenylmethylsulfonyl fluoride (PMSF) are organophosphorous inhibitors, which are potentially And irreversible serine proteolytic elements (eg trypsin and chymotrypsin). Additional inhibition of acetylcholinesterase by these compounds would increase their toxicity in unregulated delivery settings. As another candidate for future inhibition, 4- (2-aminoethyl) -benzenesulfonyl fluoride (AEBSF) has inhibitory activity compared to DFP and PMSF, but its toxicity is significantly less. Phenyl) -methanesulfonyl fluoride is another inhibitor of trypsin but toxic in an unregulated setting. In contrast to these inhibitors, 4- (4-isopropylpiperadinoadinocarbonyl) phenyl 1, 2, 3, 4, -tetrahydro-1-naphthoate methanesulfonate (FK-448) is a low toxicity substrate. , A potential and characteristic inhibitor of chymotrypsin. More representative of nonproteinaceous inhibitor candidates and also exhibiting low toxicity are chamostat mesylate (N, N'-dimethylcarbamoylmethyl-p- (p'-guanidino-benzoyoxy) phenylracetate methane-sulfonate )to be.

Also other forms of enzyme inhibitors used within the scope of the methods and compositions of the present invention are modified amino acids involved in the enzymatic degradation of amino acids and certain therapeutic compounds. The amino acids and modified amino acids used in the present invention can be produced in a relatively nontoxic and low cost. However, because amino acids are small in size and soluble, they are easily diluted and easily absorbed into the mucosal environment. Nevertheless, under suitable conditions, amino acids can act reversibly as competitive inhibitors of proteases. Some amino acids show very strong inhibitory activity. Preferred modified amino acids in the present invention are known as 'transition-state inhibitors'. The strong inhibitory activity of these compounds is due to their structural similarity to the substrate in the transitional structure, which is generally chosen to have a much higher affinity for the active site of the enzyme than the substrate itself. Metastatic state inhibitors are reversible and competitive inhibitors. Examples of inhibitors of this type are α-aminoboronic acid derivatives such as boro-leucine, boro-valine and boro-alanine. The boron atoms in these derivatives can take the form of tetrahedral bronate ions, which are thought to be similar to the transition state of proteins during hydrolysis by aminopeptidase. These amino acid derivatives are reversible and competitive inhibitors of aminopeptidase. Boro-leucine has been shown to be 100 times more inhibitory than bestatine and 1000 times more effective than puromycine. Another modified amino acid known to have strong protease inhibitory activity is N-acetylcysteine, which inhibits the enzymatic activity of aminopeptidase N. This additive also exhibits mucolytic properties that are used to reduce the effects of mucosal diffusion being impeded within the scope of the methods and compositions of the present invention.

To date, other enzyme inhibitors useful for use within the scope of comparable administration methods and binding formulations of the present invention have been selected from peptides and modified peptide enzyme inhibitors. An important and representative of the inhibitor group is cyclic dodecapeptide, bacitracin obtained from Bacillus licheniformis. In addition to these peptide types, some dipeptides and tripeptides show weak, non-specific inhibitory activity towards some proteases. By analogy with amino acids, their inhibitory activity can be enhanced by chemical modification. For example, phosphinic acid dipeptid analogs are also 'transition-state' inhibitors with strong inhibitory activity against aminopeptidase. Reportedly, they have been used to stabilize leucine enkephalin administered intranasally. Another example of a transition state analog is modified pentapeptide pepstatin, which is a very potential pepsin inhibitor. Structural analysis of pepstatin by testing the inhibitory activity of many synthetic analogues reveals key structural-functional features representative of the inhibitory activity of the molecule. Another particular form of modified peptides includes inhibitors in which an aldehyde group is located at the end of their structure. For example, the molecular sequence benzyloxycarbonyl-Pro-Phe-CHO, which meets the first and second specificities known as the requirements of chymotrypsin, is known to be a potential reversible inhibitor of this target protease. The chemical structures of other inhibitors with aldehyde groups at the end, such as anti-pine, leupeptin, chymostatin and elastatin, are reversible and modified like phosphoramidone, bestatin, puromycin, and amastatin, just like any other known structure. Peptides are known in the art.

Polypeptide protease inhibitors allow for easier delivery in concentrated drug-media matrices than smaller compounds because of their relatively high molecular weight. Additives for protease inhibition within the scope of the formulations and methods of the present invention require the use of a complexing agent. These agents mediate enzyme inhibition by depriving bivalent cations, which are cofactors of many proteases, from the nasal environment (or pharmaceutical or therapeutic compositions). For example, the co-agent EDTA and DTPA as comparably administered or combinatorially formulated additives will inhibit selected proteases at appropriate temperatures to sufficiently enhance intranasal delivery of a biologically active agent according to the present invention. More representative examples of inhibitors belonging to this class are EGTA, 1, 10-phenanthroline and hydroxychinoline. Moreover, these co-agents tend to chelate divalent cations, so these and other co-agents are useful as direct absorption-promoters in the present invention.

와 Zn

와 같은 2가 양이온 복합체에 기초한다. 이 폴리머들은 상기 언급한 바와 같이 첨가적 효소 저해제에서 결합 파트너 또는 매개체로서 쓰일 수 있다는 것이 더 연구되었다. 예를 들면, 키토산-EDTA 결합이 개발되어 왔고 이는 아연 의존적 프로테아제의 효소 활동에 대해 강한 저해 효과를 나타낸다는 점에서 본 발명의 범위 내에서 유용하다. 본 발명의 다른 효소 저해제의 공유 부착이 일어난 후에 폴리머들의 점막흡착 성질은 실질적으로 손상되지 않을 것으로 보이고, 생물학적 활성제인 전달 운반체와 같이 본 발명의 범위 내에서 감소할 것으로 기대되는 일반적인 효용성을 가지는 것도 아니다. 이에 반하여, 독성과 그에 따른 다른 부작용뿐만 아니라 바람직하지 않은 저해제의 희석 효과도 최소화하면서 공유결합 효소 저해제가 약물 전달 사이트에 농축된 형태로 유지되는 동안, 점막흡착 메커니즘에 의해 점막 표면과 전달 운반체 사이의 거리가 감소하여 활성제의 예비침투적 물질대사가 최소화될 것이다. 이에 의하여 희석 효과가 배제되기 때문에 대등하게 투여되는 효소 저해제의 효과적인 양이 감소할 수 있다.As described in more detail elsewhere herein, in particular, various polymers, such as mucoadhesive polymers, are enzyme inhibitors within the scope of comparable administration, multiprocessing formulations and / or binding formulation methods and compositions of the present invention. Has been studied for use. For example, poly (acrylate) derivatives such as poly (acrylic acid) and polycarbophil can affect the activity of proteases, including trypsin and chymotrypsin. The inhibitory effect of these polymers is Ca

And Zn

Based on divalent cation complexes such as It has been further studied that these polymers can be used as binding partners or mediators in additive enzyme inhibitors as mentioned above. For example, chitosan-EDTA binding has been developed and is useful within the scope of the present invention in that it exhibits a strong inhibitory effect on the enzymatic activity of zinc dependent proteases. After covalent attachment of other enzyme inhibitors of the present invention occurs, the mucoadhesive properties of the polymers appear to be substantially intact and do not have the general utility expected to decrease within the scope of the present invention, such as a biologically active delivery carrier. . In contrast, while the covalent enzyme inhibitor remains in concentrated form at the drug delivery site while minimizing the dilution effect of the inhibitor as well as toxicity and other adverse effects, the mucosal adsorption mechanism allows The distance will be reduced to minimize preinvasive metabolism of the active agent. This can reduce the effective amount of the enzyme inhibitor administered evenly because the dilution effect is excluded.

Representative examples of mucoadhesive polymer-enzyme inhibitors useful within the scope of mucosal formulations and methods of the present invention include, but are not limited to: Carboxymethylcellulose-pepstatin (with antipeptin activity); Poly (acrylic acid) -Bowman-Birk inhibitors (anti-chymotrypsin); Poly (acrylic acid) -chymostatin (anti-chymotrypsin); Poly (acrylic acid) -elastatinal (anti-elastase); Carboxymethylcellulose-elastinal (anti-elastase); Polycarbophil-elastatinal (anti-elastase); Chitosan-antipine (anti-trypsin); Poly (acrylic acid) -bacitracin (anti-aminopeptidase N); Chitosan-EDTA (anti-aminopeptidase N, anti-carboxypeptidase A); Chitosan-EDTA-antipine (anti-trypsin, anti-chymotrypsin, anti-elastase).

Mucolytics and Mucus Removers and Methods

Effective delivery of biotherapeutics via intranasal administration must take into account the decreased drug transport rate across the mucus for nasal mucosal protection and drug loss due to the binding of glycoproteins in the mucosal layer. Normal mucus is a viscoelastic solution, such as a gel consisting of water, electrolytes, mucins, polymers, and sticky epithelial cells. It primarily serves to protect and lubricate the underlying mucosal tissue. The secreted mucus is distributed to secretory cells in the nasal epithelium and other mucosal epithelial cells. The structural unit of the mucosa is mucine. Other polymers also contribute to the viscoelasticity of mucus, but this glycoprotein is the main one that is viscoelastic. In airway mucus these polymers include locally produced secretion IgA, IgM, IgE, lysozyme, and broncotransferrin, which also play an important role in host defense mechanisms.

Comparable methods of administration of the present invention include an effective mucolytic or mucolytic agent, which facilitates the absorption of the biotherapeutics administered intranasally to dissolve, thin or eliminate mucus from the nasal mucosal surface. Within the scope of these methods, a mucolytic or demucosal agent is administered equally as an aid to enhance intranasal delivery of a biologically active agent. In addition, significant amounts of effective mucolytic or demuxing agents provide processing formulations within the scope of the multi-processing methods of the present invention to provide improved formulations for enhancing intranasal delivery of biotherapeutic compounds by reducing nasal mucosa disorder effects. Or as an additive within the scope of the combination formulations of the present invention.

Various mucolytic or mucolytic agents may be included within the scope of the methods and compositions of the present invention. Based on their mechanism of action, mucolytic or mucolytic agents can often be classified into the following groups. Proteases (eg, pronase, papain) that cleave the protein core of mucin glycoproteins; Sulfhydryl compounds that cleave mucoprotein disulfide linkages; And surfactants such as detergents that break non-covalent bonds in mucus (eg Triton X-100, Tween 20), chopping salts and sodium dioxycholate, sodium taurodioxycholate, sodium glycolate and lysophosphatidylcholine Included in, but not limited to, additional compounds of the invention.

및/또는 Mg

의 킬레이팅에의한 부분적인 후반 기능)이 있다.The effect of bile causing the structural degradation of mucus is in order of dioxycholate>taurocholate> glycocholate. Other effective agents that reduce the viscosity or adsorption of mucus for enhancing intranasal delivery in accordance with the methods of the present invention include small chain fatty acids, mucolytic agents acting by chelation, such as N-acyl collagen peptides, bile salts , And saponin (Ca, which plays an important role in mucus layer structure

And / or Mg

Partial late function by chelating).

An additional mucolytic agent used in the methods and compositions of the present invention is N-acetyl-L-cysteine (ACS), which reduces both the viscosity and adsorption capacity of bronchial mucus and is anesthetized (7.5% to 12.5%). Has been shown to significantly increase the intranasal bioavailability of human growth hormone. This and other mucolytic or mucolytic agents are typically administered into the body with biologically active agents in the concentration range of about 0.2 to 20 mM to contact the nasal mucosa to reduce the polar viscosity and / or elasticity of the nasal mucosa.

Other mucolytic or mucolytic agents can also be selected from the range of glycosidase enzymes that can break the glycosidic bonds of mucous glycoproteins. α-amylases and β-amylases are representative of the enzyme group, although their mucolytic effects may be limited. In contrast, bacterial glycosidase can pass through the mucous layer of a bacterial host.

Combined use of most biologically active agents within the scope of the present invention, including peptides, proteins and nonionic detergents, is also generally useful as a mucolytic or mucus remover. These agents will not modify or substantially impair the activity of the therapeutic polypeptide.

Ciliostatic Agents and Methods

By washing the mucosa cilia, tissues, such as nasal mucosa, have the ability to self-clean. Since this is necessary as a protective action against substances such as dust, allergens and bacteria, it was generally thought that this function should not be compromised by mucosal administration. Mucosal cilia in the respiratory tract are particularly important as a defense mechanism against infection. To do this, cilia in the nose and airways move along the mucous membrane to the mucus layer to remove foreign particles or microorganisms.

Within the scope of the methods and compositions of the present invention, the cilia deterrent agent contains a Y2 receptor-binding peptide, analogs and mimetics administered to the mucosa (eg, intranasal), and other biologically active agents disclosed herein. Used to increase time. Equivalent administration of one or more ciliary arresting agents or binding formulations thereof, which function to inhibit ciliary movement of mucosal cells, temporarily and reversibly increases the time the biologically active agents administered to the mucosa remain in vivo. In this respect the delivery agents within the scope of the present invention are significantly enhanced. Using the ciliary arrest factor in the present invention with the above point of view, it is characteristic or non-indirect, or an appropriate amount (depending on the concentration, duration, delivery state) can be successfully used as a ciliary arrester in the future. Thus, the cilia arrest agent results in a reversible reduction or interruption of the mucosal ciliary movement of the mucosal site at which it is administered to enhance delivery of Y2 receptor-binding peptides, analogs and mimetics, and other biologically active agents disclosed herein. There are no unacceptable side effects.

In more detail, certain ciliary arrest factors are used in combined formulations or equivalent administration protocols with one or more Y2 receptor-binding peptide proteins, analogs and mimetics, and / or other biologically active agents disclosed herein. Various bacterial ciliary arrest factors described and isolated herein can be used in embodiments of the present invention. Cilia arrest factors from bacterium Pseudomonas aerugiytosa include phenazine derivatives, pyo compounds, (2-alkyl-4-hydroxyquinolines), and rhamnolipids (known as hemolysin). The pyo compound exhibits ciliary arrest at a concentration of 50 μg / ml and there are no obvious extrastructural wounds. Phenazine derivatives also inhibited ciliary motility but substantially caused some membrane rupture at concentrations above 400 ㎍ / ml. Limited exposure of the ramolipid organ explants resulted in ciliary arrest, which is associated with a modified ciliated membrane. Further extended exposure of the rhamnoline is associated with exposure of the dynein arm from the barn.

Surfactants and Methods

In a detailed aspect of the invention, one or more of the membrane penetration enhancers is a mucosal delivery method of the invention to enhance mucosal delivery of Y2 receptor-binding peptide proteins, analogs, mimetics, and other biologically active agents disclosed herein. Can be used for formulations. In the present invention, the membrane penetration enhancer may be selected from the following. (i) surface active agents, (ii) bile salts, (ii) phospholipid additives, mixed micelles, liposomes, or mediators, (iii) alcohols, (iv) enamines, (v) NO donors Compounds, (vi) a long-chain amphipathic molecule (vii) small hydrophobic penetration enhancers; (viii) sodium or salicylic acid derivatives; (ix) glycerol esters of acetoacetic acid, (x) cyclodextrins or beta-cyclodextrin derivatives, (xi) medium chain fatty acids, (xii) chelating agents, (xiii) amino acids or salts thereof, (xiv) N-acetylamino acids Or a salt thereof, (xv) an enzyme that breaks down the selected membrane composition, (ix) an inhibitor of fatty acid synthesis, or (x) an inhibitor of cholesterol synthesis; Or (xi) a combination of the membrane penetration enhancers cited in (i)-(x) above.

Certain surface active agents are readily included in mucosal delivery formulations and methods as mucosal absorption enhancers. Such agents that can be administered equally or formulated in combination with Y2 receptor-binding peptide proteins, analogs and mimetics, and other biologically active agents disclosed herein, can be selected from a broad population of surfactants. Surfactants are generally divided into three groups.

(1) nonionic polyoxyethylene esters; (2) bile, such as sodium glycorate (SGC) and deoxycholate (DOC); (3) fucidinic acid derivatives such as sodium taurodihydrofucidite (STDHF). The reaction mechanism of the various surfactants includes the solubility of the biologically active agent. Often in clustered proteins and peptides, due to the surface activity of the absorption promoter, reactions between the proteins can result in smaller units, such as surfactants coated with monomers, to be more easily maintained in solution. Examples of other surfactants include L-α-Phospharidycholine Didecanoyl (DDPC) polysorbate 80 and polysorbate 20. I think these monomers are more transportable units than the ones that are bundled together. The second potential mechanism is to prevent the degradation of peptides or proteins by proteases in mucosal environments. As is known, bile salts and some fusidic acid derivatives are inhibited by proteolytic degradation by nasal homogenates in amounts or below that required to enhance protein absorption. This protease is particularly important for peptides with short biological half-lives.

Degrading Enzymes and Inhibitors of Fatty Acid and Cholesterol Synthesis

In aspects associated with the present invention, Y2 receptor-binding peptide proteins, analogs and mimetics, and other biologically active agents for mucosal administration may be degrading enzymes or metabolism stimulants or inhibitors of fatty acid and sterol synthesis or other epithelial compositions, Equally administered or formulated using a penetration enhancer selected from US Pat. No. 6,190,894. Degrading enzymes such as phospholipase, hyalurodinase, neuramininases, and chondroitinases, for example, do not irreversibly damage the mucosa and are Y2 receptor-binding peptides. It is used to enhance mucosal penetration of proteins, analogs and mimetics, and other biologically active agents. In one embodiment within the methods or inventions provided herein, chondroitinase is used for modification of glycoproteins, glycolipids, which are components of mucosal permeation membranes. This enhances mucosal absorption of Y2 receptor-binding peptide proteins, analogs and mimetics, and other biologically active agents disclosed herein.

Regarding inhibitors of mucosal construct synthesis, this is known as free fatty acids, which account for 20-25% of epidermal fat weight. Two rate limiting enzymes in the biosynthesis of free fatty acids are acetyl CoA carboxylase and fatty acid synthase. Inhibitors of free fatty acid synthesis and metabolism used in the methods and compositions of the present invention include, but are not limited to:

Inhibitors of acetyl CoA carboxylase, such as 5-tetradecyloxy-2-furancarboxylic acid (TOFA); Inhibitors of fatty acid synthase; Gomicin A, 2- (p-amylcinamyl) amino-4-chlorobenzoic acid, brofenacyl bromide, monoalid, 7, 7-dimethyl-5, 8-eicosadienoic acid, nisergoline, ce Blue tin, nicardipine, quercetin, dibutyryl-cyclic AMP, R-24571, N-oleoylethanolamine, N- (7-nitro-2, 1, 3-benzosadiazole-4- yl) phosphatidylserine, cycloporin A, local anesthetics, including dibucaine, prenylamine, retinoids, all-trans and 13-cis-retinoic acid, W-7, trifluoroferrazine, R-24571 (calcida) Inhibitors of phospholipase A, such as zolium), 1-hexadosyl-3-trifluoroethyl glycero-sn-2-phosphomenthol (MJ33); Calcium channel blockers including nicadipine, verapamil, diltiazem, nifedipine, and nimodipine; Antimalarial, mepacrine, chloroquine and hydroxychloroquine including quinacrine; Beta blockers, including propanalol and labetalol; Calmodulin antagonists; EGTA; Timmersol; Glucosteroids, including dexamethasone and prednisolone; And nonsteroidal anti-inflammatory drugs containing indomethacin and naproxen.

Free sterols such as cholesterol correspond to 20-25% of the weight of epithelial fat. The rate limiting enzyme of cholesterol biosynthesis is 3-hydroxy-3-methylglutaryl (HMG) CoA reductase. Inhibitors of cholesterol used within the scope of the methods and compositions of the present invention include, but are not limited to:

Cholesterol oleate, cholesterol sulfate and phosphate, and oxygenated (HMG) CoA reductase inhibitors such as 25-OH-- and 26-OH--cholesterol and simvastatin, lovastatin, fluindostatin (fluvastatin) Competitive inhibitors of (HMG) CoA reductase; Squalene synthetase inhibitors; Squalene epoxidase inhibitors; Inhibitors of DELTA7 or DELTA24 reductase, such as 22,25-diazacholesterol, 20,25-diazacholesterol, AY9944, and triparanol.

Each inhibitor of fatty acid synthesis or a sterol synthesis inhibitor is administered equally or one or more Y2 receptor-binding peptide proteins, analogs and mimetics, and other biologically active agents disclosed herein to enhance epithelial penetration of the active agent. It is formulated in combination with (s). The effective concentration range of the sterol inhibitor in therapeutic or additional formulations for mucosal delivery is generally from about 0.0001% to about 20% of the total weight, more specifically from about 0.01% to about 5%.

Nitric oxide donors and methods

In another related aspect of the present invention the nitric oxide (NO) donor comprises one or more Y2 receptor-binding peptide proteins, analogs and mimetics, and other biologically active agents disclosed herein to enhance epithelial penetration of the active agent. It is selected as a membrane penetration enhancer to enhance mucosal delivery. Various known NO donors are useful at concentrations effective within the range of the methods and compositions of the present invention. Examples of NO donors include, but are not limited to:

Nitroglycerin, nitrofluside, NOC5 [3- (2-hydroxy-l- (methyl-ethyl) -2-nitrosohalazino) -1-propanamine], NOC12 [N-ethyl-2- (1 -Ethyl-hydroxy-2-trosohydrazino) -ethanamine], SNAP [S-nitroso-N-acetyl-DL-penicylamine], NORI and NOR4.

Within the scope of the methods and compositions of the invention, the selected NO donor is administered in an effective amount equally through or into the mucosal epithelium, or with one or more Y2 receptor-binding peptide proteins, analogs. Carcasses and / or combined with other biologically active agents disclosed herein are formulated through or into mucosal epithelium.

Epithelial Junction Structures and / or Physiological Function Modulators

The present invention provides a pharmaceutical composition, the composition of which is formulated in the manufacture of a medicament for mucosal delivery, wherein the parent is combined with one or more Y2 receptor-binding peptide proteins, analogs that bind to a mucosal delivery enhancing agent as disclosed herein. Carcasses, and other biologically active agents.

Penetrants reversibly enhance mucosal epithelial intercellular transport by regulating mucosal junction structure and / or physiological function, generally at the mucosal epithelial surface of a subject. This effect involves inhibition by penetrants that are homozygous or heterozygous between epithelial membrane adsorbent proteins of neighboring epithelial cells. Target proteins suitable for such blocking of homozygous or heterozygous bonds are selected from variously linked and conjugated adhesion molecules (JAMs), occludins, claudins.

Also in a detailed embodiment, the present invention provides penetrating peptides and peptide analogs and mimetics to enhance mucosal epithelial transport. Test peptides and proteins serve to modulate the epithelial junction structure and / or physiological function of a mammalian subject within the scope of the methods and compositions of the present invention. The peptides and peptide analogs and mimetics effectively inhibit the homomorphic and / or heterozygous binding of epithelial membrane adsorption proteins extracted from conjugated adsorption molecules (JAMs), occlusins, and clarines.

A widely studied drug is a bacterial toxin derived from Vibrio cholera known as "zonula occludens toxin" (ZOT). This toxin regulates increased nasal mucosa permeability and causes diarrhea in subjects infected with this toxin. Fasano et al, Proc. Nat. Acad. Sci., U. S. A., 8: 5242-5246 (1991). When tested on the rabbit ileal mucosa, ZOT regulates tight junctions within the cells to increase permeability. It is also known that ZOT can reversibly open tight junctions of the nasal mucosa. U.S. Patent 5,908,825.

Various ZOT analogs and mimetics that act as ZOT and ZOT active agonists and antagonists within the scope of the methods and compositions of the present invention increase intracellular uptake across the nasal mucosa and into the nasal mucosa. It is useful for enhancing intranasal delivery of biologically active agents. In the present invention, ZOT is actually activated by causing a structural rearrangement of the tight junction, characterized by altered sites of the conjugate protein ZO1. Within these aspects ZOT is formulated in combination with an effective amount of a biologically active agent that can be administered equally or reversibly increases nasal mucosal permeability without substantial side effects, thereby significantly enhancing the absorption of the active agent.

Vasodilators and Methods

Still other absorption-promoting agents showing beneficial use within the bound compositions and methods and comparable administration of the invention are vasodilator compositions, more specifically vasodilators. In the present invention, the compositions are directed to Y2 receptor-binding peptides, analogs and mimetics, and (or in mucosal epithelium) (or mucosal epithelium) to specific target tissues or compartments (eg, systemic circulation or It modulates the structure and physiological function of the mucosal vasculature while increasing the transport rate of other biologically active agents to the central nervous system.

Vasodilation agents used in the present invention actually increase nitric oxide (NO) to reduce calcium in the cytoplasm or to inhibit the myosin photokinase kinase to relax submucosal vessels. These are generally divided into nine categories. Calcium antagonists; Potassium channel openers; ACE inhibitors; Angiotensin-II receptor antagonists; α-adrenergic and imidazole receptor antagonists; β1-adrenergic antagonists, phosphodiesterase inhibitors, eicosanoids and NO donors.

Despite chemical differences, the pharmacokinetics of calcium antagonists are similar. Due to its high absorption in the system cycle, these drugs pass first in the liver, with varying individual differences in pharmacokinetic power. The half-life of calcium is short, with the exception of the newer drugs of the dihydropyridine type (e.g., amlodipine, felodipine, isradipine, nilvadipine, nisoldipine and nitrendipine). Thus, maintaining their effective drug concentrations will require delivery by multiple dose, or controlled-release, release formulations described elsewhere herein. Potassium channel opener minoxidil treatment will have limited method and dosage levels due to potential side effects.

ACE inhibitors are most effective when they prevent the conversion from antagonist I to antagonist II and increase the renin product. Because ACE is the same as kinase-II, which inactivates the potential endogenous vasodilator bradykinin, ACE inhibition reduces bradykinin degradation. ACE inhibitors have the additional effect of relieving cardiac fibrosis and ventricular hypertrophy in animal models, helping the heart and repairing the heart. A potent elimination pathway for ACE inhibitors is through renal excretion. Therefore, kidney damage is associated with reduced clearance and dose reduction of 25% to 50% is required for patients with moderate to severe kidney disease.

In the context of NO donors, they are useful in the present invention in the additional effect of mucosal penetration. In addition to the above-mentioned NO donors, the NO-nucleophilic NO complex, called NO / nucleophiles or NONOates, releases NO naturally and non-enzymatically when degraded in aqueous solutions at physiological pH. . NONOate releases a limited amount of NO and the predictable rate range is <3 minutes for diethylamine / NO and about 20 hours for diethylenetriamine / NO (DETANO).

Some of the inventions and compositions of the present invention may be administered equivalently (eg, systemically or intranasally, simultaneously or in effective temporary binding), or the subject's target tissue or body compartment (eg, One or more Y2 receptor-binding peptides, analogs and mimetics, and other biologically active agents, in an amount effective to enhance mucosal absorption of the active agent to reach the liver, portal vein, central nervous system tissue or body fluid, or serum). S) are formulated in combination.

Selective Transport Enhancers and Methods

The compositions and methods of the present invention suitably add a selective transport enhancer that facilitates the transport of one or more biologically active agents. These transport enhancers are used in combinatorial formulations or one or more Y2 receptor-binding peptide proteins, analogs and mimetics disclosed herein. And the transport enhancers may be used to enhance the delivery of one or more additional biologically active agents across the mucous membrane, such as within a compartment of the target tissue or subject (eg, mucosal epithelium, liver, central nervous system tissue or body fluid, or serum). It is used to enhance mucosal delivery so that it can reach. Alternatively, the transport enhancer may be used in combination formulations. Or in an equivalent dosing protocol to directly enhance mucosal delivery of one or more Y2 receptor-binding peptide proteins, analogs and mimetics with or without enhanced additive biologically active agent.

Representative examples of selective transport enhancers used in the present invention include, but are not limited to the following. Binders such as glycosides, molecules containing sugars, and especially lectin binders known to react with epithelial transport membranes. For example, certain "bioadhesive" ligands are present in various plant and bacterial lectins, which bind to some of the sugars on the cell surface by receptor-mediated reactions. Such binding is used as a mediator or conjugated transport mediator used to enhance mucosal delivery (eg, intranasal mucosal delivery) of the biologically active agents described herein. Certain biosorbent ligands used in the present invention will regulate the transmission of biological signals to epithelial target cells causing selective uptake of the adsorbent ligand by differentiated cell transport processes (endocytosis, transcytosis). Thus these transport modulators stimulate or selectively uptake one or more Y2 receptor-binding peptide proteins, analogs and mimetics, and other biologically active agent (s) into and / or through the mucosal epithelium. It can be used as a "carrier system" to indicate absorption. These and other selective transport enhancers significantly enhance mucosal delivery of polymeric biologics (particularly peptides, proteins, oligonucleotides, and polynucleotide vectors) in the present invention. Lectin is a plant protein that binds to the glycoproteins of eukaryotic cells and certain sugars found on the surface of glycolipids. High concentrations of lectin solutions have a "mucotractive effect" and various studies have shown rapid receptor mediated rapid mediated lectin and lectin conjugates (eg, concanavalin A conjugated with colloidal gold particles). endocytocis; RME). In addition, studies have reported that absorption of lectin mechanisms may be useful for intestinal drug targeting in vivo. In some of these studies, polystyrene nanoparticles (500 nm) have been reported to have improved systemic absorption after covalent bonding with tomato lectins and oral administration to rats.

In addition to plant lectins, microbial adsorbates and penetration factors are abundant candidate sources that can be used as adsorbent / selective transport media within the scope of the mucosal delivery methods and compositions of the invention. Two substances required for the bacterial adsorption process are 'adhesin' (adsorption or transplantation factor) and receptors on the host cell surface. Bacteria that cause infection of the mucous membranes need to penetrate the mucous layer before they attach themselves to mucosal epithelial cells. This attachment is mediated by the process of the bacteria's processes or glandular structures, even if other cellular components are involved in the process. Adsorbent bacteria transplant mucosal epithelium by amplifying and transducing (with or without help from toxins) to initiate a continuous biochemical reaction in the target cell. In connection with this infiltration mechanism, various types of biosorbent proteins (eg, invacin, internalin) are first produced by a variety of known bacteria or viruses. These proteins allow extracellular adsorption by microorganisms with specific selectivity to host cells and even to specific target tissues. Signals transmitted by these receptor-ligand responses allow live microorganisms that are not damaged by endo- or transcytotic processes to migrate into the epithelial cells (and ultimately through the epithelial cells). Such spontaneous phenomena can be used in accordance with the methods set forth herein to enhance the delivery of biologically active agents to or through epithelium, or across the epithelium, and / or to a target site where a drug reaction may occur (e.g., By complexing of biological synthesis agents such as Y2 receptor-binding peptides with adhicins).

Various bacterial and plant toxins that also bind to epithelial surfaces in specific and lectin-like ways are useful within the scope of the methods and compositions of the present invention. For example, diphtheria toxin (DT) enters host cells rapidly by RME. Similarly, E. Coli's B subunit is a very specific and lectin-like method that lifts unstable toxin masses up to the intestinal epithelial cell membrane. Absorption of this toxin and transcytosis of enterocytes into the basolateral side have been reported both in vivo and in the inanimate state. Other studies have shown the transmembrane domain of diphtheria toxin in E. coli as a high-Mw-poly-L-lysine in combination with maltose-binding fusion proteins. The resulting complex has been successfully used to mediate the uptake of reporter genes in the inanimate state. In addition to these examples, Staplaylococcus aureus is a protein set that acts as a superantigen and toxin (eg, staphylococcal enterotoxin A (SEA), SEB, toxic shock syndrome toxin 1) syndrome toxin 1 (TSST-1)>]. In studies involving these proteins, dose-dependent, promoted transcytosis of SEB and TSST-I in Caco-2 cells has been reported.

Viral haemagglutinins include another form of transport agent for promoting mucosal delivery of biologically active agents within the scope of the methods and compositions of the present invention.

The initial stage of many viral infections is the binding of surface proteins (hamaglutinin) in mucosal cells. These binding proteins are found in most viruses, including rotaviruses, varicella zoster virus, semliki forest virus, adenoviruses, and potato leafol viruses. potato leafroll virus), and reovirus. These and other representative viral haemagglutinins can be used in binding formulations (eg, mixed or conjugated formulations). Or one or more Y2 receptor-binding peptides, equivalent preparation protocols consisting of analogs and mimetics disclosed herein. Or to equally enhance mucosal delivery of one or more additional biologically active agents. In contrast, viral haemagglutinin directly enhances mucosal delivery of one or more Y2 receptor-binding peptide proteins, analogs and mimetics, with or without enhanced delivery of the added biologically active agent. For combination formulations or equivalent formulation protocols.

In various places in the body, selective transport-mediating factors can be used within the scope of the present invention. Mammalian cells have generated well-established mechanisms to promote specific substrates and corresponding target uptake. Collectively, these membrane modification processes are called 'endocytosis' and include phagocytosis, pinocytosis, receptor-mediated endocytosis (Clratin-mediated RME), and photocytosis. (Clratin mediated RME). REM is a very specific cellular and enzymatic biological process whereby, as the name implies, various ligands bind to the cell surface and continue to be absorbed and sacrificed within the cell. In many cells, the endocytosis process is active and takes less than 30 minutes to absorb and replace the entire membrane surface. Two types of receptors are proposed based on their origin in the cell membrane. The amino terminus of the type 1 receptor is located outside the cell membrane, while the terminal of the type 2 receptor is present in the cell.

Another embodiment of the present invention utilizes transferrin as a mediator or stimulant of a biologically active agent RME delivered to the mucosa. Transferrin is an 80 kDa iron-carrying glycoprotein that is effectively taken up by cells by RME. Transferrin receptors are mostly found on the surface of proliferating cells, those raised from erythrocytes, and many types of fields. Transcytosis of the transferrin (TF) and transferrin conjugates is reportedly enhanced in the presence of Brepedlin A (BFA), a fungal metabolite. In another study, BFA treatment has been reported to rapidly increase the apical endocytosis of lysine and HRP in MDCK cells. Thus, other agents that stimulate BFA and receptor-mediated transport are used to be formulated in combination within the scope of the methods of the present invention. And / or as a medicament administered equally to enhance receptor-mediated transport of biologically active agents, including Y2 receptor-binding peptide proteins, analogs and mimetics.

Polymer Delivery Vehicles and Methods

In some aspects of the invention, Y2 receptor-binding peptide proteins, analogs and mimetics, other biological agents disclosed herein, and the delivery-enhancing agents described above, exhibit biocompatible polymer functions as mediators or bases. Included individually or in combination within the range of formulations administered to the mucosa (eg nasal mucosa). Such polymer mediators include delivery media of polymer powder, matrix or microparticles, among other polymer forms. The polymer may be plant, animal, or synthetic. Often the polymer is crosslinked. In addition, in these delivery systems Y2 receptor-binding peptide proteins, analogs and mimetics may be covalently bound to the polymer and not separated from the polymer even by simple washing. In other embodiments, the polymer may be chemically modified with enzyme inhibitors or other agents that degrade the biologically active agent (s) and / or delivery enhancer (s). In some formulations the polymer is partially or wholly insoluble in water but is a water swellable polymer such as a hydrogel. The polymers useful in the present invention are preferably hydrophilic in nature so that they can react with and / or absorb significant amounts of water. The polymers often take the form of a hydrogel in contact with water or other moisture media for a sufficient time to reach equilibrium with water. In a more detailed embodiment, the polymer is a hydrogel that absorbs at least twice the weight of water at equilibrium at room temperature when contacted with excess water. US Patent No. 6,004,583.

Drug delivery systems based on biodegradable polymers are preferred in many biomedical technologies because these systems are broken by enzymatic reactions to hydrolytic or nontoxic molecules. The rate of degradation is controlled by manipulation of the composition of the biodegradable polymers matrix. Therefore, these system types can be used in settings for long term release of certain biologically active agents. Biodegradable polymers such as poly (glycolic acid) (PGA), poly- (lactic acid) (PLA), and poly (D, L-lactic-co-glycolic acid) have drawn attention as possible drug delivery carriers. The reason is that the degradation products of these polymers are nontoxic. During normal metabolism, these polymers break down into carbon dioxide and water. These polymers also show very good biocompatibility. In order to prolong the biological activity of Y2 receptor-binding peptides, analogs and mimetics, as well as other biologically active agents disclosed herein, as well as selective delivery enhancers, these agents are polyorthoesters, polyanhydrides. ), Or a polymer matrix such as polyester. This makes it possible to maintain the activity and to release something like the active agent determined by the decomposition of the polymer matrix. Although encapsulation of biotherapeutic molecules with synthetic polymers can stabilize them during storage and delivery, the biggest obstacles to polymer-based release techniques are often during formulations involving heat, sonication, and organic solvents. Is the impaired activity of therapeutic molecules.

Absorption-promoting polymers studied for use in the present invention include naturally occurring or synthetic polymers, resins, resins, and other agents, as well as derivatives of the aforementioned polymers and mixtures of each of these materials or other polymers. Chemically or physically modified versions are included. It does not adversely affect the desired properties such as water absorption, hydrogel formation, and / or chemical stability suitable for useful techniques while their alteration, modification or mixing takes place. In a more detailed aspect of the invention, polymers such as nylon, acrylan, and other normally hydrophobic synthetic polymers can be sufficiently modified by reactions to become water expandable and / or to form a stable gel in an aqueous medium.

Absorption-promoting polymers of the present invention can include polymers of homo- or copolymer groups based on various combinations of vinyl monomers, such as: Acrylic acids and methacrylic acids, acrylamide, methacrylamide, hydroxylethyl acrylate or methacrylate, vinylpyrrolidones and polyvinyl alcohols and their co- and terpolymers, polyvinylacetates, CO- and terpolymers with the monomers described above and 2-acrylamido-2methyl-propanesulfonic acid (AMPS '). Very useful are copolymers of the monomers described above with the ability to copolymerize, such as acrylic or methacrylamide acrylate or methacrylate esters with ester groups derived from straight or branched chain alkyl, aryl. The straight or branched chain alkyl, aryl, is a 1 to 6 carbon alkyl substituent-steroid, sulfate, phosphate, or N, N-dimethylaminoalkyl (meth) acrylamide, dimethylaminoalkyl (meth) acrylate, ( It has up to four aromatic rings which may include cationic monomers such as meth) acryloxyalkyltrimethylammonium chloride, (meth) acryloxyalkylmethylbenzylammonium chloride.

The additional absorption-promoting agents used in the present invention are dextran, dextrin, natural substances such as natural gums and resins, processed collagen, chitin, chitosan, ullalan, zuglan ), Alginate and modified alginates, such as Kelcoloid (modified alginate polypropylene glycol) "gellan gum," and "Kelcool" Xanthan gum such as "Keltrol", estastin, alpha hydroxy butyrate, and its copolymers, hyaluronic acid and its derivatives, such as polylactic acid and glycolic acid It comes from natural polymers.

The type of polymer applicable in the present invention is an olefin-unsaturated carboxylic acid, which contains at least one activated carbon-carbon olefin double bond and at least one carboxy group. That is to say an acid or a functional group such as: an olefin which can be easily polymerized because it is present in the monomer molecule, in the alpha-beta position relative to the carboxy group, or as part of the terminal methylene grouping. It means that it can be easily converted to an acid containing a double bond. Such olefin-unsaturated acids include acrylic acid, alpha-cyano acrylic acid, betamethylacrylic acid (crotonic acid), beta-acryloxypropionic acid, p-chloro exemplified by acrylic acid itself. Such as c-chloro cinnamic acid, 1-carboxy-4-phenylbutadiene-1,3-itaconic acid, citraconic acid, mesaconic acid, glutamic acid, aconitic acid, fumaric acid, malic acid, tricarboxy ethylene Contains substances.

As used herein, the term "carboxylic acid" includes polycarboxylic acids and their anhydrides, such as maleic anhydride, wherein the anhydride groups remove one water molecule from two carboxylic groups located on the same carboxylic acid molecule. It is formed by removing.

Representative acrylates used as absorption-promoters in the present invention include methyl acrylate, ethyl acrylate, propyl acrylate, isopropyl acrylate, butyl acrylate, isobutyl acrylate, methyl methacrylate, methyl acrylate, ethyl Methacrylate, octyl acrylate, heptyl acrylate, octyl methacrylate, isopropyl methacrylate, 2-ethylhexyl methacrylate, nonyl acrylate, hexyl acrylate, n-hexyl acrylate, and the like. . Higher numbers of alkyl acrylic acids include decyl acrylate, isodecyl methacrylate, lauryl acrylate, styryl acrylate, behenyl acrylate, mesyl acrylate, and methacrylate versions accordingly. Mixtures of two, three or more long chain acrylates will polymerize successfully with one of the carboxy monomers. Other comonomers include olefins containing alpha olefins, vinyl ethers, vinyl esters, and mixtures accordingly.

Other vinylidene monomers, including acrylonitriles, include the delivery of one or more Y2 receptor-binding peptide proteins, analogs and mimetics, and other biologically active agent (s) within the scope of the methods and compositions of the present invention. Used to enhance absorption. Enhancing delivery of the active agent (s) to a subject's target tissue or to liver, portal vein, central nervous system tissue or body fluids, serum, and the like. Useful alpha, beta-olefin unsaturated nitriles are preferably monoolefin unsaturated nitriles having 3 to 10 carbon atoms such as acrylonitriles, methacrylonitriles, and the like. Most preferred are acrylonitrile and methacrylonitriles. Acrylamides containing 3 to 35 carbon atoms can also be used, including monoolefin unsaturated amides. Representative amides include acrylamide, methacrylamide, Nt-butyl acrylamide, N-cyclohexyl acrylamide, higher number acrylamide, alkyl groups on nitrogen having from 8 to 32 carbon atoms, N-methylol acrylamide, N-propanol acrylamide, N-methol acrylamide, N-methol methacrylamide, N-methol maleimide, N-methol maleamic acid esters, N-methol-p-vinyl benzamide, other N-alkylol amides of alpha, beta-olefin unsaturated carboxylic acids, including those having 4 to 10 carbon atoms, and the like.

Additionally useful absorption promoting materials are alpha-olenes containing 2 to 18 carbon atoms, more preferably 2 to 8 carbon atoms; Dienes containing 4 to 10 carbon atoms; Vinyl esters such as vinyl acetate and allyl esters; Vinyl aromatics such as styrene, methyl styrene, chloro styrene; Ketones such as vinyl and allyl ether and vinyl methyl ether and methyl vinyl ketone; Chloroacrylate; Cyanoalkyl acrylates such as alpha cyanomethyl acrylate, and alpha-, beta-, gamma-cyanopropyl acrylates; Alkoxyacrylates such as metooxy ethyl acrylate; Haloacrylates such as chloroethyl acrylate; Vinyl halides and vinyl chloride, vinylidene chloride and the like; Divinyls, diacrylates, and other multifunctional monomers such as divinyl ether, diethylene glycol diacrylate, ethylene glycol dimethacrylate, methylene-bis-acrylamide, alipentaerythritol, and the like; And bis (beta-haloalkyl) alkenyl phosphonates, such as bis (beta-chloroethyl) vinyl phosphonate and others known in the art. The carboxyl copolymers containing monomers are secondary components, and other vinylidene monomers are readily prepared for the methods disclosed herein as the main components.

에서 C

을, 바람직하게는, C

에서 C

, 직선과 가지 친 사이클릭 C

에서 C

; (메트)가 사용되는 곳은 포함된 메틸 그룹을 가진 및 가지지 않은 모노머들을 의미한다. 다른 매우 유용한 하이드로겔 폴리머들은 팽창성이 있으나 폴리(비닐 피롤리돈)스타치, 카르복시메틸 셀룰로오즈 그리고 폴리비닐 알코올의 가용성 버전들이다. Hydrogels used as absorption promoters within the scope of the present invention are acrylic groups, methacrylic acids, acrylamide, hydroxymethylacrylate (HEA) or methacrylate (HEMA), and vinyl that reacts with water and has water expandability It can consist of synthetic copolymers made from pyrrolidones. Examples of useful polymers are the following polymer types, particularly for delivering peptides and proteins. (Meth) acrylamide and 0.1 to 99 wt% (meth) acrylic acid; (Meth) acrylamides and 0.1-75 wt% (meth) acryloxyethyl trimethiammonium chloride; (Meth) acrylamide and 0.1-75 wt% (meth) acrylamide; Acrylic acid and 0.1-75 wt% alkyl (meth) acrylates; (Meth) acrylamide and 0.1-75 wt% AMPS. RTM. (Lubrizol Corporation trademark); (Meth) acrylamide and 0 to 30 wt% alkyl (meth) acrylamides and 0.1-75 wt% AMPS. RTM .; (Meth) acrylamide and 0.1-99 wt. % HEMA; (Meth) acrylamide and 0.1 to 75 wt% HEMA and 0.1 to 99% (meth) acrylic acid; (Meth) acrylic acid and 0.1-99 wt% HEMA; 50 mole% vinyl ether and 50 mole% maleic anhydride; (Meth) acrylamide and 0.1 to 75 wt% (meth) acryloxyalkyl dimethyl benzylammonium chloride; (Meth) acrylamide and 0.1 to 99 wt% vinyl pyrrolidone; (Meth) acrylamide and 50 wt% vinyl pyrrolidone and 0.1-99. 9 wt% (meth) acrylic acid; (Meth) acrylic acid and 0.1 to 75 wt% AMPS. RTM. And 0.1-75 wt% alkyl (meth) acrylamide. In the above examples, alkyl is C

C

Is preferably C

C

, Straight and pruned cyclic C

C

; Where (meth) is used means monomers with and without the included methyl group. Other very useful hydrogel polymers are expandable but soluble versions of poly (vinyl pyrrolidone) starch, carboxymethyl cellulose and polyvinyl alcohol.

Additional polymeric hydrogel materials useful within the scope of the present invention include (poly) hydroxyalkyl (meth) acrylates: anionic and cationic hydrogels: poly (electrolight) composites; Poly (vinyl alcohols) with low acetate residues: carboxymethyl cellulose crosslinked with a swellable mixture of crosslinked agar: an expandable composition constituting methyl cellulose mixed with a poorly crosslinked agar; Water expandable copolymers produced by dispersion of well separated copolymers with styrene, ethylene, propylene, orobutylene; Water expandable polymers of N-vinyl lactams; Expandable sodium salts of carboxymethyl cellulose; And the like.

Within the scope of the present invention, polymers which absorb and retain secretions, which can be useful for forming hydrophilic hydrogels suitable for mucosal delivery of biological agents, are pectin; Polysaccharides such as agar, acacia, karaya, tragacenth, algins and sugars and their crosslinked versions; Acrylic acid polymers, copolymers and salt derivatives, polyacrylamides; Water expandable water indine maleic anhydride polymers; Starch fusion copolymers; Acrylate type polymers and copolymers having water absorption of about 2 to 400 times its own weight; Diesters of polyglucan; Mixtures of crosslinked poly (vinyl alcohol) and poly (N-vinyl-2-pyrrolidone); Polyoxybutylene-polyethylene block copolymer gels; Carob gum; Polyester gels; Poly urea gels; Polyether gels; Polyamide gels; Polyimide gels; Polypeptide gels; Polyamino acid gels; Poly cellulosic gels; Crosslinked indine-maleic anhydride acrylate polymers; And polysaccharides.

Synthetic hydrogel polymers used in the present invention can be made by unlimited combination of many monomers in various ratios. The hydrogels can be crosslinked and generally have an absorbent capacity to absorb body fluids and expand to an expanded or expanded equilibrium. Hydrogels actually swell or increase in volume by absorbing up to about 2-5, 5-10, 10-50, 50-100 times or more of their weight when delivered to the nasal mucosal surface. The appropriate degree of expandability of a given hydrogel is determined by different biological agents, which are molecular weight, size, solubility, and diffusion of the drug mediated by the polymer, encapsulated or encapsulated in the polymer and encapsulated in the specific space and in each polymer. They are characterized by factors such as cooperative chain motion combined with them.

Hydrophilic polymers useful in the present invention are insoluble in water but are water expandable. Polymers expanded by absorbing such moisture are typically referred to as hydrogels or gels. Such gels can be conveniently produced from a polymer that can be dissolved in water via a crosslinking process of the polymer with a suitable crosslinking agent. However, stable hydrogels can also be formed from certain polymers according to methods known in the art under defined pH, temperature and / or ion concentration conditions. In practice, the polymers are crosslinked until they have good hydrophilicity to improve physical integrity (compared to non-crosslinked polymers of the same or similar type) and are important while maintaining the ability to release the drugs at the proper location and time. It enhances the ability to maintain biological compositions and additive compositions for administration, such as cytokines or enzyme inhibitors, within the gel network.

In general, the hydrogel polymers used in the present invention are crosslinked, wherein an amount of 0.01 to 25% of the weight of the crosslinking agent based on the weight of the monomer forming the copolymer is such that the two functional groups crosslink and more preferably 0.1 to 20%, more generally 0.1 to 15% crosslinking. Another useful amount of crosslinking agent is 0.1 to 10% of its weight. Tri, tetra or more multifunctional crosslinking agents may also be used. When such a reagent is activated, a lower amount may be needed to obtain sufficient network properties to effectively contain the level of crosslinking density at equilibrium—ie the degree of crosslinking or the biologically active agent.

상기 교차결합들은 체액을 포함한 물에서 팽창할 수 있는 폴리머를 가지고 공유결합, 이온결합, 수소결합을 할 수 있다. 이러한 크로스링커들과 크로스링킹 반응은 이 분야에서 숙려된 것으로 알려졌고 많은 경우에 폴리머 시스템에 의존한다. 이러한 교차결합된 네트워크는 불포화된 모노머들의 자유 라디컬 코폴리머화에 의해 형성될 수 있다. 폴리머 하이드로겔은 알코올들, 산들, 글리옥살과 같은 그룹을 가지는 아민들, 포름알데히드 또는 글루타르알데히드, 비스 무수물들 그리고 기타 같은 종류의 폴리머들에서 발견되는 기능기 그룹들의 반응에 의해 크로스링킹하는 폴리머들에 의해 형성될 수 있다.

The cross-links can be covalent, ionic, and hydrogen-bonded with a polymer that can expand in water, including body fluids. These crosslinkers and crosslinking reactions are known in the art and in many cases depend on the polymer system. Such crosslinked networks can be formed by free radical copolymerization of unsaturated monomers. Polymer hydrogels are polymers that crosslink by reaction of functional groups found in alcohols, acids, amines with groups such as glyoxal, formaldehyde or glutaraldehyde, bis anhydrides and other types of polymers. It can be formed by the.

The polymers can also be crosslinked with, for example, the following polyenes. Decadiene ortrivinyl cyclohexane; Acrylamides such as N, N-methylene-bis (acrylamide); Multifunctional acrylates such as trimethylol propane triacrylate; Or a multifunctional vinylydinene monomer comprising at least two terminal CH 2 <groups (including, for example, divinyl benzene, divinyl naphthlene, allyl acrylate, etc.). In some embodiments, the cross-linking monomers used to prepare the copolymer are polyalkenyl polyesters with one or more alkenyl ether groupings per molecule, which are terminal methylene groupings (eg, at least two carbon atoms). And an alkenyl group with an oletin double bond present to attach to the polyhydroxy alcohol, which is formed by etherification of a polyhydric alcohol having two hydroxy groups. Compositions belonging to this class can be produced by reaction with an alkenyl halide such as allyl chloride or allyl bromide in an aqueous alkaline solution of one or more polyhydric alcohols. The product can be a polyether composite with various ether groups. The polyester cross-linking agent increases the number of potential polymerized groups of the molecule. In practice, polyesters containing an average of one or more alkenyl ether groupings per molecule are used. Other cross-linking monomers include the following. Diallyl esters, dimethallyl esthers, allyl or metallyl acrylates and acrylamides, tetravinyl silanes, polyalkenyl methanes, diacrylates Diacrylates and dimethacrylates, divinyl compounds such as divinyl benzene, polyallyl phosphate, diallyloxy compounds and phosphite esters and Etc. Typical agents include allyl pentaerythritol, allyl sucrose, trimethylolpropane triacrylate, 1,6-hexanediol diacryllate, trimethylolpropane diallyl ether, pentaerythritol triacrylate , Tetramethylene dimethacrylate, ethylene diacrylate, ethylene dimethacrylate, triethylene glycol dimethacrylate, etc. Allyl pentaerythritol, trimethylolpropane diallyl ether and allyl sucrose provide suitable polymers. When there is a cross-linking agent, the polymer mixtures may contain about 0.01 to 20%, for example, 1%, 5%, or 10% or more of the weight of the cross-linking monomers combined with the total carboxylic acid monomer and other monomers. It always includes

In a more detailed aspect of the invention, mucosal delivery of Y2 receptor-binding peptides, analogs and mimetics, and other biologically active agents disclosed herein, results in slow release of the medicament or an enzymatic or physiological protective media or carrier (eg, For example, as a hydrogel that protects drugs from enzymatic degradation activity). In some embodiments, the active agent is bound to the carrier or vehicle by chemical means, and is also additionally mixed or bound to additional agents such as enzyme inhibitors, cytokines, and the like. The active agent may alternatively be very physically trapped in a mediator or mediator, such as a polymer matrix, resulting in loss of mobility.

Polymers, such as hydrogels useful in the present invention, may be combined with functionally linked agents such as glycosides chemically bound to the polymer to enhance the nasal bioavailability of the formulated active agent. Examples of such glycosides are as follows. Fructosides, galactosides, arabinosides, malomsides and their alkyl substituted derivatives with albutin, phlorizin, amygdalin, Natural glycosides such as digitonin, saponin, and indican. There are various ways in which typical glycosides can be combined with a polymer. For example, hydrogen of hydroxy groups of glycosides or other similar carbohydrates may be replaced by alkyl groups from the hydrogel polymer to form ethers. In addition, the hydroxy group of glycosides can be reacted to esterify the carboxy groups of the polymer hydrogel to form the polymer ester in situ. Another approach is to use the condensation of acetobromoglucose with cholest-5-ene-3beta-ol of the maleic acid copolymer. N-substituted polyacrylamides can be synthesized by reaction of activated polymers with omega-aminoalkylglycosides: (1) (carbohydrate-spacer) (n) -polyacrylamide, pseudopolysaccharides ; (2) (carbohydrate spacer) (n) -phosphatidylethanolamine (m)-polyacrylamide, neoglycolipids, derivatives of phosphatidylethanolamine; (3) (Carbohydrate-spacer) (n)-Biotin (m)-Polyacrylamide. These biootinylated derivatives can attach to the lectins on the surface of the juma to promote the uptake of biologically active agents such as polymer-encapsulated Y2 receptor-binding peptides.

In a more detailed aspect of the invention, one or more Y2 receptor-binding peptides, analogs and mimetics, and / or other biologically active agents (protease inhibitor (s), cytokin (es), intercellulars disclosed herein) Optionally including secondary active agents such as additive modulators of linkage physiological function) or are bound to polymeric media or matrices. For example, this is accomplished by chemical binding of the peptide or protein active agent and other optional agents within the scope of the crosslinked polymer network. Chemical modifications are also possible that separate the polymer from reactants such as molecules containing glycosides. In some aspects, the biologically active agent (s), and optional secondary active agent (s), can be functionalized where appropriate reaction groups are present or where the active agent (s) are chemically added. In most cases ethylenic polymerizable groups are added and functionalized actives are then copolymerized with monomers and crosslinking agents, which can be polymerized in solution (generally in water), emulsions, suspensions or It is used in standard polymerization methods such as dispersion polymerization. Often functionalizing agents have a high enough concentration of functional groups or polymerizable groups so that many of the active agents can be functionalized. For example, in a polypeptide having 16 amine sites, it is generally preferred to be functionalized at at least 2,4,5,7, and 8 or more sites.

After functionalization, the functionalized drug (s) is mixed with a crosslinking drug consisting of drugs that form monomers and important polymers. Polymerization is then induced to make a polymer comprising the active agent (s) bound in this medium. The polymer is then washed with water or other solvents or purified to remove impurities. And if necessary, grind or crush it by physical means such as stirring, passing through a mesh, or sonicating to obtain the desired size. Solvents such as water are then removed to prevent degradation or denaturation of the active agent (s). Preferred methods are lyophilization or other methods that may be useful and used (eg vacuum drying, air drying, spray drying, etc.).

The present invention will react with other reactive groups with electrophiles containing effective amino, hydroxyl, thiol, and unsaturated groups to induce polymerizable groups of peptides, proteins and other active agents. Can be. For example, unsaturated monomers containing N-hydroxy succinimidyl groups, active carbonates such as p-nitrophenyl carbonate, tresylate, oxycarbonylimidasols, epoxy Epoxides, isocyanates and aldehydes, and unsaturated carboxy methyl azides and unsaturated orthopyridyl-disulfides belonging to this class of drugs. Examples of unsaturated agents include allyl glycidyl ether, allyl chloride, allyl bromide, allyl iodide, acryloyl chloride, allyl isocyanate, allylsulfonyl chloride, maleic Acid anhydrides, copolymers of maleic anhydride and allyl ethers, and the like.

All lysine derivatives except aldehydes can generally react with other amino acids, such as imidazole groups of histidine, hydroxy groups of tyrosine and thiol groups of cystine. Can react.

Aldehydes including agents that function are lysine specific. Types capable of reacting with lysines, citrates, tyrosine and effective groups are widely documented herein and are known to those skilled in the art.

In the case of biological agents containing amino groups, they can easily react with acylloyl chlorides such as acryloyl chloride and induce a polymerizable acrylic group in the agent. The functionalizing active agent that then passes through the acrylic group is bound to the polymer while preparing the polymer, such as during the crosslinking of the acrylamide and acrylic acid copolymer.

In another additional aspect of the invention, a biological agent comprising peptides, proteins, nucleic acids, and other molecules that are bioactive in vivo, may include some hydrophilic agent in which one or more agent (s) are linear polyalkylene glycols. And bond-stabilize covalently with a polymer consisting of a phosphorus polymer moiety and some lipophilic polymer moieties (see US Pat. No. 5,681, 811). In some aspects, the biologically active agent is covalently linked with a polymer consisting of: (i) some linear polyalkylene glycols and (ii) some lipophilic linear polyalkylene glycols in the active agent, and some lipophilic molecules Arranged in combination in relation to each other so that the active therapeutic agent has enhanced in vivo resistance to enzymatic degradation (ie, about the stability of the biological therapeutic agent under conditions similar to the unconjugated form in which the polymer is not paired). In another aspect, the conjugated-stabilized formulations have a three-dimensional arrangement with the biologically active agent covalently linked to the polysorbate composite consisting of: The polysorbate composites comprise (i) some linear polyalkylene glycols and (ii) some lipophilic molecules in the active agent, some linear polyalkylene glycols and some linear polyalkylene glycols and lipophilic molecules in combination relative to each other. So that (a) the lipophilic molecule is used externally in a three-dimensional array, and (b) the active agent in the composition has enhanced in vivo resistance to enzymatic degradation.

Looking deeper, a multiliganded conjugated complex of biologically active agents covalently bound to a portion of the triglyceride backbone is provided through a polyalkylene glycol spacer group bonded to a carbon atom of a portion of the triglyceride backbone, wherein at least one fatty acid portion is It is directly attached to a carbon atom of a portion of the triglyceride backbone or covalently linked through a portion of a polyalkylene glycol spacer (see US Pat. No. 5,681, 811). In such multiligand conjugated therapeutic complexes, the alpha 'and beta carbon atoms of some bioreactive triglycerides have a portion of fatty acids attached covalently attached directly or indirectly through a portion of the polyalkylene glycol spacer. Alternatively, if the bioreactive therapeutic agent is covalently linked to the gamma carbon atoms of the triglyceride backbone portion directly or indirectly through a portion of the polyalkylene spacer, the fatty acid portion is directly or through the polyalkylene spacer portion. It may be covalently attached to the alpha and alpha'carbons of some of the triglyceride backbones. It will be appreciated that a variety of structural, compositional, and arrangements are possible that are suitable for the multiligand therapeutic complex that constitutes part of the triglyceride backbone within the scope of the present invention. Further mentioning, such multiligand conjugated therapeutic agent complexes, biologically active agent (s) may be covalently linked to a portion of the modified backbone of triglycerides via alkyl spacer groups, or other acceptable spacer groups in the scope of the present invention. . As used herein, acceptable spacer groups are steric, compound and end use using specific acceptability.

In an additional aspect of the invention, junction-stabilized composites are provided. Conjugation-stabilizing complexes constitute a polysorbate complex consisting of a polysorbate moiety comprising a triglyceride backbone covalently linked with functional groups of alpha, alpha 'and beta carbon atoms. The above-mentioned functionalized groups include (i) fatty acid groups; And (ii) a polyethylene glycol group in which a biologically active agent or part thereof is covalently bound (e.g., combined with a suitable functional group of the polyethylene glycol group). Such covalent bonds can be, for example, directly at the hydroxy end functional group of the polyethylene glycol group, or the polyethylene glycol group capped by the capping reaction of the hydroxy end of the polyethylene glycol group with the carboxyl functional group spacer group. This terminal carboxyl functional group is made indirectly such that a biologically active agent or a portion thereof can be covalently bound.

In another additional aspect of the present invention, a stable and water soluble junction-stabilized composite is provided. This complex may be linked to one or more Y2 receptor-binding peptide proteins, analogs and mimetics, and / or to other physiologically compatible polyethylene glycols (PEGs) that modify a portion of glycolipids disclosed herein. Consisting of biologically active agent (s). In such complexes, the biological agent (s) can be covalently linked to a physiologically compatible PEG that modifies a portion of glycolipids by allowing free amino acid groups to facilitate covalent bonds, wherein the readily covalent bonds are biochemical in vivo. Can be separated by enemy hydrolysis and / or protein hydrolysis. Physiologically compatible PEGs that modify some of the glycolipides include monopalmitates, monolaurates, dipalmitates, monolaurates, dilaurates, trilaurates, monoliates, dioleates, trioleates, Polysorbate polymers, such as polysorbate polymers composed of fatty acid ester groups selected from the group consisting of monosterate, distirate, and tristerate, can be advantageously constructed. In this complex, the physiologically compatible PEG that modified some of the glycolipids is polyethylene glycol esters of fatty acids and polyethylene glycols of fatty acids such as fatty acids extracted from lauric acid, palmitic acid, oleic acid, stearic acid, for example. The polymer selected from the group consisting of esters can be suitably constituted.

Storage of substances

In some aspects of the invention, the binding formulations and / or equivalent methods of administration present an effective amount of peptides and proteins, which peptides and proteins attach to the charged glass in a container to reduce the effective concentration. Can be. Silanized containers, such as silanized glass containers, reduce the uptake of the polypeptide or protein in the glass container to store the finished product.

Also in an additional aspect of the invention, a kit for treating a mammalian patient consists of a stable pharmacological composition of one or more Y2 receptor-binding peptide composition (s) formulated for delivery to a patient's mucosa. Wherein the composition effectively alleviates symptom (s) such as obesity, cancer, or malnutrition or destruction associated with cancer. Another part of the kit is a medicament containing one or more Y2 receptor-binding peptide compositions. Medicine bottles are made of pharmaceutical polymers, glass and other suitable materials. The medicament bottle is, for example, a silanized glass bottle. Another part of the kit is a delivery hole capable of delivering the composition to the mucosal surface of the patient. The delivery hole is made of pharmaceutical polymer, glass or other suitable material. The delivery hole is for example silanized glass.

Special cleaning techniques suitable for the surface to be silanized through the silanization process at low pressures are combined with the silanization technique. Silane is gaseous and hot at the surface to be silanized. The method provides a renewable surface having a silane layer of a stable, uniform and functional monolayer. The silanized surface prevents the polypeptides or mucosal delivery enhancers of the invention from binding to the glass.

O)로 세척한다. 실란 접시는 그 다음에 95%EtOH로 씻고 아세톤 접시는 아세톤으로 씻는다. 제약병은 아세톤에서 10분간 초음파처리한다. 아세톤 초음파 처리 후에, 약제병은 ddH

O로 최소한 두 번 씻는다. 약제병들은 0.1M NaOH에서 10분간 초음파 처리한다. 약제병이 NaOH에서 초음파 처리되는 동안 실란 용액이 후드에서 만들어진다. (실란 용액: 95% 에탄올 800 mL; 빙산 초산 96 L ; 글리시드옥시프로필트리메트옥시 실란 25 mL). NaOH 초음파 처리 후에, 약제병은 ddH

O 접시에서 최소한 두 번 씻는다. 상기 약제병은 살린 용액에서 3분에서 5분 동안 초음파처리된다. 상기 약제병은 100% EtOH 접시에서 씻는다. 약제병은 미리 정제한 N

가스로 건조하고 사용 전에 100℃ 오븐에 2시간 동안 저장한다.This procedure is useful for preparing silanized pharmaceutical bottles for maintaining the Y2 receptor-binding peptide composition of the present invention. Glass trays are water distilled twice before use (ddH).

O) The silane dish is then washed with 95% EtOH and the acetone dish with acetone. Pharmaceutical bottles are sonicated for 10 minutes in acetone. After acetone sonication, the medicinal bottle is ddH

Wash at least twice with O. Drug bottles are sonicated for 10 minutes in 0.1 M NaOH. Silane solution is made in the hood while the vial is sonicated in NaOH. (Silane solution: 800 mL of 95% ethanol; 96 L glacial acetic acid; 25 mL glycidoxypropyltrimethoxy silane). After NaOH sonication, the medicinal bottle is ddH

O Wash at least twice in the dish. The medicine bottle is sonicated for 3 to 5 minutes in the saline solution. The vial is washed in a 100% EtOH dish. Drug Bottles N Pre-Refined

Dry with gas and store in 100 ° C. oven for 2 hours before use.

Biosorption Delivery Carriers and Methods

Some aspects of the invention provide that combination formulations and / or equivalent methods of administration present an effective amount of nontoxic biosorbent material, wherein the biosorbent material is an accessory for enhancing mucosal delivery of one or more biologically active agent (s). Composition or media. Biosorbents in the present invention generally or specifically adsorb to the surface of one or more compositions or target mucosa. Biosorbents maintain desirable concentration gradients of biologically active agents to allow large molecules such as peptides and proteins to penetrate the mucosal epithelium or through the mucosal epithelium. Indeed, in the compositions and methods of the present invention, biosorbents have been used to increase the ability of peptides and proteins to penetrate mucosal epithelium or through mucosal epithelium 2 to 4 times, often 5 to 10 times. Enhancement of such mucosal penetration allows large molecules to be effectively intermucosal delivered, for example mucosal penetration from the base to the nasal epithelium or adjacent extracellular compartments or serum or central nervous system tissue or body fluids.

This enhanced delivery represents a dramatically improved effect of bioactive peptides, proteins and other polymeric therapeutic materials. This result will depend on the hydrophilic portion of the composition so that hydrophilic materials will exhibit very high permeability compared to compositions that are insoluble in water. In addition to these effects, the use of biosorbents to enhance drug persistence at the mucosal surface can lead to a storage mechanism suitable for prolonged drug delivery, whereby the compositions not only penetrate the mucosal tissue but also at the mucosal surface. As the composition begins to disappear, it diffuses back to the mucosal surface.

Various adsorbents suitable for oral administration have been disclosed in the art, see US Pat. Nos. 3,972, 995; 4,259, 314; 4,680, 323; 4,740, 365; 4,573, 996; 4,292, 299; 4,715, 369; 4,876, 092; 4,855, 142; 4,250, 163; 4,226, 848; 4,948, 580; U. S. Patent No. 3,093, where it finds use within the scope of new compositions and methods of the present invention. Within the scope of the methods and compositions of the present invention, various potential bioadsorbent polymers as mucosal delivery platforms, such as nasal mucosa, have their capacity to react with mucosal surfaces after binding of biologically active agents and retention of Y2 receptor-binding peptides. It can be easily evaluated by the release capacity. In addition, well known methods will be applied to determine the biocompatibility of selected polymers with mucosal administration site tissue. When the target mucosa is covered with mucus (ie, without mucolytic or demux), the biosorbents can act as connecting links under the mucosal epithelium. Therefore, the term "bioadsorbent" as used herein refers to the raw but adsorbed mucosal tissues of the present invention, but the adsorption to mucosal tissues is caused by incomplete or temporary adhesion between the mucus layer and the underlying tissue, such as the surface of the nose where rapid mucus removal occurs. May be limited. In this regard, mucin glycoproteins continue to be secreted and form viscoelastic gels as soon as they are released from cells or glands. The luminal surface of the adsorption gel layer, however, continues to wear out by mechanical, enzymatic and / or cilia movement. In areas where such activity is more dominant or where longer adsorption times are desired, equivalent methods of administration and combination formulation methods of the present invention, as disclosed herein, may further incorporate mucosal and / or ciliary arrest methods or agents. will be.

Indeed, mucoadhesive polymers used in the present invention are natural or synthetic molecules that adhere to wet mucosal tissue surfaces by nonspecific composite mechanisms. In addition to these mucoadsorbent polymers, the invention also shows methods and compositions comprising biosorbents that directly adsorb to the cell surface rather than mucus by reaction, including specific, receptor-mediated ones. One example of a biosorbent with specific functions is a group of mixtures known as lectins. They can react with specific molecules such as glycoproteins, glycoproteins, glycolipids and become part of the nasal epithelial cell membrane to be recognized as "lectin receptors."

In some aspects of the invention, the biosorbent for enhancing intranasal delivery of a biologically active agent consists of a hydrophilic polymer matrix or mixture of polymers, such as water soluble or water expandable, that can adsorb on wet mucosal surfaces. These adsorbents can be formulated into ointments, hydrogels (see above), thin films, and other external forms. Often, these adsorbents have a mixed biologically active agent that enables local delivery or slow reaction of the active agent. Some are formulated with additional ingredients to facilitate penetration of the active agent into the nasal mucosa, such as in the individual's circulatory system.

Various natural and synthetic polymers make important bonds to mucosal and / or mucosal epithelial surfaces under physiological conditions. The intensity of this reaction can be easily measured by mechanical peeling or cutting tests. When applied to a wet mucosal surface, many dry substances will adhere at the same time, at least lightly. After this initial contact some hydrophilic materials begin to draw water by absorption, expansion or capillary forces. And if this water is absorbed from the underlying substrate or from polymer-tissue, the adsorption will fully achieve the purpose of enhancing the mucosal absorption of the biologically active agent. This "adsorption by hydration" will be very strong, but formulations adapted to use this mechanism must catch the swelling ability to continue the deformation of what is taken in the hydrated mucus state. This is intended for many of the hydrocolloids of the present invention, especially some cellulose derivatives which are generally non-adsorbable in the pre-hydration state. Nevertheless, biosorbent drug delivery systems for mucosal administration are very effective in the present invention when the biosorbent drug is used in dry polymer powder, microspheres, or membrane type delivery forms.

Other polymers adhere to mucosal surfaces not only in dry conditions but also fully hydrolyzed with excess water. Thus, the selection of mucoadsorbents requires proper examination of physiological conditions as well as physiological-chemical conditions, and under those conditions adhesion to tissue will be formed and maintained. In particular, the amount of moisture or water that is always present at the site where adsorption is intended, and the dominant pH, are known to have a significant effect on the mucoadhesion strength of different polymers.

Many polymer biosorbent drug systems have been studied for the past 20 years but have not always been successful. Various mediators, however, have recently been important for dentistry, ophthalmology, orthopedics, and surgery. Acrylic-based hydrogels, for example, are widely used as bioabsorbers. Acrylic-based hydrogels are well suited for bioabsorption, partly because they are fluid and non-abrasive in their expanded state, reducing damage that causes abrasion of tissues upon contact. Moreover, their high permeability in the expanded state allows unreacted monomers, uncrosslinked polymer chains, and initiators to be washed out of the matrix after polymerisation, which is an important feature in the selection of biosorbent materials for the present invention. to be. Acrylic based tools show very high adsorption bonds. For controlled mucosal delivery of peptides and protein drugs, the methods and compositions of the present invention optionally include polymeric delivery vehicles that partially have the function of protecting biological agents from proteolysis, while simultaneously passing through the nasal mucosa or nasal mucosa. Provided for enhanced penetration of peptides or proteins through mucous membranes. Biosorbent polymers in the present invention have demonstrated significant potential for enhancing oral drug delivery. For example, 9-desglycine amide, 8-arginine vasopressin (DGAVP), injected with 1% (W / V) saline and mucoadsorbed poly (acrylic acid) derivative polycarbophil, is injected into the duodenum of rats. Availability is increased by 3-5 times compared to the aqueous peptide solution without this polymer.

Poly (acrylic acid) -type mucoadhesive polymers are potential inhibitors of some intestinal proteolysis. Enzyme inhibition mechanisms are due to the strong affinity for divalent cations such as calcium and zinc of these types of polymers, which are essential cofactors of metallo-proteinases such as trypsin and chymotrypsin. . Removal of proteases by poly (acrylic acid) in these regulators has been reported to induce irreversible structural alteration of enzyme proteins followed by loss of enzymatic activity. At the same time, other mucoadhesive polymers (eg, some cellulose derivatives and chitosan) will not inhibit proteolytic enzymes under certain conditions. In contrast to other enzyme inhibitors (e.g., aprotinin, bestatin) studied for use in the present invention, these are relatively small molecules, and in terms of their size, intranasal absorption of the inhibitor polymers is minimized, Side effects will be eliminated. Thus, mucoadhesive polymers will act as both absorption-promoting adsorbents and enzyme protectors to enhance the controlled delivery of peptide and protein drugs, especially when particularly safe devices such as poly (acrylic acid) -types are considered.

In addition to the prevention of enzymatic degradation, the biosorbents and other polymeric or nonpolymeric absorption-promoters used in the present invention will directly increase the mucosal permeability of the biologically active agent. In order to facilitate transport across the nasal epithelial membrane of large hydrophilic molecules, mucoadhesive polymers and other biological agents are required to enhance the penetration effect except caused by prolonged residence time before coming to the mucosa in mucosal delivery systems. Has been. The time course of drug plasma concentrations reportedly teaches that biosorbent microspheres violently but temporarily increase the permeability of insulin across the nasal mucosa. Reportedly, other mucoadhesive polymers, such as, for example, chitosan, used in the present invention enhance the permeability of mucosal epithelium even when used in aqueous or gel form. Another mucoadhesive polymer that directly affects epithelial penetration is hyaluronic acid and its ester derivatives. Biosorbents which are particularly useful in the range of comparable administration and / or combination formulation methods and compositions of the present invention are chitosan and analogs and derivatives thereof. Chitosan is a nontoxic, biocompatible and biodegradable polymer that is widely used in pharmaceuticals and medicines because of its low toxicity and good biocompatibility. Natural polyaminosaccharides are prepared from chitin by N-acetylation with alkalis. As used in the methods and compositions of the present invention, chitosan increases retention of Y2 receptor-binding peptide proteins, analogs and mimetics, and other biologically active agents disclosed herein. This method of administration can also improve patient compliance and acceptability. As further provided herein, the methods and compositions of the present invention optionally comprise a new chitosan derivative or a chemically modified chitosan form. The new derivative used in the present invention is named (3- [1-4] -2-guanidino-2-dioxy-D-glucose polymer (poly-GuD). Chitosan is the N-diacetylate of chitin. As a naturally occurring polymer, it has been widely used in the preparation of microspheres in oral or nasal formulations Chitosan polymers have also been proposed as soluble mediators of parenteral drug delivery. o-methylisourea has been used to convert chitosan amines to some of its guanidinium, which is, for example, an equi-normal of chitosan and o-methylisourea above pH 8. ) Is prepared by reaction between solutions.

The guanidium product is-[14] -guanidino-2-dioxy-D-glucose polymer. In the present invention, abbreviated as Poly-GuD. (Monomer F. W. = 161 of chitosan amine; monomer F. W. = 203 of Poly-GuD guanidium).

Poly-GuD preparation method used in the present invention includes the following protocol.

Solutions:

Preparation of 0.5% Acetic Acid Solution (0.088N):

Pipette 2.5 mL glacial acetic acid into a 500 mL volumetric flask and dilute with purified water.

Preparation of 2N NaOH Solution:

Place about 20 g of NaOH pellets in a beaker with about 150 mL of purified water. Dissolve and cool at room temperature. Place this solution in a 250 mL volumetric flask and dilute with purified water.

O-methylisourea sulfite (0.4N urea group equivalent):

Transfer about 493 mg of O-methylisourea sulfite into a 10 mL volumetric flask to dissolve and dilute with purified water.

The pH of this solution is 4.2.

Preparation of barium chloride solution (0.2M):

Add about 2.086 g of barium chloride to a 50 mL volumetric flask and dilute with purified water.

Preparation of Chitosan Solution (0.06 N Amine Equivalent):

Add approximately 100 g of chitosan to a 50 mL beaker and add 10 mL of 0.5% acetic acid (0.088N). Stir until completely dissolved.

The pH of this solution is about 4.5.

Preparation of O-Methylisourea Chloride Solution (0.2 N Urea Group Equivalent):

Pipette 5.0 mL of O-methylisourea sulfite solution (0.4 N urea group equivalent) into a beaker and place 5 mL of 0.2 M barium chloride solution into the beaker. A precipitate is formed. Continue to mix the solution for 1 minute. Filter the solution with a 0.45 m filter and discard the precipitate. The concentration of O-methylisourea chloride solution in the supernatant is 0.2 urea group equivalents.

The pH of this solution is 4.2.

How to :

Add 1.5 mL of 2N NaOH to 10 mL of the prepared chitosan solution (0.06 N amine equivalent) as described in section 2.5. The pH of the solution is adjusted from about 8.2 to 8.4 with 2N NaOH. Stir this solution for 10 minutes. 3.0 mL of the O-methylisourea chloride solution (0.2 N urea group equivalent) described above is added. Stir the solution overnight.

The pH of the solution is adjusted with 0.5% acetic acid (0.088 N).

Dilute the solution with purified water to a volume of 25 mL.

The concentration of Poly-GuD in the solution is 5 mg / mL, 0.025 N (guanidium group) equivalents.

The additional substances classified as biosorbents in the present invention act to mediate specific reactions, which are in fact classified as "receptor-ligand reactions" between the complementary structures of the biosorbent composition and the components of the mucosal epithelial surface. . As can be seen from examples such as lectin-sugar reactions, many natural examples suggest the appearance of biosorbents with specific binding.

Many plant lectins have been studied as absorption promoting agents. One plant lectin is Phaseolusvulgaris hemagglutinin (PHA), which shows more than 10% oral biocompatibility when fed to rats. Tomato (Lycopersicon esculeutum) lectin (TL) is safe for various methods of administration.

In summary, the aforementioned biosorbents are useful in methods of administration comparable to the combination formulations of the present invention. The present invention includes forms and effective amounts of biosorbents for prolonging persistence, otherwise enhancing the mucosal absorption of one or more Y2 receptor-binding peptide proteins, analogs and mimetics, and other biological agents. . In some embodiments, the biosorbent acts as an 'adhesive agent', while in other embodiments, adjunct delivery or combination formulation of the biosorbent enhances the contact of the biologically active agent with the nasal mucosa. Increasing epithelial permeability to promote the reaction of epithelial cell “receptors” with specific receptor-ligands or to significantly increase drug concentration gradients measured at target sites (such as liver, serum, or central nervous system tissue or body fluids) where delivery occurs. To strengthen the contact. In addition, the additional biosorbents used in the present invention serve to inhibit enzymes (eg, proteases) in order to enhance the stability of the biotherapeutics administered to the mucosa in combination formulations or equivalent delivery.

Liposomes and Micellar Delivery Vehicles

Equivalent methods of administration and combination formulations of the present invention may optionally include Y2 receptor-binding peptide proteins, analogs and mimetics, including fats or fatty acids based on mediators, processing agents, or delivery mediators, And provide improved formulations to enhance mucosal delivery of other biologically active agents. For example, various formulations and methods are provided for mucosal delivery, which enhances the chemical and physiological stability of biologically active agents and increases the half-life of these agents (eg, protein hydrolysis, chemical modifications) in mucosal delivery. And / or active agents such as peptides or proteins mixed or encapsulated by a liposome, a medium in the form of mixed micelles to reduce sensitivity to denaturation).

In some aspects of the invention, differentiated delivery systems of biological agents consist of small fat carriers known as liposomes. These are actually naturally occurring, biodegradable, nontoxic, and immune fat molecules, and efficiently bind drug molecules, including peptides and proteins, across or over the membranes of these molecules. Liposomes as peptides and protein delivery systems of the present invention are further enhanced by the fact that the liposome membrane can maintain an aqueous solution environment preferred by the encapsulated protein within the carriers while protecting the encapsulated protein from proteolytic or other denaturing factors. Its attraction increases. Although all known liposome preparation methods do not lend themselves to encapsulation of peptides and proteins because of the physical and chemical properties of peptides and proteins, many methods allow for encapsulation of these polymers without substantial inactivation.

Various liposome preparation methods that can be used in the present invention are US Pat. Nos. 4,235, 871,4, 501,728, and 4,837, 028. Using liposome delivery, the biologically active agent is captured to or bound to the liposome or fat carrier.

Like liposomes, unsaturated long chain fatty acids can also enhance mucosal absorption activity to form closed carriers with structures similar to bilayer structures (so called ufasomes). These can be formed using oleic acid to capture biologically active peptides and proteins suitable for delivery of mucous membranes such as nasal mucosa in the present invention.

Other delivery systems used in the present invention combine the use of polymers and liposomes to combine the advantages of both polymers and liposomes, such as encapsulation in natural polymer fibrin. In addition, the release of biotherapeutic materials in this delivery system can be controlled by the use of covalent crosslinking and the addition of antifibrin degradation agents to the fibrin polymer.

Further simplified delivery systems for use in the present invention are the use of cationic fats as delivery carriers or mediators, which catalyze electrostatic reactions between fatty media and charged biologically active agents such as proteins and polyanionic nucleic acids. Effectively used. This allows the potent drug bundle to be in the proper form in the compartments in mucosal administration and / or continuous delivery systems.

Additional delivery vehicles for use in the present invention include long and medium chain fatty acids as well as surfactants mixed with fatty acid micelles. Most lipids that occur naturally in the ester form have an important relationship with their own transport capacity across the mucosal surface. Monoglycerides of free fatty acids and free fatty acids to which polar groups are attached proved to act as penetration enhancers of intestinal surface membranes in mixed micelle form. The discovery of membranes that alter the function of free fatty acids (carboxylic acids having chains of varying lengths from 12 to 20 carbon atoms) and their polar derivatives have prompted extensive research to use these agents as mucosal absorption enhancers.

Long chain fatty acids, in particular soluble fatty acids (unsaturated fatty acids and monoglycerides such as oleic acid, linoleic acid, monoolein) for use in the method of the present invention are disclosed as Y2 receptor-binding peptides, analogs and mimetics, and disclosed herein. It provides a useful medium for enhancing mucosal delivery of other biologically active agents. Medium chain fatty acids (C6 to C12) and monoglycerides have also been shown to enhance intestinal drug absorption and can be adapted for use in the mucosal delivery formulations and methods of the present invention. Moreover, sodium salts of medium and long fatty acids are effective delivery vehicles and absorption enhancers suitable for mucosal delivery of the biologically active agents of the invention. Thus, the fatty acids can be used in the form of aqueous solutions of sodium salts or by the combination of non-toxic supernatants such as polyoxyethylated hydrogenated castor oil, sodium taurocholate and the like. Other fatty acids and formulations in mixed micelle form useful in the present invention include, but are not limited to: A selective combination of bile salts such as Na caprylate (C8), Na caprate (C10), Na laurate (C12) or Na oleate (C18), glycocholate and taurocholate.

Pegylation

Additional methods and compositions provided within the scope of the present invention include chemical modification of biologically active peptides and protein weaves, such modifications of polymers such as dextran, polyvinyl, glycopeptides, polyethylene glycols and poly amino acids. By covalent bonds of the sex substances. The resulting peptides and proteins retain their biological activity and solubility upon mucosal administration. In another embodiment, Y2 receptor-binding peptide proteins, analogs and mimetics, and other biologically active peptides and proteins are conjugated with polyalkylene oxide polymers, in particular polyethylene glycol (PEG). US Patent No. 4,179, 337.

Amine-reacted PEG polymers for use in the present invention include SC-PEG and 20000 having a molecular weight of 2000,5000, 10000,12000; U-PEG-10000; NHS-PEG-3400- biotin; T-PEG-5000; T-PEG-12000; And TPC-PEG-5000. PEGylation of biologically active peptides and proteins is accomplished by modification of carboxy sites (eg, aspartic acid or glutamic acid groups in addition to the carboxy terminus). The utility of PEG-hydrazide in the selective modification of carbodiimide-activated protein carboxy groups under acidic conditions is described. Alternatively, PEG modifications of two functions of biologically active peptides and proteins can be used. In some processes, charged amino residues, including lysine, aspartic acid and glutamic acid, have a clear tendency to have solvent access at the protein surface.

Other Modifications for Stabilization of Active Agents

In addition to pegylation, biologically active agents such as peptides and proteins used in the present invention enhance the circulation of half-life by protecting the active agent in conjunction with other protective or stabilizing compositions, for example one or more immunoglobulins. By a method of making a mixed protein with an active peptide, analog or mimetic linked to one or more mediator proteins such as chains.

Formulation and Dosing

Mucosal delivery formulations of the invention consist of Y2 receptor-binding peptides, analogs and mimetics, substantially one or more pharmaceutically acceptable mediators and optionally other therapeutic ingredients. The mediator (s) must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the formulation and must not produce a deleterious effect that is unacceptable to the patient. Such mediators are those described above or otherwise well known in the pharmaceutical arts. Preferably, the formulation should not contain substances such as enzymes or oxidants that are known to be incompatible when administered with a biological agent. The formulations may be formulated by methods well known in the art of pharmacy.

Within the scope of the compositions and methods of the present invention, the Y2 receptor-binding peptide proteins, analogs and mimetics, and other biologically active agents disclosed herein are oral, rectal, vaginal, intranasal, pulmonary, skin It may be administered to a patient by a variety of mucosal administration methods, including through or local delivery methods on the eyes, ears, skin or other mucosal surfaces. Optionally, Y2 receptor-binding peptide proteins, analogs and mimetics, and other biological agents disclosed herein may be intramuscular, subcutaneous, intravenous, intraarterial, intraarticular, intraperitoneal, or parenteral routes. It may be administered equally or auxiliary by nasal mucosal routes, including. In another embodiment, the biologically active agent (s) is an ex vivo tissue or organ therapeutic formulation composition comprising the biologically active agent in a suitable liquid or solid medium, for example in a mammalian patient directly in vitro. It can be administered to the cells, tissues or organs from which they are derived.

The compositions according to the invention are often administered in aqueous solutions for spraying in the nose or lungs and formulated in spray form by various methods known in the art. Preferred systems for making preparations for spraying the nose are US Pat. No. 4,511,069. The formulations may be in a multi-dose container, such as, for example, a sealed dispensing system disclosed in US Pat. No. 4,511,069. In addition, air delivery forms such as compressed air, jets, ultrasonics, and piezoelectric nebulizers may be included, which deliver biological agents suspended or dissolved in pharmaceutical solvents such as water, ethanol, or mixtures thereof. .

The solution sprayed into the nose and lung of the present invention consists of a drug or delivery drug optionally formulated with a nonionic surfactant (eg, polysorbate-80), and one or more buffers. In some embodiments of the invention, the nasal spray solution also consists of a propellant. The pH of the solution sprayed into the nose is optionally between about pH 3.0 and 6.0, preferably 5.0 ± 0.3. Suitable buffers for use in the compositions are those described above or otherwise known in the art. Other compositions may be added to enhance or maintain chemical stability. Such compositions include preservatives, surfactants, dispersants or gases. Suitable preservatives include, but are not limited to, phenol, methyl parabens, parabens, m-cresol, thiomersal, chlorobutanol, benzyl alcoholic chloride, and the like. Suitable surfactants include, but are not limited to, oleic acid, sorbitan trioleate, polysorbate, lecithin, phosphatidyl choline, and various long chain diglycerides and phospholipids. Suitable dispersants include, but are not limited to, ethylenediamineetheracetic acid, and the like. Suitable gases include, but are not limited to, nitrogen, helium, carbon fluorocarbons (CFCs), hydrocarbon fluorocarbons (HFCs), carbon dioxide, air, and the like.

In another embodiment, mucosal formulations are administered in dry powder formulations, which formulations comprise the biologically active agent in a dried form, usually lyophilized, within a size range suitable for intranasal delivery. The minimum particle size suitable for deposition within the range of the nose or lung is usually about 0.5 μ mass median equivalent aerodynamic diameter (MMEAD), typically about 1 μMMEAD, and more practically about 2 μMMEAD to be. The maximum size suitable for deposition within the range through the nose is about 10 μMMEAD, generally about 8 μMMEAD, more practically about 4 μMMEAD. Nasal respirable powders within this size range can be produced by convenient techniques such as jet milling, spray drying, solvent precipitation, supercritical fluid concentration, and the like. Dry powders with the appropriate MMEAD can be administered to the patient via a convenient dry powder inhalant (DPI), which is by breathing the patient based on lung or nasal inhalation to disperse aerosolized amounts of powder. Alternatively, the dry powder may be administered through an air assisted device, using an external power source such as a piston pump to disperse the aerosolized amount of powder.

Tools for dry flour actually require about 1 mg to 20 mg of flour to produce an aerosolized dose ("puff"). If the amount of the required or desired biologically active agent is less than this, then the powdered active agent can actually be mixed with the pharmaceutical dry bulking powder to match the total powder mass. Preferred bulking powders are sugar, lactose, dextrose, mannitol, glycine, trehalose, human albumin serum (HSA), and starch. Other suitable dry bulking flours are cellobiose, dextran, maltotriose, pectin, sodium citrate, and the like.

In order to formulate compositions for mucosal delivery within the scope of the present invention, various pharmaceutically acceptable additives may be combined with the active agent as well as a medium or salt for the dispersion of the biologically active agent. Preferred additives are pH adjusting agents such as arginine, sodium hydroxide, citric acid and the like, but are not limited to the examples described above. In addition, local anesthetics (e.g. benzyl alcohol), isotonic agents (e.g. sodium chloride, mannitol, sorbitol), absorption inhibitors (e.g. Tween 80), solubility enhancers (e.g. cyclodextrins and their derivatives), stabilizers (Eg albumin serum), and reducing agents (eg glutathione). When the mucosal delivery composition is a liquid, the tonicity of the formulation can be measured with reference to the tension of the 0.9% (W / V) physiologically alive solution to maintain consistency with the formulation, which is administered The elongate device can be practically adjusted until there are no substantial and irreversible side effects that can be induced at the nasal mucosa site. In general, the tension of the solution is about one-third to three, more practically one-half to two, and most often three-fourth to 1.7.

The biologically active agent may be dispersed in a base or carrier, which base or carrier will consist of hydrophilic compositions having the ability to disperse the agent and other desired additives. The base will be selected from a variety of suitable mediators. Mediators include copolymers of polycarboxylic acids or their salts, carboxylic anhydrides with other monomers (e.g. methyl (meth) acrylate, acrylic acid, etc.) (e.g. maleic anhydride), hydrophilic vinyls such as polyvinyl acetate Polymers, polyvinyl alcohol, polyvinylpyrrolidone, cellulose derivatives such as hydroxycellulose, hydroxypropylcellulose, etc., and natural polymers such as chitosan, collagen, sodium alginate, gelatin, hyaluro Acids, and therefore non-toxic metal salts, including but not limited to. Often, biodegradable polymers can be selected as the base or mediator, for example, polyacetic acid, poly (lactic acid-glycolic acid) copolymer, polyhydroxybutyric acid, poly (hydroxybutyric acid-glycolic acid) copolymer and Mixtures accordingly. Alternatively or additionally, synthetic fatty acids such as polyglycerol fatty acid esters, sucrose fatty acid esters can be used as a vehicle. Hydrophilic polymers and other media may be used alone or in combination. And by enhancing structural integrity, the media can be distributed by partial crystallization, ionic bonding, crosslinking, and the like. The media may be provided in various forms such as body fluids, viscoelastic solutions, gels, doughs, flours, microspheres, and membranes to directly contact the nasal mucosa. The use of mediators selected in the present invention will facilitate the uptake of the biologically active agent.

The biologically active agent can be combined with the base or the carrier according to various methods. And release of this active agent will be by combining with diffusion, dispersion of the media or formulation with moisture channels. In some cases, the active agent is dispersed in microcapsules (microspheres) or nanocapsules (nanospheres) formulated with a suitable polymer, such as isobutyl 2-cyanoacrylate, in a biocompatible dispersion medium that contacts the nasal mucosa. Dispersed, which exhibits sustained delivery and biological activity over extended periods of time.

To further enhance mucosal delivery of the pharmaceutical agents of the present invention, formulations of the active agents may include such substances as bases or additives that are constituents having a hydrophilic low molecular weight. It provides a passing medium for these hydrophilic low molecular materials, which are water soluble activators such as physiologically active peptides or proteins that diffuse through the base to the body surface where the active agent is absorbed. Hydrophilic low molecular weight materials selectively absorb moisture from mucus or air upon administration to dissolve the water soluble active peptide. The molecular weight of the hydrophilic low molecular weight material is 10000 or less and preferably 3000 or less. Representative hydrophilic low molecular materials are oligo-, di- and sucrose, mannitol, sorbitol, lactose, L-arabinose, D-erythrose, D-ribose, D-xylose, D-mannose, trihalose, D-galactose , Monosaccharides such as lactulose, cellobiose, gentiose, glycerin and polyethylene glycols. Other examples of hydrophilic low molecular materials useful as mediators in the present invention include N-methylpyrrolidone, and alcohols (eg, oligovinyl alcohol, ethanol, ethylene glycol, propylene glycol, etc.). These hydrophilic low molecular materials can be used alone or in combination with each other or in combination with other active or inert compositions of nasal formulations.

Compositions of the present invention will alternatively include pharmaceutically acceptable mediators as are necessary for suitable physiological conditions, including pH adjusting agents, buffers, tension adjusting agents, wetting agents, and the like. For example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and the like. Pharmaceutically acceptable mediators may be conveniently used in solid compositions. Examples include pharmaceutical grade mannitol, lactose, starch, magnesium stearate, saccharin sodium, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like.

The therapeutic composition of the biologically active agent to be administered may be formulated in a well-equipped structure suitable for solution, microemulsion, or high concentration of active ingredient. The media may be solvents or dispersion media including such as water, ethanol, polyols (eg glycerol propylene, glycols, and liquid polyethylene glycols, and the like), and mixtures thereof. Proper fluidity of the solution can be maintained by the following examples, such as by the maintenance of the desired particle size in the case of dispersible formulations or by coating with a lecithin or by the use of a surfactant. will be. In many cases, it is desirable to include isotonic agents, such as sugars, polyacohols (mannitol, sorbitol, or sodium chloride) in the composition. Absorption of biological agents can be prolonged by retarding the absorption of substances such as monostearic acid salts, gelatin in the composition.

In some embodiments of the invention, the biologically active agent is administered in a time-release formulation. For example, the composition includes a polymer with slow release. The active agent can be formulated in media with a polymer, microencapsulated delivery system or biosorbent gel. In various compositions of the present invention, agents that delay absorption such as aluminum stearate hydrogel and gelatin can be included in the composition to prolong the delivery of the biologically active agent. When a biologically active agent is desired to take effect slowly over time, it includes a binder that slowly exhibits a reaction suitable for use in the present invention, wherein the binder provides a biocompatible reaction that inactivates the active agent and binds the biologically active agent. Slowly appearing substances. Many of these materials are known in the art. Useful as slow-responsive binders are substances that are metabolized slowly under physiological conditions after they have been delivered intranasally (eg, when they are in a physical fluid state after nasal mucosal surface or intermucosal delivery). Suitable binders include, but are not limited to, biocompatible polymers and copolymers previously used in the sustained release formulation art. These biocompatible compositions are nontoxic and inert to surrounding tissues and do not cause nasal inflammation, immune response, inflammation, and other serious side effects. They are also metabolized to metabolites that are biocompatible and easily released by the body.

Examples of polymeric materials used in the present invention include, but are not limited to, polymer matrices derived from copolymers having hydrolysable ester linkages and homopolymer esters. Many such polymeric materials are known in the art to be biodegradable, non-toxic and produce less degradation products. Examples of polymers include polyglycolic acids (PGA) and polylactic acids (PLA), poly (DL-lactic acid-co-glycolic acid) (DL PLGA), poly (D-lactic acid-coglycolic acid) (D PLGA) and poly (L-lactic acid-co-glycolic acid) (L PGA). Other useful biodegradable or biowearable polymers include, but are not limited to: Poly (epsilon-caprolactone), poly (epsilon-aprolactone-CO-lactic acid), poly (E-aprolactone-CO-glycolic acid), poly (beta-hydroxy butyric acid), poly (alkyl-2-cyano Acrylates), poly (hydroxyethyl methacrylate) hydrogels, polyamides, poly (amino acids) (ie, L-leucine, glutamic acid, L-aspartic acid and the like), poly (ester urea), Poly (2-hydroxyethyl DL-aspartamide), polyacetal polymers, polyorthoesters, polycarbonates, polymaleamides, polysaccharides and thus copolymers. Many ways of formulating these formulations are generally known in the art. Other useful formulations include slow-effecting compositions such as microcapsules, US Pat. Nos. 4,652, 441 and 4,917, 893, lactic acid-glycolic acid copolymers useful for making microcapsules and other formulations, US Pat. 4,677, 191 and 4,728,721, and sustained-release compositions for water soluble peptides, US Pat. No. 4,675, 189.

Sterile solutions are prepared by combining the required amount of active agent in an appropriate amount of solvent consisting of a combination of one or more of the components listed above, which occurs after the filtered sterilization process. In general, dispersions are prepared in combination with an active agent, aseptic mediators including the basic dispersion media and some of the ingredients listed above. The activity of microorganisms can be prevented by various antibacterial or antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

Mucosal administration according to the present invention can be effectively administered by the patient himself if there are sufficient safety measures to control and monitor the dosage and side effects. Mucosal administration can also overcome the weaknesses of other dosage forms, such as infusion, which can be painful and expose the patient to infection, which may present a problem of drug bioavailability. For nasal and pulmonary delivery, systems for controlling the dispersion of the sprayed therapeutic fluid in the air are well known. In one embodiment, a metered amount of active agent is delivered by a specially designed mechanical pump (US Pat. No. 4,511,069).

Administration (dose)

For prophylactic and therapeutic purposes, the biologically active agents disclosed herein are administered to a patient in a single dose to be delivered. By continuous delivery over extended periods of time (e.g., continuous intradermal, mucosal, or intravenous delivery) or by repeated dosing protocols (e.g., hourly, daily, or weekly, repeated dosing protocols) It is by. In the present invention, the dosage of a biologically active agent effective for treatment includes repeated administrations in an extended prophylactic or treatment plan, which is important for alleviating one or more symptoms or perceived conditions associated with the disease or condition listed above. Produces results. Determination of the effective dosage in the present invention was based on animal models prior to actually treating humans, and was dictated by an effective amount that would significantly reduce the occurrence or intensity of the subject's symptoms or conditions of the disease. Suitable models in this regard are, for example, murines, rats, pigs, cats, non-human primates, and other acceptable animal subjects known in the art. Alternatively, effective dosages can be determined using ex vivo models such as immunological and histopathological analysis. Even with these models, only general calculations and adjustments can be used to determine the amount of biologically active agent that is effective for treatment (e.g., intranasally effective to achieve the desired effect, effective for intradermal delivery, effective intravenously, effective in mucus) Practically necessary to determine the amount and concentration for the administration of the sheep).

The actual dosage of the biologically active agent may be in addition to the patient's disease symptoms or particular condition (eg, the patient's age, size, health condition, extent of symptoms, infectious agent, etc.), time or route of administration, and other drugs or therapeutic agents currently being administered. It will of course vary depending on the specific pharmacology of the biologically active agent to produce the desired activity or biological response in the patient. Dosage regimens may be adjusted to produce the appropriate prophylactic or therapeutic response. The amount of biologically active agent effective for treatment is also more important than any toxic or side effects of the active agent. The unlimited range of Y2 antagonists effective for treatment within the scope of the methods and formulations of the present invention is from 0.7 μg / kg to 25 μg / kg. To promote weight loss, intranasal doses of Y2 receptor-binding peptides are administered in amounts that are high enough to promote satiety and do not cause unwanted side effects such as nausea. The preferred dose of PYY _3-36 is about 1 μg-10 μg / kg of the patient's body weight, most preferably 3 μg / kg at about 1.5 μg / kg of the patient's body weight. The average dose a patient will receive is from 50 μg to 1600 μg, more preferably from 75 μg to 800 μg, most preferably from 100 μg, 150 μg, 200 μg to about 400 μg. In contrast, the range of amounts of biologically active agent effective for unrestricted treatment ranges from about 50.01 pmol / weight (kg) to about 10 pmol / weight (kg), from about 0.1 pmol / weight (kg) to about 5 pmo / kg (kg). Between about 0.5 pmol / kg in weight and about 1.0 pmol / kg in weight. At doses within this range, one or several can be administered, for example, several times per day or weekly. Each dose, it is desirable to administer at least 1 microgram of biologically active agent (eg, one or more Y2 receptor-binding peptide proteins, analogs and mimetics, and other biological agents) to the average human patient. More practically between about 10 μg and 5.0 μg, and in some embodiments about 100 μg and 1,0 or 2.0 mg. In some oral administrations, a high dose of 0.5 mg / kg will be required to obtain adequate plasma levels. It has been reported that specific dosages should be evaluated and controlled over time according to the patient's individual needs and expert judgment to control or supervise the administration of peptides and other biologically active agents that can penetrate. Intranasal doses of PYY range from 50 μg to 1600 μg, preferably from 75 μg to 800 μg, more preferably from 100 μg to 400 μg and most preferably from 100 μg to 200 μg with the highest effect at 150 μg. I think it will be. Repetitive doses of intranasal formulations of the present invention are between 0.1 hours and 24 hours, preferably between 0.1 and 2.0 hours, wherein the Y2 receptor-binding peptides have a therapeutic effect while minimizing the risk of overexposure and side effects. Standards will be maintained to ensure that maximal efficacy is sustained. It can be taken several times a day and preferably promotes satiety when taken an hour and a half before meals or when hungry.

Dosages of Y2 antagonists, such as PYY, can vary widely by clinician or patient observations, if administered on their own without a prescription, and maintain the desired concentration at the target site.

In another embodiment, the present invention provides compositions and methods for intranasal delivery of Y2 receptor-binding peptide, wherein the Y2 receptor-binding peptide composition (s) is repeatedly administered in an effective amount intranasally, which The Y2 receptor-binding peptide is administered to the patient several times during the daily or weekly unit in order to alleviate and keep the pulsation of the pulse due to the Y2 receptor-binding peptide effective for an extended period of time for a prolonged period of time. The compositions and methods provide Y2 receptor-binding peptide composition (s), which are administered by the patient himself through the nose at intervals of two to six times daily, such that the Y2 receptor is extended for an extended period of 8 to 24 hours. Pulses attributable to binding peptides are alleviated and kept low to be effective for treatment.

Kits

The invention also includes kits, containers, and multicontainers comprising the pharmaceutical compositions, active ingredients, and / or dosage means used to prevent and treat diseases and other conditions in mammalian patients as described above. do. In summary, these kits or containers may contain a combination of one or more Y2 receptor-binding peptide proteins, analogs and mimetics, and / or other biologically active agents formulated and formulated for mucosal delivery. Contains the containing container or formulation.

Intranasal formulations of the present invention may be administered using any spray bottle or syringe. An example of a non spray bottle is "Non Spray Pump w / Safety Clip, Pfeiffer SAP # 60548" which delivers 0.1 mL volume per spray and has a core length of 36.05 mm. It is available from Pfeiffer, Princeton, NJ. Nasal doses of Y2 receptor-binding peptides such as PYY range from 0.1 μg / kg to about 1500 μg / kg. When administered as a nasal spray, a suitable particle size for spraying is suitably 10-100 μm (microns), preferably 20-100 μm.

To promote weight loss, a single dose of Y2 receptor-binding peptide PYY is administered high enough to promote satiety and low enough not to induce side effects such as nausea. Preferred dosages of Y2 receptor-binding peptides such as PYY (3-36) are about 3 μg-10 μg / kg per patient body weight, most preferably about 6 μg / kg per patient body weight. The standard patient dose is from 50 μg to 800 μg, more preferably from 100 μg to 400 μg and most preferably from 150 μg to about 200 μg. Y2 receptor-binding peptides such as PYY (3-36) are appropriately administered at least 10 minutes to 1 hour before meals except 12 to 24 hours before meals to prevent nausea. The patient is recommended to take at least once a day before all meals until the desired weight loss is indicated. The patient then receives an amount that can maintain a reduced weight at least once a week, preferably once a day.

As the data of the examples below show, PYY (3-36) reduces appetite when administered intranasally in humans using the Y2 receptor-binding peptide formulation of the present invention. The above examples also show that for the first time postprandial physiological levels of PYY peptides can be reached via the intranasal route by intranasal administration using the Y2 receptor-binding peptide formulation of the invention wherein PYY (3-36) is a Y2 receptor-binding peptide. Show that you can.

The following examples are provided by way of example and not by way of limitation.

Example 1

A typical formulation for enhancing non-mucosal delivery of peptide YY as the teaching of immediate character is prepared and evaluated is as follows.

TABLE 1

Example 2: Non Mucosal Delivery-Infiltration Kinetics and Cytotoxicity

1. Organic Type Model

The following methods are used in various methods disclosed herein for the rate and side effects of peptide YY in formulations and formulations of the present invention, such as determining non-mucosal delivery parameters, efficacy, and cooperative formulation or equivalent administration with peptide YY. It is usefully used to evaluate the characteristics of the delivery-enhancing agent.

Penetration kinetics and cytotoxicity are also useful for determining the efficacy and characterizing the various mucosal delivery-enhancing agents disclosed herein for peristaltic formulations or equivalent administration with peptide YY. In one typical protocol the lack of infiltration response and unacceptable cytotoxicity is demonstrated by the intranasal delivery-enhancing agents previously disclosed in combination with biologically active therapeutic agents and exemplified by peptide YY.

The Epiairway system was developed by MatTek Corp (Ashland, MA) as a model of surface weighted epithelial respiratory tract. Epithelial cells were grown in porous cells based on cell culture inserts at the air-liquid interface, resulting in a dividing form of the cells. The apical space becomes cilia with microvilli microstructures and mucus produced by epithelial cells (the presence of mucins identified by immunoblotting). The insert has a diameter of 0.875 cm and provides a space area of 0.6 cm ² . The cells are plated onto the inserts in the factory for about three weeks before shipping. One kit consists of 24 units.

A. Upon arrival, the unit is placed on a sterile bed within a 6-well microplate. Each well receives 5 mL of proprietary media. This DMEM-based fluid has no serum but is added to epidermal growth factor and other factors. The solution was always tested for the endogenous level of any cytokine or growth factor and considered for intranasal delivery but was free for any cytokines and growth factors studied so far except for insulin. The 5 mL volume would be sufficient to provide only contact with the bottom of the units on their location, but allow the space at the apex of epithelial cells to leave room for direct contact with air. Sterile tweezers are used in this step and in all the minor steps involving moving the units to the well containing the liquid which is sure to be trapped without air between the bottom of the units and the liquid.

B. Units in plates are maintained at 37 ° C. for 24 hours in an incubator with an atmosphere of 5% CO ₂ in air. At the end of this time the solution is replaced with a new solution and the units are returned to the incubator for 24 hours.

2. Experimental Protocol—Infiltration Response

A. A kit of 24 airway units can be routinely consumed to evaluate five different formulations, each applied to a quad well. Each well is consumed for determination of infiltration reaction rate (4 time points), epithelial resistance, mitochondrial reductase activity measured by MTT reduction, and cell lysis measured by LDH release. An additional set of wells is used as a control group, and it is treated as a sham during the determination of the penetration kinetics but otherwise treated identically as the units for the determination of resistance and viability with epithelial pain containing the test sample. . Decisions on the control group are also routinely made up of quadruple units but sometimes consumed triple units for regulation and the remaining four units in the kit for measuring epithelial pain and resistance and viability on untreated units. It has units for determination of total LDH levels that serve as a reference for frozen or frozen and thawed 100% cell lysis.

B. In all experiments, the non-mucosal delivery formulation studied is applied to the space of the vertices of each unit in a volume of 100 μl, which is sufficient to cover the entire space of the vertices. Appropriate volumes of test formulations (typically not requiring more than 100 μl) at concentrations applied to the spaces of the vertices are ignored for subsequent determination of the concentration of the active substance by ELISA or other scheduled assays.

C. Units are placed in 6 well plates except for being placed for the experiment: each well is sufficient to contact the bottom of the porous cell membrane of the unit but 0.9 so that no significant increase in hydrostatic pressure on the unit occurs. Include mL of liquid.

D. In order to minimize the potential cause of error and avoid the formation of any concentration gradients, units are transferred from one containing 0.9 mL well to another at each time point in the study. This transition is made at the following time points based on 0 hours in a 100 μl volume of test material applied to the space of the apex: 15 minutes, 30 minutes, 60 minutes, and 120 minutes.

E. Units in their plates between time points are maintained in a 37 ° C. incubator. Plates containing 0.9 mL of liquid per each well are also maintained in the incubator to minimize the change in temperature that occurs over a short period of time when the plates are removed and units are transferred from one well to another using aseptic tweezers.

F. At the completion of each time point, the solution is removed from the wells in each transferred unit and for determination of the concentration of the penetration test substance if the test substance is from cytotoxicity, release of cytoplasmic enzymes, lactic acid dehydrogenase, epithelium. Separate into two tubes (one tube receives 700 μl and the other receives 200 μl). These samples are kept in the refrigerator if the analysis is performed for 24 hours or the samples are kept at −80 ° C. until they are incidentally fractionated and thawed once for analysis. Repeated freeze-thaw cycles should be avoided.

G. Label all tubes, plates, and wells before testing to minimize errors.

H. At the end of the 120 minute time point, units are transferred from the wells containing the last 0.9 mL to 24-well microplates containing 0.3 mL liquid per well. This volume is again sufficient to contact the bottom of the units but does not allow any significant increase in hydrostatic pressure on the units. Units are placed in an incubator prior to measurement of epithelial pain and resistance.

3. Experimental Protocol-Epithelial Penetration Resistance

A. Respiratory Tracheal Epithelial cells form dense joints that limit the flow of solutes through tissues in vivo, such as in vitro. These joints give several hundred ohms × cm ² of epithelial pain and resistance in the injured airway tissue. In MatTek airway units, the epithelial penetration resistance (TER) is typically required to be around 1000 ohms x cm ² by the manufacturer. In a slightly low (700-800 ohms × cm ² ) penetration study, the TER was found to control the ER airways units exposed during the turn of the steps, but this value is important for penetration because the penetration of small molecules is proportional to the inverse of the TER. The supply of obstruction is still high enough. Porous membrane-bottom units excluding the cell, in contrast, provide only minimal infiltrating membrane resistance (5-20 ohms x cm ² ).

B. Accurate measurement of TER requires that the electrodes of the ohmmeter are located above and below the critical surface space above and below the cell membrane, and that the distance of the electrodes from the cell membrane is regeneratively controlled. The method for TER determination recommended by MatTek and used for all experiments is described here by the "EVOM" ^TM epithelial voltmeter and the "ENDOHM" ^TM tissue resistance chamber from World Precision Instruments, Inc., Sarasota, FL. Need).

C. The chamber is initially filled with Dulbecco's phosphate buffered saline (PBS) to equilibrate the electrode for at least 20 minutes prior to TER determination.

D. The determination of TER consists of 1.5 mL of PBS in the chamber and 350 μl of PBS in the cell membrane-bottom unit measured. The topmost electrode is adjusted over the cell membrane of a unit that does not contain only cells (but which contains 350 μl of PBS) and then fixed to a certain regenerative position. The resistance of a cellless unit is typically 5-20 ohms × cm ² (“deskage resistance”).

E. Once the chamber is ready and the background resistance is recorded, the units in the 24-well plate that were only used for infiltration crystals are removed from the incubator and placed in the chambers individually for the TER determination.

F. Each unit is initially transferred to a petri dish containing PBS to confirm that the membrane bottom is damp. Fractionation of 350 μl PBS is added to the unit and then carefully aspirated with a labeled tube to clear the apex space. A second wash of 350 μl PBS is then applied to the unit and drawn into the same collection tube.

G. The unit is slowly freed of dirt on its outer surface with excess PBS prior to being placed in the chamber (including fractionation of fresh 1.5 mL PBS). Fractionation of 350 μl PBS is added to the unit before the top electrode is placed on the chamber and the TER is read by the EVOM meter.

H. After the unit's TER has been read in the ENDOHM chamber, the unit is removed, the PBS is sucked and stored, and the unit is returned to a 24-well plate containing 0.3 mL of liquid per well with an air interface on top of the apex.

I. Units are read in the following sequence: by all cross-controlled controls, then by all formulation-treated samples, then by a second TER read of each of the cross-controlled controls. . After all TER crystals have been completed, the units in the 24-well microplates are returned to the incubator to determine viability by MTT reduction.

4. Experimental Protocol—Viability by MTT Reduction

MTT is reduced by mitochondrial dehydrogenase activity and is non-mitochondrial NAD (P) from cells that are capable of producing respiratory rupture or colored without being dissolved by cells capable of growing with intact mitochondrial function. Tetrazolysone salts that can be cell-penetrated by H dehydrogenase activity. Formation of formazan is a good indicator of the viability of epithelial cells because these cells do not produce significant respiratory bursts. We used MTT reagent kits prepared by MatTek Corp for their units to access viability.

A. The MTT reagent is supplied in concentrated form and diluted into an appropriate DMEM-based diluent on the day of the viability assay (typically the penetration reaction rate in the afternoon of the day and TER in the morning). Insoluble reagents are removed by simple centrifugation prior to use. The final MTT concentration is 1 mg / mL.

B. The final MTT solution is added to wells of 24-well microplates at a volume of 300 μl per well. As emphasized above, this volume is sufficient to contact the cell membranes of the airway units, but without the significant amount of hydrostatic pressure on the cells.

C. The units are removed from their 24-well plate after TER measurement, and after removing any excess liquid from the outer surface of the units they are transferred to the plate containing the MTT reagent. The units in the plate are then placed for 3 hours at 37 ° C. in an incubator with an atmosphere of 5% CO ₂ in air.

D. After 3 hours of incubation, the units containing viable cells will visually turn purple. Insoluble formazan must be extracted from the cells in their units to a extent that is in the range of MTT reduction in quantity. Extraction of formazan is accomplished by transfer to a 24-well microplate containing 2 mL extract per well of units, after which excess liquid is removed from the outer surface of the units as before. This volume is sufficient to completely cover both the cell membrane and the space of the apex of the units. The extraction is allowed to proceed overnight in a light-resistant chamber at room temperature. MTT extracts traditionally contain high concentrations of detergent and destroy cells.

E. After extraction, the fluid from each unit and the fluid around the well are combined and then fractionated into a 96-well microplate (200 μl fractionation is appropriate) and 570 on a VMax multi-well microplate spectrophotometer. Transfer to tube for determination of absorption at nm. To ensure turbidity from debris from extracted units that do not contribute to absorption, absorption at 650 nm is also determined for each well in the VMax and is automatically removed from absorption at 570 nm. A "blank" for the determination of formazan absorption is a 200 μl aliquot of the extract with no units revealed. This absorption value is believed to constitute zero viability.

F. Two units from each kit of 24 airway units are left untreated during the determination of penetration reaction rate and TER. These units are used in places such as quantitative control for 100% cell viability. In all of the studies we performed, there was no statistically significant difference in the viability of cells in these untreated units versus cells in transversely processed and TER determinations performed for infiltration kinetics for infiltration reaction rates. Absorption of all units treated with the test formulations is considered to be linearly proportional to the percent viability of the cells in the units at incubation time with the MTT. It should be emphasized that this analysis typically should be performed no more than 4 hours after introduction of the test material into the peak space, followed by cleaning of the peak space of the units during the TER determination.

5. Determination of viability by LDH release

During the measurement of mitochondrial reducing activity by MTT reduction, a sensitive probe of cell viability, the assay requires destroying the cells and thus can only be performed at the end of each study. When necrosis lysis of the cells is performed, their cytoplasmic content flows into the periphery, and cytoplasmic enzymes such as lactic acid dehydrogenase can be detected in these fluids. Analysis of the LDH in the liquid can be performed on samples of the liquid removed at each time point of the two hour determination of the penetration reaction rate. Thus, the cytotoxic effects of formulations that have not been developed over a critical time period can be detected as the effects of formulations that first induce exposure to airway epithelium for several minutes.

The recommended LDH assay for the evaluation of cell lysis of A. airway units is based on the conversion of lactic acid to pyruvate with the production of NADH from NAD. NADH is then reoxidated following simultaneous reduction of the tetrazolysin salt INT and promoted by crude “diaphorase” preparation. Since formazan formed from the reduction of INT is soluble, a full analysis of LDH activity can be performed in a homogeneous aqueous solution containing lactic acid, NAD, diaphorase, and INT.

B. Assays for LDH activity are performed on 50 μl aliquots from samples of the “supernatant” fluid surrounding the airway unit and collected at each time point. These samples are stored in the refrigerator for no more than 24 hours, or thawed after freezing for several hours after collection. Each Epiairway unit produces samples of supernatant collected 15 minutes, 30 minutes, 1 hour, and 2 hours after application of the test substance. Aliquots are all transferred to 96 well microplates.

C. 50 μl of aliquot, which has never been revealed as a unit, is provided with negative control of “blank” or 0% cytotoxicity. We note that the apparent level of this "endogenous" LDH after the reaction of the analytical reagent mixture with the undisclosed solution is all of the handled control units beyond the 2-hour total time course needed to conduct infiltration reaction studies between experimental errors. We found something like the apparent level of LDH released by. Thus, between experimental errors, these cross-treated units do not show cell lysis of epithelial cells over a time course of invasion kinetics.

D. Within the protocol for the determination of the rate of penetration permeation in a unit that had no previous handling, to prepare a sample of the supernatant reflecting the level of LDH after 100% of the cells were released in the unit with dissolution. Add wells containing the same 0.9 mL of liquid. The plate containing the unit is frozen at −80 ° C. and the volume of the well is thawed next. This freeze-thaw cycle effectively thaws cells and releases their cytoplasm, including LDH, into the supernatant. 50 μl of aliquots from frozen and thawed cells are added to 96-well plates with control of the amount reflecting 100% cytotoxicity.

E. 50 μl aliquots of LDH assay reagent are added to each well containing an aliquot of the supernatant. The plate is then incubated for 30 minutes in the dark.

F. The reaction is stopped by the addition of a "stop" solution of 1 M acetic acid and between one hour of addition of the stop solution the absorption of the plate is determined at 490 nm.

G. Calculation of percent cell lysis is obtained from a solution given alone as a reference for cell lysis, and a linear relationship between uptake and cell lysis obtained from the solution surrounding the freezing and thawing unit given as a reference for 100% cell lysis. Is based on the assumption.

6. ELISA Decisions

The process for determining the concentrations of a biologically active agent, such as a test substance for assessing increased penetration of the active agent in conjunction with the implementation of an equivalent mucosal delivery-enhancing agent or in combination formulations of the present invention, is generally as described above and known methods. And the specific fabrication instructions of the ELISA kit used for each unique assay. Penetration kinetics of a biologically active agent are generally multiple times after the biologically active agent comes into contact with the apex epithelial cell surface (perhaps simultaneously or sequentially, exposure of the apex cell surface to mucosal delivery-enhancing agent (s)). Determined by points (eg 15 minutes, 30 minutes, 60 minutes, and 120 minutes).

The process for determining the concentrations of peptide YY neuropeptide Y, and pancreatic peptide, central nervous system (CNS) tissues or body fluids, cerebrospinal fluid (CSF), or other tissues or body fluids of a mammalian patient in the serum may be peptide YY neuropeptide Y And by immunological analysis on pancreatic peptide. Pancreatic peptides or the combination formulations of the present invention, such as test materials for the process for determining the concentrations of peptide YY neuropeptide Y and for the enhanced penetration assessment of active agents in conjunction with equivalent administration of mucosal delivery-enhancing agents, are generally described above, Radioimmunoassay (RIA), enzyme immunoassay (EIA), and immunohistochemistry or antibody reagents for immunofluorescent antibodies to peptide YY, neuropeptide Y, and pancreatic peptide (Bachem AG (King of Prussia, PA)) Consistent with the known methods and the instructions of the particular manufacturer.

Epiair ^™ tissue cell membranes are cultured in phenol red and hydrocortisone free medium (MatTek Corp., Ashland, Mass.). Tissue cell membranes are incubated for 48 hours at 37 ° C. to allow tissues to equilibrate. Each tissue cell membrane is placed in individual wells of a 6-well plate containing 0.9 mL of serum free medium. 100 μl of the formulation (test sample or control) is applied to the space of the apex of the cell membrane. Samples replicated in triplicate or quadruple of each test sample (biologically active agent, mucosal delivery-enhancing agent bound to peptide YY) and control (biologically active agent, peptide YY, alone) are evaluated in each assay. At each time point (15, 30, 60 and 120 minutes) the tissue cell membranes are transferred to new wells containing fresh medium. Samples based on 0.9 mL medium are obtained at each time point and stored at 4 ° C. for use in ELISA and lactic acid dehydrogenase (LDH) analysis.

ELISA kits are typical two-step sandwich ELISAs: The immunoactive forms of the studied drug are first "captured" by immobilized antibodies on 96-well microplates, then wash off unbound material out of the wells, and "detect". The antibody allows to react with the bound immunoactive agent. This detection antibody is typically paired with an enzyme (mostly blush and oxidase) and the amount of enzyme bound to the plate in the immune complex is measured in combination with the pigmented agent. In addition to the samples of the supernatant medium collected at each time point in the penetration kinetics study, the appropriately diluted samples of the formulation applied to the space of the apex of the unit at the beginning of the active study (biologically active test agent Subject matter, etc.) are also analyzed in ELISA plates according to a set of manufacturer provided standards. Each supernatant medium sample is typically ELISA (quadrant units are required for each formulation in the penetration rate determination and generate a total of 16 samples of supernatant medium collected at all four time points. Is analyzed in replicated wells).

A. Obvious concentrations of the active test agent for samples of supernatant or diluted samples of material applied to the space of the apex of units passing outside the region of concentrations of the standards after completion of the ELISA are common. Non-concentrations of the substance present in the experimental samples are determined by prediction above the concentrations of the criteria; To some extent, samples are appropriately diluted with the resulting concentrations of test substance that can be more accurately determined by insertion between standards in repeated ELISA.

B. ELISA for Biological Activity Test Agents, eg, peptide YY, is unique within its design and preferred protocol. Unlike most kits, ELISA is for capturing one and two monoclonals that are directly linked to non-overlapping determinants for biologically active test agents (this antibody is paired with blush and oxidase). Use an antibody. Experimental samples are given to the extent of the concentrations of peptide YY located below the upper limit of the assay, and an assay protocol that allows the culturing of samples on the ELISA with two antibodies given simultaneously to the ELISA plate is used for the manufacturer's instructions. When the peptide YY levels in the sample are significantly higher than the upper limit, the immunoactive peptide YY levels will probably increase the amount of antibody in the culture mixture and will not have detection antibody binding while no peptide YY with detection antibody binding is captured. Any peptide YY that does not will be captured on the plate.

This leads to a significant underestimation of peptide YY levels in the sample (which indicates peptide YY levels in any sample located significantly below the upper limit of analysis). The analysis protocol has been modified to eliminate this possibility:

B.1. Samples diluted in the absence of any detection antibody are incubated for one hour on an ELISA plate containing the antibody that was initially immobilized. After one hour incubation, the wells are washed away from unbound material.

B. 2. Detection Antibodies are incubated with the plate for one hour to effect the formation of immune complexes with all captured antigens. The concentration of detection antibody is sufficient to react with the highest level of peptide YY bound by the captured antibody. The plate is then washed again to remove any unbound detection antibody.

B. 3. Peroxidase substrate is added to the plate and incubated for 15 minutes to allow color development to occur.

B. 4. "Stop" solution is applied to the plate and absorption is read at 490 nm as well as 450 nm in the VMax microplate spectrometer. The absorption of the colored product at 490 nm is much lower than that at 450 nm, but the absorption at each wavelength is still proportional to the concentration of the product. Two readings were linear with the amount of bound peptide YY beyond the working area of the VMax device (although the device was reported to be accurate beyond the 0 to 3.0 OD range, but we routinely limit the area from 0 to 2.5 OD). Make sure it is related to the enemy. The amount of peptide YY in the samples is determined by inserting between the OD values obtained for the other standards included in the ELISA. Samples are rediluted and run with out-of-domain OD readings obtained for standards in a repeated ELISA.

result

Measurement of Epithelial Penetration Resistance by TER Analysis:

After the last analysis time points, the cell membranes were placed in individual wells of a 24-well culture plate in 0.3 mL of clean medium and epithelial pain and electrical resistance (TER) were measured by the EVOM epithelial voltmeter and the Endohm chamber (World Precision Instruments, Sarasota, FL). Is measured using. The top electrode is adjusted to be close to the top surface of the cell membrane but not to contact it. Tissues are removed from their individual wells at one time and the basal surfaces are washed by immersion in clean PBS. Apex spaces are slowly washed twice with PBS. The tissue unit is placed in an Endohm chamber, additionally 25 0 μL of PBS is inserted, the top electrode is repositioned and the resistance is measured and recorded.

After the measurement, the PBS is discarded and the tissue insert is returned to the culture plate. All TER values are reported as a function of the surface area of the tissue.

The final numbers are calculated as follows:

TER = (membrane and insertion resistance (R)-blank insertion R) of cell membranes × cell membrane area (0.6 cm ² ).

The effect of pharmaceutical formulations consisting of peptide YY and intranasal delivery-enhancing agents on TER assay penetrating the Epiairway ^TM cell membrane (mucosal epithelial cell layer) is shown in FIG. 1. decrease in TER value relative to the control value (regulation = about 1000 ohms-cm ² ; normalized to 100) indicates a decrease in cell membrane resistance and an increase in mucosal epithelial cell permeability.

The typical peptide YY formulation, Formulation P, shows a tremendous decrease in cell membrane resistance. (Table 2). The results indicate that the resistance of the cell membranes is reduced such that a typical formulation (eg, formulation P) is below 1% of the regulation at the tested concentration. What these values show is the triple average of each formulation. Formulations A and B are controls prepared by restored peptide YY (Bachem AG, King of Prussia, PA) comprising 60 μg peptide Y _3-36 in 100 ml of phosphate buffered saline at pH 7.4 or 5.0. Peptide YY, except for mucosal delivery enhancers, does not reduce resistance.

The results indicate that a typical formulation for enhanced intranasal delivery of peptide YY (eg, Formulation P) reduces cell membrane resistance and significantly increases the permeability of mucosal epithelial cells. Typical formulations will enhance intranasal delivery of peptide YY to serum or central nervous system tissue or body fluids. The results indicate that these typical formulations have a significant increase in mucosal epithelial cell permeability to peptide YY when in contact with mucosal epithelial yield.

[Table 2]

Penetration Reaction by ELISA Analysis:

The effect of the pharmaceutical formulations of the present invention consisting of peptide YY and intranasal delivery-enhancing agents on the penetration of peptide YY through the epiair cell cell membrane (mucosal epithelial cell layer) is measured as described above. The results are shown in Table 3. Penetration of peptide YY across the Epiairway cell membrane is measured by ELISA assay.

For typical intranasal formulations (eg, Formulation P) of the present invention, a significant increase in peptide YY penetration occurs in Formulation P as shown in Table 3. The process uses an ELISA assay to determine the concentration of biologically active peptide YY that has penetrated epithelial cells into the surrounding medium on multiple time points. The results were compared to Formulation A or B (Peptide YY Control Formulation; Formulation P compared to 60 μg Peptide YY _3-36 in 100 ml of phosphate buffered saline at pH 7.4 or 5.0; Bachem AG, King of Prussia, PA). Shows increased penetration of peptide YY. The average of a gradual increase in penetration with a typical intranasal formulation Formulation P in 120 people is 1195 times much larger than formulations A or B adjustments.

Table 3

MTT analysis:

MTT analysis is performed using the MTT-100, MatTek kit. 300 mL of MTT solution is added to each well. Tissue inserts are slowly wiped with clean PBS and placed in MTT solution. The samples are incubated at 37 ° C. for 3 hours. After incubation, the cell culture inserts are then submerged in 2.0 mL of extract solution per well to completely cover each insert. The extraction plate is covered and sealed to reduce evaporation. Extraction is carried out overnight at room temperature under dark conditions. After the extraction period is complete, the extraction solution is mixed and pipetted into a 96-well microtiter plate. Triplets of each sample are loaded together with the extraction blank. The optical density of the samples is then measured at 550 nm on a plate reader (Molecular Devices).

Typical formulations in MTT assays for enhanced intranasal mucosal delivery of peptide YY compared to regulatory formulations (Formulations A or B) and following the teaching of instantaneous features (eg Formulation P) are shown in Table 4. Shown. Results for formulations consisting of peptide YY and one or more intranasal delivery enhancers, for example, Formulation P (experiment performed in three complexes), are of the typical embodiment of the viability of mucosal epithelial tissue. It has a minimal toxic effect.

Table 4

LDH analysis:

LDH analysis on a typical formulation for enhanced intranasal mucosal delivery of peptide YY following the teaching of instant features (eg Formulation P) is shown in Table 5. Results for the three complexes of Formulation P indicate that this typical embodiment of the viability of mucosal epithelial tissues has a minimal toxic effect.

Table 5

Example 3: Formulation P (Peptide YY) of the invention in combination with triamcinolone acetonide corticosteroids improves cell viability

Current examples are the permeability of epithelial mucositis of intranasally administered peptide YY, eg, human peptide YY, in combination with, for example, triamcinolone acetonide, and more in combination with one or more intranasal delivery-enhancing agents. In vitro analysis is provided to determine the reduction. The assay includes reduction in epithelial mucositis as determined by determination of epithelial cell permeability by TER assay and cell viability in MTT assay by application of an embodiment consisting of peptide YY and triamcinolone acetonide.

Formulation P (shown in Table 1 above) is combined with the formulation with triamcinolone acetonide at doses of 0.5, 2.0, 5.0, or 50 μg. The typical dose of triamcinolone acetonide (Nasacort ^® , Aventis Pharmaceuticals) for seasonal allergic rhinitis is 55 μg per spray. Formulation P improves cell viability as measured by MTT assay, while maintaining epithelial cell permeability as measured by TER and ELISA assay combined with triamcinolone acetonide corticosteroid.

According to the methods and formulations of the present invention, the determination of the permeability of formulation P in the presence or absence of triamcinolone acetonide is performed by infiltrating epithelial electronic resistance (TER) analysis in Epiairway ^TM cell membranes. TER analysis of Formulation P plus triamcinolone acetonide at concentrations of 0.5, 2.0, 5.0, or 50 μg per spray shows that peptide YY permeability is not reduced and is identical to that of Formulation P alone. Formulation P plus triamcinolone acetonide, typically between 0 and 50 μg of triamcinolone acetonide concentration per spray, is typically at least 10 to 100 times greater than the permeability of formulations A or B (peptide YY control).

According to the methods and formulations of the invention, the determination of the permeability of formulation P in the presence or absence of triamcinolone acetonide is performed by ELISA assay in Epiairway ^TM cell membranes. Similar to the TER assay, ELISA analysis of Formulation P plus triamcinolone acetonide at concentrations of 0.5, 2.0, 5.0, or 50 μg per spray shows that peptide YY permeability is not reduced and is identical to that of Formulation P alone. Formulation P plus triamcinolone acetonide between 0 and 50 μg triamcinolone acetonide concentrations per spray is typically much greater than the permeability of formulations A or B (peptide YY control).

According to the methods and formulations of the present invention, the MTT assay measures the cell viability of formulation P in the presence or absence of triamcinolone acetonide. Typically, the addition of triamcinolone acetonide (at a concentration of 0.5, 2.0, 5.0, or 50 μg per spray) to formulation P improves cell viability compared to formulation P in the absence of triamcinolone acetonide.

The addition of triamcinolone acetonide to formulation P increases cell viability and maintains epithelial permeability as measured by the TER assay compared to formulation P in the absence of triamcinolone acetonide.

Reduction in epithelial mucosal inflammation of peptide YY administered intranasally is typically one or more steroid or corticosteroid compound (s) that are not only high potency compounds or formulations but also some examples of medium potency, or low potency compounds or formulations. Is achieved with an intranasal formulation of peptide YY in combination with Overall, high potency (equivalent dose), medium, and low potency steroids are given. Typically, intranasal formulations of peptide YY that bind high potency steroid constructs include, but are not limited to, betamethasone (0.6 to 0.75 mg dose), or dexamethasone (0.75 mg dose). In selective formulations, the intranasal formulation of peptide YY, which binds to the media potency steroid composition, is not limited but methylprednisolone (4 mg dose), triamcinolone (4 mg dose), or prednisolone (5 mg dose) Include. In more selective formulations, the intranasal formulation of peptide YY that binds a low potency steroid construct includes, but is not limited to, hydrocortisone (20 mg dose) or cortisone (25 mg dose).

Example 4: Preparation of PYY Formulation Free of Protein Stabilizers

PYY formulations suitable for intranasal administration of substantially free PYY of a protein stabilizer are prepared with the formulations listed below.

1. Add approximately 3/4 of water to the beaker, stir with a stir bar on a stir plate and add sodium citrate until it is completely dissolved.

2. Next add EDTA and stir until it is completely dissolved.

3. Next, add citric acid and stir until it is completely dissolved.

4. Methyl-β-cyclodextrin Stir until it is completely dissolved.

5. Next add DDPC and stir until it is completely dissolved.

6. Next add lactose and stir until it is completely dissolved.

7. Next add sorbitol and stir until it is completely dissolved.

8. Next add chlorobutanol and stir until it is completely dissolved.

9. Add PYY3-36 and stir slowly until it melts.

10. Measure the pH to ensure that the pH is 5.0 ± 0.25. Dilute HC1 or dilute NaOH is added to adjust the pH.

11. Add water to final volume.

Table 6

Formulation pH 5 +/- 0.25

Osmolality ~ 250

Example 5

A second formulation prepared as above except for the concentration of PYY3-36 is shown in Table 7 below.

Table 7

Formulation pH 5 +/- 0.25

Example 6: Determination of Optimal pH of PYY

Determination of pH for PYY _3-36 Stability at 40 ° C for 5 Days

A. Protocol for Formulating PYY (3-36) / pH Stability Assay Samples

Osmolality: Target 250 mM

Uses citrate / sodium citrate, tri-basic buffer, 10 mM

Osmolality = particle count × molarity = (1 + 4) × 10 mM = 50 mM

Thus, the osmolality is brought to 250 mM with 100 mM NaCl (2 particles).

B. Make a stable sample as follows (3500 μl)

Final concentration

Citrate buffer 1400 μl 25 mM l0 mM

PYY 700 μl 1.5 mg / mL 300ug / ml

Chlorobutanol 350 μl 2.5%, 0.25%

NaCl, 350 μl 1.0 M, l00 mM

Check the pH and adjust if necessary.

3500 μL of Q. S. with water.

fair:

120 μl sample inserted into 200 μl liquid in autosampler vial

3 pulls / hour point

Samples are incubated at 40 ° C. for 5 days.

C. Comparison of the target and actual final pH of the safety mixture

Target pH Actual pH

3.0 2.99

3.5 3.47

4.0 3.90

4.5 4.42

5.0 4.90

7.0 7.38

D. HPLC Process

Column: Waters C18 bondapak 10 μm 4.6 × 300 mm

HPLC System: Waters Alliance 2690

Detector: Dual Wavelength at Waters 2487 220 nm

Flow rate: l ml / mim

Injection volume: 30 μl

Column temperature. 30 ℃

Mobile phase:

Buffer A: 0.1% TFA, 1% acetonitrile in water

Buffer B: 0.11% TFA in acetonitrile

slope:

Minutes % A % B

0 75 25

   17 42 58

   19 75 25

   28 75 25

E. Results and Conclusions:

The results show that the appropriate pH for stability under the unique conditions used in this assay is 4,90. As pH increases from 2.99 to 4.90, stability increases from 76 to 87%.

At high pH, there is a large drop in stability of PYY _3-36 , for example with a time zero remaining of 7.38, only 15%.

Example 7: Intranasal Formulation Development

Peptides and proteins are relatively weak molecules compared to low molecular weight therapeutics. The purpose of formulation development is to identify candidate formulations suitable for intranasal delivery. To achieve this goal, a number of candidates are tested to confirm the formulation with proper drug stability, delivery across the nasal mucosa, toxicity and storage effects.

Initially, the effect of pH was verified. 1 shows the stability of PYY _3-36 at various pH of 7.4 and high temperature (40 ° C.) at pH 3.0. At physiological pH, there is a potential loss of drug when the temperature rises. The highest stability is achieved at about pH 5.0. This pH is chosen for further optimization of the formulation.

To further optimize stability, various stabilizing agents are tested for their ability to facilitate the transport of drugs across the nasal mucosa. The enhancers tested were selected based on their ability to open the dense joint with limited cellular toxicity. To achieve this, a basic human epithelial cell model (Epiairway, MatTek, Inc., AshlandMA) is needed. This cell line forms a virtual mesothelial epithelial cell layer with dense joints similar to the respiratory epithelium found in the nose. Drug formulations are placed on the lateral end of the tissue layer and drug quantification is performed for the baseline conditions. The extent of dense joint opening is measured by a decrease in the infiltration epithelial electrical resistance. Cell viability and cytotoxicity are monitored by MTT and LDH assays, respectively. Data from representative screening experiments are depicted in FIGS. 2-5.

2 shows data for TEER. In some instances, there is no small or no reduction in the TEER compared to the control, and it still represents a closed joint. In other examples, it is potentially dropped to TEER, which indicates a tight joint opening. The results indicate that the in vitro cell model can differentiate the ability of other formulations to open dense joints.

Candidate formulations were tested for good cell viability and low cytotoxicity.

In total, over 200 different formulations are tested to reflect the high completion nature of the in vitro screening model. Using all available data, multivariate analyzes affect each formulation configuration on each of 7 output variables (drug permeability, osmolality, stability under frozen and accelerated conditions, TEER, and MTT and LDH analyzes). This is done to reveal the effect. Multivariate analyzes consist of initial analyzes of each formulation configuration for a level that correlates with output parameters (p <0.1). In conjunction with the identified parts, a linear degenerate or stepwise logical selection model is used. Results indicate that one excipient is related to osmolality and toxicity (r ² = 0.91 and 0.27, respectively), two are related to PYY _3-36 infiltration (r ² = 0.44), and three are stability (r ² = 0.24). ), And five suggest periphery resistance (r ² = 0.55). The best formulations are determined by a process with at least a 30-75-fold increase in PYY _3-36 delivery compared to this simple buffer solution.

Based on these analyzes, an optimized PYY _3-36 formulation is selected for further development. This optimized formulation includes two stabilizers, two penetration enhancers, one chelating agent, and a preservative in one pH 5.0 sodium acetate buffer.

This formulation passes the USP Preservative Effectiveness Test. The synergistic contribution of various configurations on drug penetration is shown in FIG. 5.

Compared to simple buffer formulations at the same osmolality, the optimized formulation shows a 100-fold increase in drug penetration.

Finally, preclinical and clinical batches of optimized formulations are safely prepared and placed at 5 ° C. and 25 ° C. in the final product packaging. The basic data depicted in Table 8 reveals no inherent loss of drug for one month at 5 ° C or 25 ° C.

In summary, our process of formulation development produces PYY _3-36 formulations with adequate drug stability, delivery through the nasal mucosa, and deliverable toxicity and deliverable conservation of 4 kD peptides.

Preclinical studies

A series of six preclinical studies were completed in mice, rabbits, and dogs per day. Plasma PYY _3-36 levels in all species are determined by valid commodity radioimmunoassays.

Bioavailability in mice (molar fraction of drug divided by the amount administered intranasally in plasma) is determined to be about 6% and in rabbits about 8%. These values are probably said to be less than the actual bioavailability, such as degenerate after sampling despite the presence of any peptide degradation, or protease inhibitor in plasma before sampling, and the measured bioavailability will decrease.

Nasal toxicity was measured in rat and rabbit models for 14 consecutive days at the expected human clinical doses based on 50 × mg / kg. There were no microscopic or gross pathological findings associated with the test paper. There were no clinical observations.

Structural toxicity following intravenous administration was assessed in rat and rabbit models. The doses in IV reached approximately 160 × the expected human dose (400 μg / kg in rats and 205 μg / kg in rabbits) and there were no microscopic or macroscopic findings associated with the test paper.

Cardiovascular toxicity is assessed in anesthetized dogs with a dose domain study design. The highest dose is an infusion of PYY _3-36 at 24 ug / kg for at least 60 minutes consistent with the expected human dose based on 33 x body surface area. The resulting plasma levels, 30 ng / mL, are about 380 × base canine plasma levels. At this plasma level, there is no effect of arterial blood pressure, only a small change in the femoral blood rate, or QTc and cardiac rate (increased in 123 and 148 bpm method) and respiratory rate (decreased from 54 to 36) becomes important.

Pharmacokinetic data is collected from these preclinical studies. From one study in rats, plasma levels after intranasal administration at various doses are shown in FIGS. 6, 7 and 8. Figure 6 shows PYY _3-36 seen in plasma at 5 minute intervals, plasma concentrations (Tmax) reached at 10-15 minutes, and final elimination half-life of about 15 minutes. C _max and AUC _0-t are linear for intranasal doses.

Clinical research

Dose domain clinical trials begin with the objectives of stability, PK, and bioavailability of the intranasal formulation of PYY _3-36 . In one day, patients are initially enrolled in two of five dose groups. One patient is reported when he tastes behind his throat; There are no other opposing events in a day.

conclusion

Formulations, preclinical, and early clinical trials begin on an intranasal formulation of PYY _3-36 . Access to the formulation results in the infiltrating cell membrane permeability of this 4 kD peptide more than 100-fold without increasing cellular toxicity. Preclinical studies show safety margins to consider for structural toxicity to the nose, cardiovascular, and PYY _3-36 . Based on ongoing dose domain clinical studies, endless dose weight loss studies were planned.

Example 8a

Clinical protocol

Intranasal Peptide YY in Healthy Human Subjects _3-36 (PYY _3-36 Nasal Absorption

Purpose of this study:

The purpose of this study was to assess the uptake of PYY _3-36 administered intranasally from the nose into the bloodstream. This is a dose escalation study in Phase 1, which includes a single dose, rapid, moderate, healthy male and female volunteers. Dose increasing in the ratio of PYY _3-36 is evaluated between stability evaluation, nose resistant, and 20 ㎍ and 200 ㎍ for the absorption of PYY _3-36. Evaluation of each independent sensory disposition is also measured.

PYY _3-36 is administered to 15 healthy humans divided into 5 groups of 3 independent of each.

Group I.

The first group is administered by 20 μg of nasal spray of PYY _3-36 in 0.1 ml solution.

Group ii

The second group received 50 μg of PYY _3-36 intranasally in 0.1 ml solution.

Group iii

The third group received 100 μg of PYY _3-36 intranasally in 0.1 ml solution.

Group iv

The fourth group receives 150 μg PYY _3-36 intranasally in 0.1 ml solution.

Group v

The fifth group receives 200 μg of PYY _3-36 intranasally in 0.1 ml solution.

Blood samples are collected and plasma concentrations of PYY are determined at 0 (eg, before taking) and at 5, 7.5, 10, 15, 20, 30, 45, 60 after taking. Subjects are then absorbed and blood samples taken and the concentration of PYY determined at 30 minutes after eating. Plasma concentrations of PYY _3-36 are determined using validated analytical processes.

For each subject according to the model independent approach, after the PK parameters are calculated and whenever possible, based on the plasma concentration of PYY _3-36 :

C _max maximum observed concentration

t _max time for maximum concentration

The area under the concentration-time curve calculated by the linear trapezoidal law from AUC _ot time 0 to the time of the last measured concentration.

Subsequent parameters are calculated when the correct measurement of these parameters is performed data:

Area under the concentration-time curve that extrapolates to infinity calculated using the AUC _0-∽ scheme:

C _t is the last measured concentration and K _e is the apparent final inheritance constant.

K _{e The} apparent final phase velocity constant, K _e, is the importance of the slope of the linear degeneration of the log concentration versus time profile during the final phase.

t _1/2 apparent final phase half-life (whenever possible), t _1/2 = (1n2) / K _e .

PK calculations are performed using commercial software by WinNonlin (Pharsight Corporation, Version 3.3, or higher). The results are shown in the graph below.

Discussion and Results

background:

Each dosing group comprises three subjects administered intranasally with the formulation of the invention comprising a special dose of human PYY _3-36 synthetic and pyrogen free. Five dosing groups are organized with increasing special doses of PYY _3-36 in the formulation. Blood samples are drawn at special intervals with blood collection tubes containing lithium heparin (to prevent coagulation) and aprotonin (to preserve PYY _3-36 ). Plasma from each blood sample is collected by centrifuge and preserved in frozen purified water. One frozen purified water of each blood sample is sent to Nastech Analytical Services and arrives frozen. Each sample is stored frozen while analyzed for PYY concentration by radioimmunoassay (RIA).

Observations:

Group 1: This group of subjects received 20 micrograms of PYY _3-36 . Plasma PYY concentrations for a subject vary from a minimum of "lower than 20 pg / ml" (lower than the quantitative low limit of radioimmunoassay) to a maximum of 159 pg / ml. The trend of the measured concentrations is inconsistent with the significant absorption of the drug into the blood of the study subject.

Group 2: The group of this subject received 50 micrograms of PYY _3-36 . Plasma PYY concentrations for the subject varied from a minimum of "lower than 50 pg / ml" (lower than the quantitative low limit of radioimmunoassay) to a maximum of 255 pg / ml. The trend of the measured concentrations is inconsistent with the significant absorption of the drug into the blood of the study subject.

Group 3: This group of subjects received 100 micrograms of PYY _3-36 . Plasma PYY concentrations for the subject varied from a minimum of "lower than 87 pg / ml" (lower than the quantitative low limit of radioimmunoassay) to a maximum of 785 pg / ml. The trend of the measured concentrations is inconsistent with the significant absorption of the drug into the blood of the study subject.

Group 4: The subject's group received 150 micrograms of PYY _3-36 . Plasma PYY concentrations for the subject varied from a minimum of "lower than 45 pg / ml" (lower than the quantitative low limit of radioimmunoassay) to a maximum of 2022 pg / ml. The trend of the measured concentrations is inconsistent with the significant absorption of the drug into the blood of the study subject.

Group 5: This subject's group received 200 micrograms of PYY _3-36 . Plasma PYY concentrations for the subject varied from a minimum of "lower than 48 pg / ml" (lower than the quantitative low limit of radioimmunoassay) to a maximum of 1279 pg / ml. The trend of the measured concentrations is inconsistent with the significant absorption of the drug into the blood of the study subject.

These results are inconsistent with the uptake of dose independent PYY _3-36 .

Additional observations and data:

Summary of findings:

At intranasal doses of 50 ug-200 ug, there is a dose independent PYY absorbing plasma.

With the elimination half-life at 55 minutes, the persistence of rising plasma concentrations is significantly longer than expected.

C _max and AUC _0-t show a good linear relationship with dose.

● There is significant inward-target variability at a given dose.

Surprisingly, this study failed to measure the post-prandial rise in PYY even if the amount of food eaten was too small and the observations were explained, although the actual amount of food eaten was not measured.

The visual-signal scale hunger question suggests an increase in dose of PYY and a decrease in hunger.

Nausea and dizziness associated with very high plasma concentrations of PYY.

• Values based on the analysis account for those features that appear 2-3 times higher than previously reported in the literature.

Remark:

Some examples pMol / L are used with the PYY measurement units; In other assays, pg / mL is used. The conversion factor was pmol / L * 4.05 = pg / mL.

In some instances, a 150 minute time point is displayed in the plot. Strictly speaking, this is a postprandial data point, and perhaps eventually confuses PK evaluation. However, unforeseen findings explain that the more detailed below is the 30-minute postprandial time point and this does not differ from the baseline value.

PYY plasma concentrations:

The PYY assay explains this feature as evidence for his own PYY plasma concentration analysis. Using this analysis, samples from each time point are analyzed in triplicate. Note that, out of 180 data points, three (1.6%) appear to be biologically uncertain "outsiders." The data from this basic analysis uses a dataset with these three data points removed.

Table 9

A test in the sense of the PK plot suggests a dose response from 50-200 ug doses, but the 20 ug dose goes into noise. Thus many collateral analyzes will be data based on only 50-200 ug doses. We also suggest that PYY is an endogenous molecule and AUC0-t is more related than AUC0-inf.

Tmax and Tl / 2 (removal of half-life):

Tmax of 24 minutes is typical for droplet products. The elimination of a half-life of 55 minutes is considerably longer than expected. References show a typical t 1/2 of 5-10 minutes. The elimination of half-life is probably also affected by formulation constructs with some subsequent absorption from the nasal mucosa and peptide metabolic effects. Optionally, because the analysis explains these features that require an extraction process and the analysis will analyze the basic free portion without using extraction, while the analysis will capture both free and protein bound PYY.

From this analysis of the significance of VAS change from baseline (10, 30, and 60 minute values mean minus baseline) versus dose, one observation:

VAS Q1 "How hungry do you feel?" Subjects feel less hungry after receiving high PYY doses.

Subjects to VAS Q3, "How much do you think you can eat?" Think they can eat less after receiving a high PYY dose.

However, subjects to VAS Q2 "How full do you think you are full?" Feel less full after receiving a high PYY dose.

This suggests that feeling after PYY administration does not include fullness, satiety, or high contraction by gas.

Example 8a

PYY Human Dose and Weight Loss

The PYY intranasal formulation is created as follows.

Formulation pH 5 +/- 0.25

One or two sprays were administered to human subjects daily over a 10-day period and a weight loss of 2.5 pounds was recorded. The subject's hunger reduction was recorded after administration in the 10 minute to 12 hour region over the period.

Example 9

Ball formulation of PYY3-36 (prediction)

A biphasic tablet is prepared by the following method. The adhesive layer is prepared by 70 parts by weight of polyethylene oxide (Polyox 301N; Union Carbide), 20 parts by weight of polyacrylic acid (Carbopol 934P; BF Goodrich), and 10 parts by weight of compressible xylitol / carboxymethyl cellulose filter (Xylitab 200; Xyrofin). . These ingredients are mixed in a container for 3 minutes. The mixture is then transferred to an evaporating dish and quickly moistened the granules with anhydrous ethanol to a half-dough-viscosity. This mass is applied immediately and quickly through a 14 mesh (1.4 mm open) stainless steel screen to which the wet granules adhere. The screen is covered with perforated aluminum foil, and the wet granules are dried overnight at 30 ° C. The dried granules are removed from the screen and then applied through a 20 mesh (0.85 mm open) screen to further reduce the size of the granules. Particles that do not pass through the 20 mesh screen are briefly ground with a grinder and pestle to minimize the amount of fines and are then passed through the 20 mesh screen. The resulting particles are placed in a mixing vessel, 0.25 parts by weight of steric acid and 0.06 parts by weight of mint flavor are added and then blended into granules.

The final parts by weight of components are 69.78% polyethylene oxide, 9.97% compressible xylitol / carboxymethyl cellulose filter, 19.94% polyacrylic acid, 0.25% steric acid, and 0.06% mint flavor. An amount of 50 mg of this mixture is placed in a 0.375 inch diameter die and voltage-compressed in Carver Press Model C for a three second drag time for the formation of an adhesive layer.

The active layer is prepared from 49.39 parts by weight of mannitol, 34.33 parts by weight of hydroxypropyl cellulose (Klucel LF; Aqualon, Wilmington, Del.) And 15.00 parts by weight of sodium tarocholate (Aldrich, Milwaukee, Wis.), And in the container for 3 minutes Turn to mix. The mixture is then transferred to an evaporating dish and quickly moistened the granules with anhydrous ethanol to a half-dough-viscosity. This mass is applied immediately and quickly through a 14 mesh (1.4 mm open) stainless steel screen to which the wet granules adhere. The screen is covered with perforated aluminum foil, and the wet granules are dried overnight at 30 ° C. The dried granules are then passed through 20, 40 (0.425 mm open), and 60 (0.25 mm open) mesh screens to further reduce particle size. Particles that do not pass through the screen are briefly ground with a grinder and pestle to minimize the amount of fines and then pass through a 20 mesh screen. Screened particles are weighed, and then 0.91 parts by weight of PYY3-36 and 0.06 parts by weight of FD & C yellow # 6HT aluminum lake dye are blended with the particles sequentially dried by geometric dilution. The dyed granules are then placed in a mixing vessel and blended with 0.25 parts by weight of magnesium sterate (lubricating oil) and 0.06 parts by weight of mint flavor by spinning for 3 minutes. A 50 mg sample of this material is placed on top of a partially compressed tacky layer and the two layers are then compressed to a pressure of 1.0 ton for a three second drag time to obtain a bilayer tablet that facilitates ball delivery.

This process results in tablets of gums where the active layer comprises 0.91 parts by weight of PYY3-36, 15 parts by weight of NaTC, and 84.09 parts by weight of filters, lubricants, colorants, formulation aids, or perfume agents.

Example 10

The study was performed by comparing the ability of non-endotoxin-free PYY3-36A to penetrate into bronchial epithelium following the process of endotoxin-free PYY3-36 versus Example 1. It determines for twice the amount of endotoxin-free PYY3-36 that penetrates the bronchial epithelium as compared to the PYY3-36 formulation with endotoxin.

Both formulations include 2.5 mg / ml chlorobutanol, 2 mg / ml DDPC, 10 mg / ml albumin, 1 mg / ml EDTA and 45 mg / ml M-B-CD. One formulation includes endotoxin-free PYY3-36 and another formulation comprising 70 EUs or amounts of endotoxin.

 The mean MTT of the PYY3-36 formulation with endotoxin was 91.72% while the endotoxin-free PYY3-36 formulation had an average MTT of 100.16%.

The mean penetration of the PYY3-36 formulation with endotoxin is 5.36% while the mean penetration of the endotoxin-free PYY3-36 formulation is 10.75%.

Numerous known mucosal delivery enhancing excipients are effectively associated with endotoxin-free Y2 receptor binding peptides, particularly endotoxin-free PYY3-36, and can be used to improve nonabsorbable formulations, especially oral delivery. Some excipients are included in the following patent applications, which are incorporated by reference: US Patent Nos. 20030225300, 20030198658, 20030133953, 20030078302, 20030045579, 20030012817, 20030012817, 20030008900, 20020155993, 20020127202, 20020120009, 20020119910, 20020065255, 20020052422, 20020040061, 20020013497, 20020001591, 20010039258, 20010003001.

Oral Formulation of Y2 Receptor-binding Peptides

Oral formulations of Y2 receptor-binding peptides can be prepared according to the following procedure. Preferred formulations for oral delivery include about 0.5 mg / kg endotoxin-free PYY and one or more mucosal delivery enhancing excipients between 100 and about 200 mg / kg.

(prediction)

Example 11

Preparation of N-cyclohexanoylphenylalanine:

Dissolve phenylalanine methyl ester (1 g., 0.0046 moles) in 5 mL of pyridine. Cyclohexanoyl chloride (0.62 mL) is added and the mixture is stirred for 2 hours. The reaction mixture is poured into hydrochloric acid (1N) and ice. The aqueous mixture is extracted twice with toluene. The toluene extract was concentrated in vacuo to yield 1.1 g of crude N-cyclohexanoyl phenylalanine methyl ester.

N-cyclohexanoyl phenylalanine methyl ester (0.5 g) is dissolved in ethylene glycol dimethyl ether (20 mL). The solution is cooled to −70 ° C. and diisobutylaluminum hydride (2.04 mL of a 1.5M solution in toluene) is added. The resulting reaction mixture is stirred at -70 ° C. The reaction is stopped by dropping 2N hydrochloric acid. The mixture is extracted with cold ethyl acetate. Ethyl acetate solution is washed with brine and dried over sodium sulfate. Concentrate in vacuo and finally give a white solid by silica gel chromatography.

¹ H NMR (300MHz, DMSO-d6): 9.5 (s, 1H), 8.2 (d, 1H), 7.2 (m, 5H), 4.2 (m, 1H), 3.2 (d, 1H), 2.7 (d, 1H), 2.1 (m, 1H), 1.6 (br. M, 4H), 1.2 (br. M, 6H). IR (KBr): 3300, 3050, 2900, 2850, 2800, 1700, 1600, 1500 cm ^-1 .

Mass Spec .: M + 1 m / e 261.

Example 12

Preparation of N-Acetylphenylalanine Aldehyde:

N-acetylphenylalanine methyl ester (4.2 g, 19 mmol) is dissolved in ethylene glycol dimethyl ether. The solution is cooled to −70 ° C. and diisobutylaluminum hydride (25.3 mL of a 1.5 M solution in toluene, 39 mmol) is added. The resulting reaction mixture is stirred at -70 ° C. The reaction is stopped by dropping 2N hydrochloric acid. The mixture is extracted four times with cold ethyl acetate and four times with toluene. Ethyl acetate solution is washed with brine and dried over sodium sulfate. Concentrate in vacuo and finally give 2.7 g of a white solid by silica gel chromatography. NMR is reported in Biochemistry, 18: 921-926 (1979).

Example 13

Preparation of 3-acetamido-4- (p-hydroxy) phenyl-2-butanone (N-acetyltyrosine):

A mixture of tyrosine (28.9 g, 16 mmol), acetic anhydride (97.9 g, 96 mmol) and pyridine (35 g, 16 mmol) is heated to 100 ° C. for 1 hour. The reaction mixture is concentrated in vacuo to finally give a yellow oil. The oil was distilled off under reduced pressure to finally give 29.9 g of oil.

¹ H NMR (DMSO-d6): NMR (d6-DMSO); 8.2 (d, 1H), 7.3 (d, 2H), 7.0 (d, 2H), 4.4 (m, 1H), 3.1 (dd, 1H), 2.7 (dd, 1H), 2.3 (s, 3H), 1.8 (s, 3H)

Example 14

Preparation of 3-acetamido-7-amino-2-butanone (N-acetyllysinone):

Lysine is converted to N-acetyllysinone according to the process of Example 3.

¹ H NMR (DMSO-d6): 8.1 (d, 1H), 7.8 (br. M. 1H), 4.1 (m, 1H), 3.0 (m, 2H), 2.0 (s, 3H), 1.9 (s, 3H) and 1.3 (br. M, 6H).

Example 15

Preparation of 3-acetamido-5-methyl-2-butanone (N-acetyllucisinone):

According to the process of Example 3, leucine is converted to N-acetyllucisinone.

¹ H NMR (DMSO-d6): 8.1 (d, 1H), 4.2 (m, 1H), 2.0 (s, 3H), 1.8 (s, 3H), 0.8 (d, 6H).

Example 16

Modification of 4- (4-aminophenyl) butyric acid with benzene sulfonyl chloride:

Dissolve 4- (4-aminophenyl) butyric acid (20 g 0.11 moles) in 110 mL of 2N aqueous sodium hydroxide solution. After stirring for 5 minutes at room temperature, benzene sulfonyl chloride (14.2 mL, 0.11 moles) is added dropwise to the amino acid solution for about 15 minutes. After stirring for about 3 hours at room temperature the mixture is acidified to pH 2 by addition of hydrochloric acid. Separation by a filter finally yields a light brown precipitate. The precipitate is washed with warm water and dried. Melting point is 123-25 ℃.

If necessary the altered amino acid is altered by recrystallization or chromatography.

Example 17

Modification of 4- (4-Aminobenzoic Acid) with Benzene Sulfonyl Chloride:

The 4-aminobenzoic acid is converted to 4- (phenylsulfonamido) benzoic acid according to the process of Example 6.

Example 18

Modification of 4-Aminophenylacetic Acid, 4-Aminohydric Acid, and 4-Aminomethylbenzoic Acid with Benzene Sulfonyl Chloride:

According to the process of Example 6, 4-aminophenylacetic acid, 4-aminohypuriic acid, and 4-aminomethylbenzoic acid were respectively 4- (phenylsulfonamido) phenylacetic acid, 4- (phenylsulfonamido) hypuriric acid And 4- (phenylsulfonamidomethyl) benzoic acid.

Example 19

Changes in Benzene Sulfonyl Chloride and Amino Acids:

A mixture of 16 amino acids was prepared with previous chemical alterations. The components of the mixture are summarized in the table below. 65 g of the amino acid mixture (total concentration of [--NH2] group = 0.61 mol) are dissolved in 760 mL of 1N sodium hydroxide solution (0.7625 equiv) at room temperature. After stirring for 20 minutes, benzene sulfonyl chloride (78 ml, 1 equiv) is added for about 20 minutes. The reaction mixture is then stirred for 2.8 hours without heating. If some precipitation occurs, additional NaOH solution will be added to reach pH 9.3. The reaction mixture is stirred overnight at room temperature. The mixture is then acidified with dilute hydrochloric acid (38%, 1: 4) and the creamy substance precipitates out. The precipitated product is separated by pouring and is dissolved in sodium hydroxide (2N). This solution is then reduced under vacuum and dried in a lyophilizer to yield a yellow solid.

Table 10

Example 20

Modification of 5 Amino Acid Mixtures with Benzene Sulfonyl Chloride:

An amino acid mixture of 86.1 g (0.85 moles of NH ₂ ) (shown in the table below) is dissolved in 643 mL of 2N aqueous sodium hydroxide solution (1.5 equiv). After stirring for 30 minutes at room temperature, benzene sulfonyl chloride (108 mL, 0.86 moles) is partially added to the amino acid solution for about 15 minutes. After stirring for 2,5 hours at room temperature, the pH of the reaction mixture (pH 5) is adjusted to pH 9 with additional 2N sodium hydroxide solution. The reaction mixture is stirred overnight at room temperature. The pH of the reaction mixture is then adjusted to pH 2.5 with additional dilute aqueous hydrochloric acid solution (4: 1, H ₂ 0: HC1) to form precipitates of altered amino acids. The upper layer is discarded and the resulting yellow solid is separated by pouring, washed with water and dissolved in 2N sodium hydroxide solution. The solution is reduced under vacuum and lyophilized overnight to yield a yellow solid.

Table 11

Example 21

Modification of a 5 Amino Acid Mixture with Benzoyl Chloride:

An amino acid mixture of 86.1 g (0.85 moles of NH ₂ ) (shown in the table of Example 20) is dissolved in 647 mL of 2N aqueous sodium hydroxide solution (1.5 equiv). After stirring for 10 minutes at room temperature, benzoyl chloride (99 mL, 0.85 moles) is added to the amino acid solution partially for about 10 minutes. After stirring for 2,5 hours at room temperature, the pH of the reaction mixture (pH 12) is adjusted to pH 2.5 with additional aqueous dilute hydrochloric acid solution (4: 1, H ₂ 0: HC1) to form precipitates of altered amino acids. After precipitation for 1 hour, the resulting precipitate is separated by pouring, washed with water and dissolved in 2N sodium hydroxide solution. The solution is reduced in vacuo to yield an altered amino acid that is not purified to a white solid (expected yield 220.5 g).

Example 22

Modification of L-valine with Benzene Sulfonyl Chloride:

L-valine (50 g, 0.43 mol) is dissolved in 376 mL of 2N aqueous sodium hydroxide solution (0.75 equiv) by stirring for 10 minutes at room temperature. Benzene sulfonyl chloride (68.7 mL, 0.38 mol, 1.25 equiv) is then added to the amino acid solution at room temperature for about 20 minutes. Precipitation forms at room temperature for 2 hours. The precipitate is dissolved by addition of an additional 200 mL of 2N sodium hydroxide solution. After further stirring for 30 minutes, dilute hydrochloric acid aqueous solution (4: 1, H ₂ O: HCl) is added so that the pH of the reaction mixture reaches 2.6. A precipitate of altered amino acids is formed and recovered by pouring. This material is taken up in 2N sodium hydroxide solution and dried in vacuo to separate the white solid, with an expected yield of 84.6 g (77%) of unrefined modified amino acid.

Example 23

Modifications of Phenylalanine Methyl Ester with Benzene Sulfonyl Chloride:

L-phenylalanine methyl ester (15 g, 0.084 mol) is dissolved in dimethylformamide (DMF) (100 mL) and pyridine (30 mL) added thereto. A solution of hypuryl chloride (16.6 g, 0.084 mol in 100 mL DMF) is added immediately to the amino acid ester solution in two portions. The reaction mixture is stirred overnight at room temperature. The reaction mixture is then reduced in vacuo and dissolved in 1N sodium hydroxide solution. The solution is heated at 70 ° C. for 3 hours to hydrolyze the methyl ester to free carboxylic acid. The solution is then acidified to pH 2.25 using dilute hydrochloric acid aqueous solution (3: 1, H ₂ O / HCl). A precipitate, such as a gum, is formed which is recovered and dissolved in 1N sodium hydroxide solution. Reducing the solution in vacuo yields the expected 18.6 g of unpurified altered amino acid product. After recrystallization from acetonitrile, pure modified phenylalanine (expected yield 12 g) is recovered as a white powder. mp 223-225 ° C.

Example 24

Preparation of Dose Solution of PYY (3-36)

2.9 ml of 15% ethanol is added to 568 mg of acetyl phenylalanine aldehyde, 132 mg of carbomethoxy phenylalanine leucine, and 100 mg of acetyl-Phe-Leu-Leu-Arg aldehyde in the test tube. The solution is stirred and NaOH (1.0 N) is added to raise the pH to 7.2. Water is added to bring the total volume to 4.0 mL. The sample has a mediator concentration of 200 mg / mL. PYY (3-36) (800 μg) is added to the solution. The total PYY3-36 concentration is 200 g / mL.

A similar procedure prepares a second solution with 668 mg of acetyl phenylalanine aldehyde as the mediator component and 132 mg carbomethoxy phenylalanine leucine and a third solution with acetyl phenylalanine aldehyde as mediator. Each solution has an endotoxin-free PYY (3-36) concentration of 200 ug / mL.

Example 25

Preparation of Altered Amino Acids / PYY (3-36) Components

 Preparation of Modified Amino Acid Microspheres Containing Protected Endotoxin-Free PYY3-36

The modified amino acid mixture prepared in the same manner as Example 9 is dissolved in distilled water (pH 7.2) at a concentration of 100 mg / ml at 40 ° C. The solution is then filtered with a 0.2 micron filter and the temperature is maintained at 40 ° C. PYY3-36 (Bachem) is dissolved in an aqueous solution of citric acid (1.7 N) and gelatin (5%) at a concentration of 150 mg / ml. This solution is then heated to 40 ° C.

The two heated solutions are then mixed at 1: 1 (v / v). The microsphere suspension results are then filtered with glass wool and centrifuged at 1000 g for 50 minutes. The pellet is resuspended in 0.85N citric acid to be 5 to 7 times the original volume. PYY3-36 concentration of resuspended pellet is determined by HPLC. Additional microspheres are prepared according to the above process except for PYY3-36. This "empty microsphere" is used to dilute the preparation of salmon-colored PYY3-36 microspheres protected with a final dose suspension if necessary.

(b) Preparation of Soluble Modified Amino Acid Mediator / PYY3-36 System

Soluble amino acid dose preparations comprising PYY3-36 are prepared by dissolving the modified amino acid material in distilled water (pH 8) at an appropriate concentration. The solution is heated to 40 ° C. and then filtered with a 0.2 micron filter. PYY3-36 is also dissolved in distilled water and then added with a modified amino acid solution prior to oral administration.

Pulmonary Delivery of PYY3-36

(prediction)

The carrier compound prepared as described below will probably be used directly as a delivery vehicle by simply mixing one or more compounds or salts, polyamino acids or peptides with an endotoxin-free Y2 receptor-binding peptide for pulmonary delivery.

Dosage mixtures are prepared by mixing an aqueous solution of the active ingredient with an aqueous solution of the medium just prior to administration. Optionally, the mediator and the physiological or chemically active ingredient can be mixed during the manufacturing process. The solution will optionally include adducts such as phosphate buffer salts, citric acid, acetic acid, gelatin, and gum acacia.

Many known lung delivery methods can use endotoxin-free Y2 receptor-binding peptides, in particular PYY3-36, to improve the delivery of PYY to the lung. The following non-limiting patent applications are incorporated herein by reference for page delivery: US Pat. Nos. 20030223939, 20030215514, 20030215512, 20030209243, 20030203036, 20030198601, 20030183228, 200301885765, 20030150454, 20030124193, 20030094173.

Example 26

Preparation of Media

Preparation of 2- (4- (N-salicyloyl) aminophenyl) propanoic acid (mediator B)

A slurry of 500. methylene chloride with 58.6 g (0.355 mol) of 2- (4-aminophenyl) propanoic acid was treated with 90.11 ml (77.13 g. 0-710 mol) trimethylsilyl chloride and heated to reflux for 120 minutes. do. The reaction mixture is cooled to 0 ° C. and treated with 184.44 ml (107.77 g, 1.065 mol) of triethylamine. After stirring for 5 minutes, the mixture is treated with a solution of 70.45 g (0.355 mol) of O-acetylsalicyloyl chloride and 150 ml of methylene chloride. The reaction mixture is warmed to 25 ° C. and stirred for 64 hours. Volatile substances are removed under vacuum. The residue is stirred for 1 hour in 2N sodium hydroxide solution and acidified with 2M aqueous sulfuric acid solution. The solid is recrystallized twice from ethanol / water to give a brown solid. Separation by filter is within the expected yield of 53.05 g (52% yield) of 2- (4- (N-salicyloyl) aminophenyl) propanoic acid. characteristic. Solubility: 200 mg / m: 200 mg + 350 μl 2N NaOH + 650 μl H ₂ 0-pH-7.67. Analysis: C, 67.36; H, 5.3; N, 4.91.

Preparation of Sodium 2- (4- (N-salicyloyl) aminophenyl) propionate (Sodium Salt of Media B)

A solution of 53.05 g (0.186 mol) of 2- (4- (N-salicyloyl) aminophenyl) propanoic acid and 300 ml of ethanol is treated with 7.59 g (0.190 mol) of NaOH dissolved in 22 ml of water. . The reaction mixture is stirred at 25 ° C. for 30 minutes and at 0 ° C. for 30 minutes. The resulting pale yellow solid is separated by a filter to give 52.61 g of sodium 2- (4- (N-salicyloyl) aminophenyl) propionate. characteristic. Solubility: 200 mg / ml clear solution, pH = 6.85. Anal C, 60.45; H, 5. 45; N, 3.92; Na, 6.43. Melting Point 236-238 ℃

Preparation of Sodium Salt of Mediator C

A 2 L round bottom flask equipped with a magnetic stirrer and a reflux condenser are charged with a suspension of 3- (4-aminophenyl) propionic acid (15.0 g. 0.084 moles, 1.0 equiv) in dichloromethane (250 ml). Chlorotrimethylsilane (18.19 g, 0.856 moles, 2.0 equiv) is added in one portion and the mixture is heated to reflux for 1.5 h under argon. The reaction is allowed to cool to room temperature and placed in an ice vessel (internal temperature <10 ° C.). The reflux condenser is replaced with an additional funnel containing triethylamine (25.41 g, 0.251 moles, 3.0 equiv). Triethylamine is added dropwise for about 15 minutes and a yellow solid forms during the addition. The funnel is replaced by another additional funnel containing a solution (100 mL) of dimethoxybenzo chloride (8.31 g, 0.091 moles, 1.09 equiv). The solution is added dropwise for about 30 minutes. The reaction is stirred for another 30 minutes in an ice container and for 3 hours at room temperature. Dichloromethane is evaporated in vacuo to give a brown oil. The brown oil is cooled in an ice container and added to an ice-cold solution (250 ml) of saturated sodium bicarbonate. The ice container is removed and the reaction is stirred for 1 hour to obtain a clean brown solution. The solution is acidified with concentrated HCl and stored in ca SC for 1 hour. The mixture is extracted with dichloromethane (3 times, 100 mL) and dried over sodium sulfate, then the salts are filtered off and the dichloromethane is removed under vacuum.

12 g of medium C acid is dissolved in 75 mL of ethanol with warmth. To this solution is added 8.5 M sodium hydroxide (1.02 molar equivalents, 1.426 g in 4.5 mL water) solution. The mixture is stirred for 15 minutes. About 3/4 of ethanol is removed under vacuum and added to cause the formation of a precipitate in 100 mL of n-heptane oil product. The solid is dried under vacuum at 50 ° C.

Analysis: C ₁₉ H ₂₀ NO ₅ Na 0.067 H ₂ 0: C, 62.25; H, 5.54; N, 3.82; Na, 6.27.

Preparation of N- (4-methylsalicynoyl) -8-aminocaprylic acid (mediator D)

(a) Preparation of Oligos (4-Methylsalicylate)

Acetic anhydride (32 mL, 34.5 g, 0.338 mol, 1.03 eq), 4-methylsalicylic acid (50 g, 0.329 mmol, 1.00 eq), and xylene (100 mL) in 1 L, magnetic stir bar, thermometer, and It is applied to a four necked flask equipped with a condenser. The flask is placed in a sand container where the heating of the cloudy white mixture begins. The reaction mixture turns into a clear yellow solution near 90 ° C. Most volatile organics (xylene and acetic acid) are distilled in the Dean-Stark trap for more than 3 hours (135-146 ° C). The distillation continues for another hour while the vessel temperature slowly increases to 204 ° C. and allows the distilled material to slowly drip off. The residue is poured into a white, still hot aluminum tray. When cooled, a fragile yellow glass is formed. The solid remains a fine powder. Oligo (4-methylsalicylate) is obtained for use without further purification.

(b) Preparation of N- (4-methylsalicynoyl) -8-aminocaprylic acid

A 7M solution of potassium carbonate (45 mL, 43.2 g, 0.313 mol, 0.95 equiv), 8-aminocaprylic acid (41.8 g, 262 mol, 798 equiv), and water (20 mL) was added to a magnetic stir bar, condenser, Add to a 1 L round bottom flask equipped with additional funnels. The pale white mixture is treated with oligo (4-methylsalicylate) (44.7 g, 0.329 mmol 1.0 equiv) and dioxane (250 mL) with the solution added for at least 30 minutes. The reaction mixture is heated to 90 ° C. for 3 hours (at which time the reaction is determined by HPLC). The clean orange reaction mixture is cooled to 30 ° C. and acidified to pH = 2 with 50% aqueous sulfuric acid solution (64 g). The solid product is separated by a filter. The white solid is recrystallized in 1170 mL of 50% ethanol-water. The solid is recovered by the filter and dried for at least 18 hours in a vacuum oven at 50 ° C. N- (4-methylsalicynoyl) -8-aminocaprylic acid separated as a white solid (30.88 g, 52%); mp = 113-114 ° C. Analysis: C ₆ H ₂₃ NO ₄ : C, 65.51; H, 7.90; N, 4.77.

An aqueous solution of PYY (3-36) is then mixed with one or more vehicles that can be prepared and sprayed into the lungs to produce the PYY (3-36) construct. The appropriate concentration of PYY3-36 should be about 400 pg / mL for the composition result. See US Patent Application No. 20030072740.

Example 27

Total Extracted Radioimmunoassay for Determination of PYY Concentration in Plasma

1.0 Introduction:

Radioimmunoassays have been developed to determine the concentration of human peptide YY (3-36) (hPYY) in human plasma. Samples are collected and frozen with anticoagulant (EDTA) and protease inhibitor (aprotinin). Analysis is a 4 day process. On the first day samples, controls and standards are extracted with alcohol and dried. On the second day all samples are reconstituted and mixed with polyclonal rabbit antiserum, which is directly opposite hPYY. On the third day, the iodine marker hPYY is applied. On the fourth day a special precipitating agent (goat anti-rabbit IgG and normal rabbit serum) is added. The free tracer is separated by centrifugation, and the combined tracer is counted in a gamma counter. Concentration is calculated by insertion of a standard curve and assay performance is controlled by quality control samples.

2.0 Materials:

2.1 Peninsula PYY Kit (Peninsula Laboratories, Cat.No. S-2043-0001)

2.2 Alcohol Reagent (Fisher Inc., Cat.No. A995-4) (or equivalent)

2.3 Stripped human plasma (fasted, infused with lithium heparin) Golden West Biologies Inc. (Cat. No., SD1020-H) (Analytical SOP &num; A-003)

2.4 Ice Baths (Fisher, Cat No. 11-676-36) (or equivalent)

2.5 10 mL pipette (Fisher Cat.No.13-678-11E) (or equivalent)

2.6 Standard Human PYY from Nastech QC (3-36) (Bachem Cat. No. H8585)

2.7 distilled water (Milli-Q Millipore, Cat.No. ZMQ56VFT1) (or equivalent)

2.8 Triton X-100 (Sigma, Cat.No. T-9284) (or equivalent)

2.9 Aluminum Foil (Fisher, Cat. No. 01-213-3) (or equivalent)

2.10 Aprotinin (ICN Biomedicals Inc. Cat.No. 190779) (or equivalent)

2.11 2x75 mm tube (Evergreen Scientific, Cat.No. 214-2023-010) (or equivalent)

2.12 12x75 mm Tube Caps (Evergreen Scientific, Cat.No. 300-2912-G20) (or equivalent)

2.13 1.5 mL microfuge tubes (Fisher, Cat. No. 05-402-25) (or equivalent)

2.14

3.0 devices:

3.1 Wallac WIZARD 1470 Automatic Gamma Counter (Perkin Elmer, Model No. 1470-002) (or equivalent)

3.2 Isotemp Basic Freezer, -70 ° C (Kendro Laboratory Products, Model No. C90-3A31) (or equivalent)

3.3 CentriVap Thickener (Labconco, Cat.No.7810000) (or equivalent)

3.4 VX-2500 multitube vortexer (VWR, Cat.No. 58816-115) (or equivalent)

3.5 Marathon21000R centrifuge (Fisher, Cat. No. 04-977-21000R) (or equivalent)

3.6 Swing Bucket Rotor (Fisher, Cat.No. 04-976-006) (or equivalent)

3.7 Motorized Pipette-Aid (Fisher, Cat.No. 13-681-15E) (or equivalent)

3.8 Eppendorf Micropipettes

3.8.1 2 μl-20 μl (Fisher, Cat.No. 21-371-6) (or equivalent)

3.8.2 20 μl-200 μl (Fisher, Cat. No. 21-371-10) (or equivalent)

3.8.3 100 μl to 1000 μl (Fisher, Cat. No. 21-371-13) (or equivalent)

3.9 Eppendorf Repeat Pipette (Fisher, Cat.No. 21-380-9) (or equivalent)

3.10 Eppendorf Repeat Pipette Blender Tips

3.10.1 2.5 mL (Fisher, Cat.No. 21-381-331) (or equivalent)

3.10.2 25 mL (Fisher, Cat.No. 21-381-115) (or equivalent)

3.11 Quantitative Transfer Pipettes (Fisher, Cat.No. 21-169-10A) (or equivalent)

4.0 process

DAY 1

4.1 Dissolve the reagents and samples required for analysis. If a sufficient amount is not needed, prepare RIA buffer at 1 × concentration (RIAB).

4.2 Prepare standard curve samples in mixed and exfoliated human plasma. If a starting concentration of 12.8 μg / mL is used, prepare as follows.

4.2.1 Add 990 μl RIAB to Tube O.

4.2.2 Add 990 μl of infused plasma to Tube A.

4.2.3 Add 500 μL of mixed plasma with Tubes B-H.

4.2.4 Add 10 μl 12.8 g / mL standard with Tube O vortex.

4.2.5 Add 10 μl solution from Tube O with the Tube A vortex.

4.2.6 Add 500 μl solution from Tube A with the Tube B vortex.

4.2.7 Add 500 μl solution from Tube B with Tube C vortex.

4.2.8 Tube H is repeated dilution as in 4.2.7 (shown in Scheme # 1).

4.3 If necessary, dilute an unknown human plasma sample for testing. Samples should be diluted into mixed and exfoliated human plasma.

4.4 Add 1.2 mL of cold alcohol to empty tubes for NSB, TB, all standards, QC samples, and human plasma samples to be tested.

4.5 Add 400 μL of mixed and stripped human plasma to the NSB and TB tubes caps, vortex.

4.6 Individual Standard Curve Tubes H-A (shown in Scheme # 1) With a cap vortex, 400 μl of standard samples prepared from 4.2.5 to 4.2.8, respectively, are added.

4.7 μL of QC samples are added to the individual tubes cap vortex.

4.8 Sample 400 μl of the gel of each of the tested samples is added to its individual tubes cap vortex.

4.9 All samples are incubated on ice for 30-60 minutes.

4.10 Turn on the cold-trap switch on the compressor.

4.11 Centrifuge all tubes for 15 min at 3000 rpm at 4 ° C.

4.12 Transfer the supernatant from 1.3 mL to each of the three empty tubes from each supernatant sample. If not turned immediately, store in ice bath or 2-8 ℃.

4.13 Place samples on the compressor.

4.14 Samples should be run for 2 hours at 40 ° C., followed by a full 5 hours at room temperature or during drying.

4.15 Remove dried samples and store overnight at 2-8 ° C.

DAY 2

4.16 Remove the dried tubes from the 2-8 ° C cooler.

4.17 Add 100 μl of 4 ㅧ RIA buffer concentration to each tube.

4.18 Add 100 μl of 0.6% TX100 (Attachment # 1) to each tube to ensure that all extracts are fully reconstituted for 30 seconds.

4.19 All samples are incubated on ice for 30-60 minutes.

4.20 Water Add 200 μl of distilled water to each tube vortex.

4.21 Transfer 100 μl of each sample extract to individual tubes.

Note: NSB, TB, TC, standard curve samples, and QCs typically require three tubes for each sample since they are triple reactions. Human plasma samples are often tested at any fluctuation depending on sample availability.

4.22 Rabbit rabbit PYY is prepared as described in Peninsula Laboratories Kit Insert.

4.23 Add 100 μl of RIAB to each NSB tube.

4.24 Add 200 μl RIAB to each TC tube.

4.25 Add 100 μl rabbit anti PYY to all remaining tubes vortex.

4.26 Cover with foil and store overnight at 2-8 ° C.

DAY 3

4.27 Remove the tubes from the 2-8 ° C cooler.

4.28 Prepare a ¹²⁵ I-peptide YY tracer (Attachment # 2).

4.29 Add 100 μl of prepared tracer to all tubes, caps and vortex.

4.30 Store overnight at 2-8 ° C.

DAY 4

4.31 Remove the tubes from the 2-8 ° C cooler.

Prepare goat anti-rabbit IgG serum (GARGG) and normal rabbit serum (NRS) as described in 4.32 Peninsula Laboratories Kit Insert.

4.33 Add 100 μl of GARGG to each tube (except TC tubes).

4.34 Add 100 μl of NRS to each vortex (except TC tubes) and vortex.

4.35 Incubate 90-120 minutes at room temperature.

4.36 Tubes (except TC tubes), add 500 μl of RIAB to the vortex and centrifuge immediately.

Note: 500 μl of RIAB should only be added to the tubes prior to centrifugation. Only RIAB is added to the tubes prepared for centrifugation. 500 μl of RIAB should be added when additional tubes are ready for centrifugation.

4.37 tubes (containing 500 μl RIAB) are centrifuged for 15 minutes at 3000 rpm, 4 ° C. TC tubes are not centrifuged.

4.38 Suck the supernatant from the supernatant centrifuge tubes.

4.39 The tubes are placed in black racks designed for counting on a gamma counter. The first rack must be labeled with the appropriate program number. All racks then contain no program numbers. Samples should be taken in the following order:

4.39.1 NSB Tubes

4.39.2 TB tubes

4.39.3 TC tubes

4.39.4 Standard tubes (increase)

4.39.5 QC Samples (3 Concentrations)

4.39.6 Unknown human samples

4.39.7 QC Samples (3 Concentrations)

4.40 After all samples have been counted, attach a stop mark to the empty black rack.

4.41 Press 'Start' on the Gamma Counter keypad to start counting.

4.42 Press E 'on the gamma counter keypad for Enter to see the CPM results.

5.0 Evaluation of Results

5.1 The following guide applies to the identification and rejection of isolates in the analysis. As with isolates, all of the following conditions must be met to qualify the result and not include the final calculation of the result.

5.1.1 QCs and Unknown Samples:

5.1.1.1 The% CV of all replicas should exceed 20%.

5.1.1.2 There must be at least three results for evaluation.

5.1.1.3 The difference between the suspected isolate and the next closest value should not exceed 20%.

5.1.1.4 The difference between high and low of extra results should be less than 20%.

5.1.2 Standard Curve Samples:

5.1.2.1 The% CV of all replicas must be much greater than 15%.

5.1.2.2 There must be at least three results for evaluation.

5.1.2.3 The difference between the suspected isolate and the next closest value should be more than 20%.

5.1.2.4 The difference between high and low of extra results should be less than 15%.

6.0 Analysis Features

6.1 QC samples are prepared at the following concentrations. Two samples at each concentration are tested in the assay. Four of the six QC samples tested should fall within the following area (usually 30% of the concentration). At least one of the two QCs tested at any concentration should be within the scope of analysis for acceptable data.

6.1.1 QC1 (100 pg / mL) 70-130 pg / mL

6.1.2 QC2 (200 pg / mL) 140-260 pg / mL

6.1.3 QC3 (500 pg / mL) 350-650 pg / mL

6.2 Standard curve parameters require TBD.

2. Schematic # 1

PYY RIA standard:

Attachment # 1

0.6% TX-100

Reagent: 0.6% TX-100

Substance: Milli-Q Distilled Water

TX-100 Preparation: 1) Measure 50 mL of Milli-Q distilled water

             2) Add 300 μL of TX-100 using a quantitative transfer pipette.

             3) Mix the wells.

Attachment # 1

¹²⁵ I-peptide PYY Tracer

Reagent: ¹²⁵ I-peptide PYY Tracer

Substance: l × RIA buffer

¹²⁵ I-peptide PYY

Preparation: 1) Reconstitute 1 mL l × RIA buffer and tracer.

     2) Measure the amount of tracer on the gamma counter. Carry 10 μl of reconstituted tracer into the tracer tube. Place in black rack for gamma counter with program number 30 attached.

    3) Place the rack on the gamma counter with the rear stop rack.

     4) Press 'Start' to begin and then press 'E' to see CPM results.

     5) Determine the amount of tracer (X μl) and the required RIAB for preparation as follows:

6) Combine X μl of ¹²⁵ I-peptide YY with Y mL of RIAB. Mix the wells.

Example 28

Preparation of NPY Formulation Free of Protein Stabilizer

PYY formulations suitable for intranasal administration of NPY, a potential free of protein stabilizer, are prepared having the formulations described below.

12. Approximately 3/4 of water is added to the beaker and stirred on the stir bar and stirred until the sodium citrate is completely dissolved.

13. EDTA is then added and stirred until it is completely dissolved.

14. Next, citric acid is added and stirred until it is completely dissolved.

15. Methyl- (3-cyclodextrin) is added and stirred until complete dissolution.

16. Next, DDPC is added and stirred until complete melting.

17. Lactose is then added and stirred until it is completely dissolved.

18. Sorbitol is then added and stirred until it is completely dissolved.

19. Chlorobutane is then added and stirred until it is completely dissolved.

20. NPY (3-36) is then added and stirred slowly until dissolved.

21. Check that the pH is 5.0 ± 0.25. Dilute hydrochloric acid or dilute NaOH is added to adjust pH.

22. Add water to final volume.

Table 12

Formulation pH 5 +/- 0. 25

Osmolality ~ 250

Example 29

A second formulation is prepared as described above except that the concentration of NPY (3-36) is 15 mg / mL as shown in Table 13 below.

Table 13

Formulation pH 5 +/- 0. 25

Example 30

Preparation of Pancreatic Peptide (PP) Formulation Formulations of Protein Stabilizers

PYY formulations suitable for intranasal administration of PP, a potential free of protein stabilizer, are prepared having the formulations described below.

23. Approximately 3/4 of water is added to the beaker and stirred on a stir bar and stirred until the sodium citrate is completely dissolved.

24. EDTA is then added and stirred until it is completely dissolved.

25. Next, citric acid is added and stirred until it is completely dissolved.

26. Methyl- (3-cyclodextrin) is added and stirred until complete dissolution.

27. Next, DDPC is added and stirred until complete melting.

28. Lactose is then added and stirred until it is completely dissolved.

29. Next, sorbitol is added and stirred until it is completely dissolved.

30. Chlorobutane is then added and stirred until it is completely dissolved.

31. NPY (3-36) is then added and stirred slowly until dissolved.

32. Check that the pH is 5.0 ± 0.25. Dilute hydrochloric acid or dilute NaOH is added to adjust pH.

33. Add water to final volume.

Table 14

Formulation pH 5 +/- 0. 25

Osmolality ~ 250

Example 31

A second formulation is prepared as described above except that the concentration of PP (3-36) is 15 mg / mL as shown in Table 15 below.

Table 15

Formulation pH 5 +/- 0. 25

Simple modifications and variations of the present invention can be readily used by those skilled in the art, and all such variations or modifications can be considered to be included within the scope of the present invention.

<110> Nastech Pharmaceutical Company Inc.          Quay, Steven C.          Brandt, Gordon          Kleppe, Mary S.          MacEvilly, Conor J. <120> COMPOSITIONS AND METHODS FOR ENHANCED MUCOSAL DELIVERY OF Y2          RECEPTOR-BINDING PEPTIDES AND METHODS FOR TREATING AND PREVENTING          OBESITY <130> 02-04 PCT <150> US 60 / 518,812 <151> 2003-11-10 <150> US 10 / 322,266 <151> 2002-12-17 <150> US 60 / 493,226 <151> 2003-08-07 <150> US 60 / 501,170 <151> 2003-09-08 <150> US 60 / 510,785 <151> 2003-10-10 <150> US 60 / 517,290 <151> 2003-11-04 <160> 105 FastSEQ for Windows Version 4.0 <210> 1 <211> 36 <212> PRT <213> Homo sapiens <400> 1 Tyr Pro Ile Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu   1 5 10 15 Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr              20 25 30 Arg Gln Arg Tyr          35 <210> 2 <211> 34 <212> PRT <213> Homo sapiens <400> 2 Ile Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu Asn   1 5 10 15 Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln              20 25 30 Arg Tyr         <210> 3 <211> 15 <212> PRT <213> Homo sapiens <400> 3 Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr   1 5 10 15 <210> 4 <211> 33 <212> PRT <213> Homo sapiens <400> 4 Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu Asn Arg   1 5 10 15 Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg              20 25 30 Tyr     <210> 5 <211> 32 <212> PRT <213> Homo sapiens <400> 5 Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu Asn Arg Tyr   1 5 10 15 Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr              20 25 30 <210> 6 <211> 31 <212> PRT <213> Homo sapiens <400> 6 Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu Asn Arg Tyr Tyr   1 5 10 15 Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr              20 25 30 <210> 7 <211> 30 <212> PRT <213> Homo sapiens <400> 7 Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu Asn Arg Tyr Tyr Ala   1 5 10 15 Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr              20 25 30 <210> 8 <211> 29 <212> PRT <213> Homo sapiens <400> 8 Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu Asn Arg Tyr Tyr Ala Ser   1 5 10 15 Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr              20 25 <210> 9 <211> 28 <212> PRT <213> Homo sapiens <400> 9 Gly Glu Asp Ala Ser Pro Glu Glu Leu Asn Arg Tyr Tyr Ala Ser Leu   1 5 10 15 Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr              20 25 <210> 10 <211> 27 <212> PRT <213> Homo sapiens <400> 10 Glu Asp Ala Ser Pro Glu Glu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg   1 5 10 15 His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr              20 25 <210> 11 <211> 26 <212> PRT <213> Homo sapiens <400> 11 Asp Ala Ser Pro Glu Glu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His   1 5 10 15 Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr              20 25 <210> 12 <211> 25 <212> PRT <213> Homo sapiens <400> 12 Ala Ser Pro Glu Glu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr   1 5 10 15 Leu Asn Leu Val Thr Arg Gln Arg Tyr              20 25 <210> 13 <211> 24 <212> PRT <213> Homo sapiens <400> 13 Ser Pro Glu Glu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu   1 5 10 15 Asn Leu Val Thr Arg Gln Arg Tyr              20 <210> 14 <211> 23 <212> PRT <213> Homo sapiens <400> 14 Pro Glu Glu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn   1 5 10 15 Leu Val Thr Arg Gln Arg Tyr              20 <210> 15 <211> 22 <212> PRT <213> Homo sapiens <400> 15 Glu Glu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu   1 5 10 15 Val Thr Arg Gln Arg Tyr              20 <210> 16 <211> 21 <212> PRT <213> Homo sapiens <400> 16 Glu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val   1 5 10 15 Thr Arg Gln Arg Tyr              20 <210> 17 <211> 20 <212> PRT <213> Homo sapiens <400> 17 Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr   1 5 10 15 Arg Gln Arg Tyr              20 <210> 18 <211> 19 <212> PRT <213> Homo sapiens <400> 18 Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg   1 5 10 15 Gln Arg Tyr             <210> 19 <211> 18 <212> PRT <213> Homo sapiens <400> 19 Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln   1 5 10 15 Arg Tyr         <210> 20 <211> 17 <212> PRT <213> Homo sapiens <400> 20 Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg   1 5 10 15 Tyr     <210> 21 <211> 16 <212> PRT <213> Homo sapiens <400> 21 Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr   1 5 10 15 <210> 22 <211> 36 <212> PRT <213> Homo sapiens <400> 22 Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp   1 5 10 15 Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr              20 25 30 Arg Gln Arg Tyr          35 <210> 23 <211> 11 <212> PRT <213> Homo sapiens <400> 23 His Tyr Ile Asn Leu Ile Thr Arg Gln Arg Tyr   1 5 10 <210> 24 <211> 34 <212> PRT <213> Homo sapiens <400> 24 Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp Met Ala   1 5 10 15 Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg Gln              20 25 30 Arg Tyr         <210> 25 <211> 33 <212> PRT <213> Homo sapiens <400> 25 Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp Met Ala Arg   1 5 10 15 Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg Gln Arg              20 25 30 Tyr     <210> 26 <211> 32 <212> PRT <213> Homo sapiens <400> 26 Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp Met Ala Arg Tyr   1 5 10 15 Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg Gln Arg Tyr              20 25 30 <210> 27 <211> 31 <212> PRT <213> Homo sapiens <400> 27 Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp Met Ala Arg Tyr Tyr   1 5 10 15 Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg Gln Arg Tyr              20 25 30 <210> 28 <211> 30 <212> PRT <213> Homo sapiens <400> 28 Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp Met Ala Arg Tyr Tyr Ser   1 5 10 15 Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg Gln Arg Tyr              20 25 30 <210> 29 <211> 29 <212> PRT <213> Homo sapiens <400> 29 Pro Gly Glu Asp Ala Pro Ala Glu Asp Met Ala Arg Tyr Tyr Ser Ala   1 5 10 15 Leu Arg His Tyr Ile Asn Leu Ile Thr Arg Gln Arg Tyr              20 25 <210> 30 <211> 28 <212> PRT <213> Homo sapiens <400> 30 Gly Glu Asp Ala Pro Ala Glu Asp Met Ala Arg Tyr Tyr Ser Ala Leu   1 5 10 15 Arg His Tyr Ile Asn Leu Ile Thr Arg Gln Arg Tyr              20 25 <210> 31 <211> 27 <212> PRT <213> Homo sapiens <400> 31 Glu Asp Ala Pro Ala Glu Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg   1 5 10 15 His Tyr Ile Asn Leu Ile Thr Arg Gln Arg Tyr              20 25 <210> 32 <211> 26 <212> PRT <213> Homo sapiens <400> 32 Asp Ala Pro Ala Glu Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His   1 5 10 15 Tyr Ile Asn Leu Ile Thr Arg Gln Arg Tyr              20 25 <210> 33 <211> 25 <212> PRT <213> Homo sapiens <400> 33 Ala Pro Ala Glu Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr   1 5 10 15 Ile Asn Leu Ile Thr Arg Gln Arg Tyr              20 25 <210> 34 <211> 24 <212> PRT <213> Homo sapiens <400> 34 Pro Ala Glu Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile   1 5 10 15 Asn Leu Ile Thr Arg Gln Arg Tyr              20 <210> 35 <211> 23 <212> PRT <213> Homo sapiens <400> 35 Ala Glu Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn   1 5 10 15 Leu Ile Thr Arg Gln Arg Tyr              20 <210> 36 <211> 22 <212> PRT <213> Homo sapiens <400> 36 Glu Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu   1 5 10 15 Ile Thr Arg Gln Arg Tyr              20 <210> 37 <211> 21 <212> PRT <213> Homo sapiens <400> 37 Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile   1 5 10 15 Thr Arg Gln Arg Tyr              20 <210> 38 <211> 20 <212> PRT <213> Homo sapiens <400> 38 Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr   1 5 10 15 Arg Gln Arg Tyr              20 <210> 39 <211> 19 <212> PRT <213> Homo sapiens <400> 39 Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg   1 5 10 15 Gln Arg Tyr             <210> 40 <211> 18 <212> PRT <213> Homo sapiens <400> 40 Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg Gln   1 5 10 15 Arg Tyr         <210> 41 <211> 17 <212> PRT <213> Homo sapiens <400> 41 Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg Gln Arg   1 5 10 15 Tyr     <210> 42 <211> 16 <212> PRT <213> Homo sapiens <400> 42 Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg Gln Arg Tyr   1 5 10 15 <210> 43 <211> 15 <212> PRT <213> Homo sapiens <400> 43 Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg Gln Arg Tyr   1 5 10 15 <210> 44 <211> 14 <212> PRT <213> Homo sapiens <400> 44 Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg Gln Arg Tyr   1 5 10 <210> 45 <211> 13 <212> PRT <213> Homo sapiens <400> 45 Leu Arg His Tyr Ile Asn Leu Ile Thr Arg Gln Arg Tyr   1 5 10 <210> 46 <211> 12 <212> PRT <213> Homo sapiens <400> 46 Arg His Tyr Ile Asn Leu Ile Thr Arg Gln Arg Tyr   1 5 10 <210> 47 <211> 36 <212> PRT <213> Homo sapiens <400> 47 Ala Ser Leu Glu Pro Glu Tyr Pro Gly Asp Asn Ala Thr Pro Glu Gln   1 5 10 15 Met Ala Gln Tyr Ala Ala Glu Leu Arg Arg Tyr Ile Asn Met Leu Thr              20 25 30 Arg Pro Arg Tyr          35 <210> 48 <211> 11 <212> PRT <213> Homo sapiens <400> 48 Arg Tyr Ile Asn Met Leu Thr Arg Pro Arg Tyr   1 5 10 <210> 49 <211> 34 <212> PRT <213> Homo sapiens <400> 49 Leu Glu Pro Glu Tyr Pro Gly Asp Asn Ala Thr Pro Glu Gln Met Ala   1 5 10 15 Gln Tyr Ala Ala Glu Leu Arg Arg Tyr Ile Asn Met Leu Thr Arg Pro              20 25 30 Arg Tyr         <210> 50 <211> 33 <212> PRT <213> Homo sapiens <400> 50 Glu Pro Glu Tyr Pro Gly Asp Asn Ala Thr Pro Glu Gln Met Ala Gln   1 5 10 15 Tyr Ala Ala Glu Leu Arg Arg Tyr Ile Asn Met Leu Thr Arg Pro Arg              20 25 30 Tyr     <210> 51 <211> 32 <212> PRT <213> Homo sapiens <400> 51 Pro Glu Tyr Pro Gly Asp Asn Ala Thr Pro Glu Gln Met Ala Gln Tyr   1 5 10 15 Ala Ala Glu Leu Arg Arg Tyr Ile Asn Met Leu Thr Arg Pro Arg Tyr              20 25 30 <210> 52 <211> 31 <212> PRT <213> Homo sapiens <400> 52 Glu Tyr Pro Gly Asp Asn Ala Thr Pro Glu Gln Met Ala Gln Tyr Ala   1 5 10 15 Ala Glu Leu Arg Arg Tyr Ile Asn Met Leu Thr Arg Pro Arg Tyr              20 25 30 <210> 53 <211> 30 <212> PRT <213> Homo sapiens <400> 53 Tyr Pro Gly Asp Asn Ala Thr Pro Glu Gln Met Ala Gln Tyr Ala Ala   1 5 10 15 Glu Leu Arg Arg Tyr Ile Asn Met Leu Thr Arg Pro Arg Tyr              20 25 30 <210> 54 <211> 29 <212> PRT <213> Homo sapiens <400> 54 Pro Gly Asp Asn Ala Thr Pro Glu Gln Met Ala Gln Tyr Ala Ala Glu   1 5 10 15 Leu Arg Arg Tyr Ile Asn Met Leu Thr Arg Pro Arg Tyr              20 25 <210> 55 <211> 28 <212> PRT <213> Homo sapiens <400> 55 Gly Asp Asn Ala Thr Pro Glu Gln Met Ala Gln Tyr Ala Ala Glu Leu   1 5 10 15 Arg Arg Tyr Ile Asn Met Leu Thr Arg Pro Arg Tyr              20 25 <210> 56 <211> 27 <212> PRT <213> Homo sapiens <400> 56 Asp Asn Ala Thr Pro Glu Gln Met Ala Gln Tyr Ala Ala Glu Leu Arg   1 5 10 15 Arg Tyr Ile Asn Met Leu Thr Arg Pro Arg Tyr              20 25 <210> 57 <211> 26 <212> PRT <213> Homo sapiens <400> 57 Asn Ala Thr Pro Glu Gln Met Ala Gln Tyr Ala Ala Glu Leu Arg Arg   1 5 10 15 Tyr Ile Asn Met Leu Thr Arg Pro Arg Tyr              20 25 <210> 58 <211> 25 <212> PRT <213> Homo sapiens <400> 58 Ala Thr Pro Glu Gln Met Ala Gln Tyr Ala Ala Glu Leu Arg Arg Tyr   1 5 10 15 Ile Asn Met Leu Thr Arg Pro Arg Tyr              20 25 <210> 59 <211> 24 <212> PRT <213> Homo sapiens <400> 59 Thr Pro Glu Gln Met Ala Gln Tyr Ala Ala Glu Leu Arg Arg Tyr Ile   1 5 10 15 Asn Met Leu Thr Arg Pro Arg Tyr              20 <210> 60 <211> 23 <212> PRT <213> Homo sapiens <400> 60 Pro Glu Gln Met Ala Gln Tyr Ala Ala Glu Leu Arg Arg Tyr Ile Asn   1 5 10 15 Met Leu Thr Arg Pro Arg Tyr              20 <210> 61 <211> 22 <212> PRT <213> Homo sapiens <400> 61 Glu Gln Met Ala Gln Tyr Ala Ala Glu Leu Arg Arg Tyr Ile Asn Met   1 5 10 15 Leu Thr Arg Pro Arg Tyr              20 <210> 62 <211> 21 <212> PRT <213> Homo sapiens <400> 62 Gln Met Ala Gln Tyr Ala Ala Glu Leu Arg Arg Tyr Ile Asn Met Leu   1 5 10 15 Thr Arg Pro Arg Tyr              20 <210> 63 <211> 20 <212> PRT <213> Homo sapiens <400> 63 Met Ala Gln Tyr Ala Ala Glu Leu Arg Arg Tyr Ile Asn Met Leu Thr   1 5 10 15 Arg Pro Arg Tyr              20 <210> 64 <211> 19 <212> PRT <213> Homo sapiens <400> 64 Ala Gln Tyr Ala Ala Glu Leu Arg Arg Tyr Ile Asn Met Leu Thr Arg   1 5 10 15 Pro Arg Tyr             <210> 65 <211> 18 <212> PRT <213> Homo sapiens <400> 65 Gln Tyr Ala Ala Glu Leu Arg Arg Tyr Ile Asn Met Leu Thr Arg Pro   1 5 10 15 Arg Tyr         <210> 66 <211> 17 <212> PRT <213> Homo sapiens <400> 66 Tyr Ala Ala Glu Leu Arg Arg Tyr Ile Asn Met Leu Thr Arg Pro Arg   1 5 10 15 Tyr     <210> 67 <211> 16 <212> PRT <213> Homo sapiens <400> 67 Ala Ala Glu Leu Arg Arg Tyr Ile Asn Met Leu Thr Arg Pro Arg Tyr   1 5 10 15 <210> 68 <211> 15 <212> PRT <213> Homo sapiens <400> 68 Ala Glu Leu Arg Arg Tyr Ile Asn Met Leu Thr Arg Pro Arg Tyr   1 5 10 15 <210> 69 <211> 14 <212> PRT <213> Homo sapiens <400> 69 Glu Leu Arg Arg Tyr Ile Asn Met Leu Thr Arg Pro Arg Tyr   1 5 10 <210> 70 <211> 13 <212> PRT <213> Homo sapiens <400> 70 Leu Arg Arg Tyr Ile Asn Met Leu Thr Arg Pro Arg Tyr   1 5 10 <210> 71 <211> 12 <212> PRT <213> Homo sapiens <400> 71 Arg Arg Tyr Ile Asn Met Leu Thr Arg Pro Arg Tyr   1 5 10 <210> 72 <211> 36 <212> PRT <213> Rat <400> 72 Tyr Pro Ala Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu   1 5 10 15 Leu Ser Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr              20 25 30 Arg Gln Arg Tyr          35 <210> 73 <211> 36 <212> PRT <213> Pig <400> 73 Tyr Pro Ala Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu   1 5 10 15 Leu Ser Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr              20 25 30 Arg Gln Arg Tyr          35 <210> 74 <211> 36 <212> PRT <213> Guinea pig <400> 74 Tyr Pro Ser Lys Pro Glu Ala Pro Gly Ser Asp Ala Ser Pro Glu Glu   1 5 10 15 Leu Ala Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr              20 25 30 Arg Gln Arg Tyr          35 <210> 75 <211> 36 <212> PRT <213> Rat <400> 75 Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp   1 5 10 15 Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr              20 25 30 Arg Gln Arg Tyr          35 <210> 76 <211> 36 <212> PRT <213> Rabbit <400> 76 Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp   1 5 10 15 Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr              20 25 30 Arg Gln Arg Tyr          35 <210> 77 <211> 36 <212> PRT <213> Dog <400> 77 Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp   1 5 10 15 Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr              20 25 30 Arg Gln Arg Tyr          35 <210> 78 <211> 36 <212> PRT <213> Pig <400> 78 Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp   1 5 10 15 Leu Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr              20 25 30 Arg Gln Arg Tyr          35 <210> 79 <211> 36 <212> PRT <213> Cow <400> 79 Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp   1 5 10 15 Leu Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr              20 25 30 Arg Gln Arg Tyr          35 <210> 80 <211> 36 <212> PRT <213> Sheep <400> 80 Tyr Pro Ser Lys Pro Asp Asn Pro Gly Asp Asp Ala Pro Ala Glu Asp   1 5 10 15 Leu Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr              20 25 30 Arg Gln Arg Tyr          35 <210> 81 <211> 36 <212> PRT <213> Guinea pig <400> 81 Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp   1 5 10 15 Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr              20 25 30 Arg Gln Arg Tyr          35 <210> 82 <211> 36 <212> PRT <213> Sheep <400> 82 Ala Pro Leu Glu Pro Val Tyr Pro Gly Asp Asn Ala Thr Pro Glu Gln   1 5 10 15 Met Ala Gln Tyr Ala Ala Asp Leu Arg Arg Tyr Ile Asn Met Leu Thr              20 25 30 Arg Pro Arg Tyr          35 <210> 83 <211> 36 <212> PRT <213> Pig <400> 83 Ala Pro Leu Glu Pro Val Tyr Pro Gly Asp Asp Ala Thr Pro Glu Gln   1 5 10 15 Met Ala Gln Tyr Ala Ala Glu Leu Arg Arg Tyr Ile Asn Met Leu Thr              20 25 30 Arg Pro Arg Tyr          35 <210> 84 <211> 36 <212> PRT <213> Dog <400> 84 Ala Pro Leu Glu Pro Val Tyr Pro Gly Asp Asp Ala Thr Pro Glu Gln   1 5 10 15 Met Ala Gln Tyr Ala Ala Glu Leu Arg Arg Tyr Ile Asn Met Leu Thr              20 25 30 Arg Pro Arg Tyr          35 <210> 85 <211> 36 <212> PRT <213> Cat <400> 85 Ala Pro Leu Glu Pro Val Tyr Pro Gly Asp Asn Ala Thr Pro Glu Gln   1 5 10 15 Met Ala Gln Tyr Ala Ala Glu Leu Arg Arg Tyr Ile Asn Met Leu Thr              20 25 30 Arg Pro Arg Tyr          35 <210> 86 <211> 36 <212> PRT <213> Cow <400> 86 Ala Pro Leu Glu Pro Glu Tyr Pro Gly Asp Asp Ala Thr Pro Glu Gln   1 5 10 15 Met Ala Gln Tyr Ala Ala Glu Leu Arg Arg Tyr Ile Asn Met Leu Thr              20 25 30 Arg Pro Arg Tyr          35 <210> 87 <211> 36 <212> PRT <213> Rat <400> 87 Ala Pro Leu Glu Pro Met Tyr Pro Gly Asp Tyr Ala Thr His Glu Gln   1 5 10 15 Arg Ala Gln Tyr Glu Thr Gln Leu Arg Arg Tyr Ile Asn Thr Leu Thr              20 25 30 Arg Pro Arg Tyr          35 <210> 88 <211> 36 <212> PRT <213> mouse <400> 88 Ala Pro Leu Glu Pro Met Tyr Pro Gly Asp Tyr Ala Thr Pro Glu Gln   1 5 10 15 Met Ala Gln Tyr Glu Thr Gln Leu Arg Arg Tyr Ile Asn Thr Leu Thr              20 25 30 Arg Pro Arg Tyr          35 <210> 89 <211> 37 <212> PRT <213> Guinea pig <400> 89 Ala Pro Leu Glu Pro Val Tyr Pro Gly Asp Asn Ala Thr Pro Glu Gln   1 5 10 15 Gln Met Ala Gln Tyr Ala Ala Glu Met Arg Arg Tyr Ile Asn Met Leu              20 25 30 Thr Arg Pro Arg Tyr          35 <210> 90 <211> 22 <212> PRT <213> Homo sapiens <400> 90 Asp Glu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu   1 5 10 15 Val Thr Arg Gln Arg Tyr              20 <210> 91 <211> 24 <212> PRT <213> Homo sapiens <400> 91 Thr Pro Glu Glu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu   1 5 10 15 Asn Leu Val Thr Arg Gln Arg Tyr              20 <210> 92 <211> 25 <212> PRT <213> Homo sapiens <400> 92 Val Ser Pro Glu Glu Leu Asn Arg Tyr Tla Ala Ser Leu Arg His Tyr   1 5 10 15 Leu Asn Leu Val Thr Arg Gln Arg Tyr              20 25 <210> 93 <211> 26 <212> PRT <213> Homo sapiens <400> 93 Glu Ala Ser Pro Glu Glu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His   1 5 10 15 Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr              20 25 <210> 94 <211> 27 <212> PRT <213> Homo sapiens <400> 94 Asp Asp Ala Ser Pro Glu Glu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg   1 5 10 15 His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr              20 25 <210> 95 <211> 30 <212> PRT <213> Homo sapiens <400> 95 Val Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu Asn Arg Tyr Tyr Ala   1 5 10 15 Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr              20 25 30 <210> 96 <211> 31 <212> PRT <213> Homo sapiens <400> 96 Asp Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu Asn Arg Tyr Tyr   1 5 10 15 Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr              20 25 30 <210> 97 <211> 33 <212> PRT <213> Homo sapiens <400> 97 Gln Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu Asn Arg   1 5 10 15 Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg              20 25 30 Tyr     <210> 98 <211> 33 <212> PRT <213> Homo sapiens <400> 98 Arg Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu Asn Arg   1 5 10 15 Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg              20 25 30 Tyr     <210> 99 <211> 33 <212> PRT <213> Homo sapiens <400> 99 Asn Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu Asn Arg   1 5 10 15 Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg              20 25 30 Tyr     <210> 100 <211> 34 <212> PRT <213> Homo sapiens <400> 100 Val Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu Asn   1 5 10 15 Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln              20 25 30 Arg Tyr         <210> 101 <211> 34 <212> PRT <213> Homo sapiens <400> 101 Leu Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu Asn   1 5 10 15 Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln              20 25 30 Arg Tyr         <210> 102 <211> 27 <212> PRT <213> Homo sapiens <400> 102 Asp Asp Ala Ser Pro Asp Glu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg   1 5 10 15 His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr              20 25 <210> 103 <211> 31 <212> PRT <213> Homo sapiens <400> 103 Asp Ala Pro Gly Glu Asp Ala Thr Pro Glu Glu Leu Asn Arg Tyr Tyr   1 5 10 15 Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr              20 25 30 <210> 104 <211> 33 <212> PRT <213> Homo sapiens <400> 104 Asn Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Asp Glu Leu Asn Arg   1 5 10 15 Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg              20 25 30 Tyr     <210> 105 <211> 34 <212> PRT <213> Homo sapeins <400> 105 Leu Lys Pro Glu Ala Pro Gly Asp Asp Ala Ser Pro Glu Glu Leu Asn   1 5 10 15 Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln              20 25 30 Arg Tyr          

Claims (177)

delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete A water soluble peptide YY (PYY) composition consisting of methyl-β-cyclodextrin, EDTA, lactose, sorbitol, DDPC and one PYY peptide and having a pH of 3 to 6, wherein the PYY peptide is SEQ ID NOs: 2, 3 and SEQ ID NOs: A water soluble peptide YY (PYY) composition, characterized in that it consists of one amino acid sequence selected from the group consisting of 90-105. A water soluble peptide YY (PYY) composition consisting of methyl-β-cyclodextrin, EDTA, lactose, sorbitol, DDPC and peptide YY (PYY) (SEQ ID NO: 2) and having a pH between 3 and 6. 18. The water soluble peptide YY (PYY) composition according to claim 16 or 17, wherein the composition is formulated for intranasal delivery. delete delete delete delete delete delete delete delete delete delete delete delete delete Water, PYY (3-36) (SEQ ID NO: 2), chlorobutanol, sodium citrate, citric acid, methyl-β-cyclodextrin, EDTA, lactose, sorbitol and DDPC, pH 5.0 ± 0.3 PYY composition characterized by. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete

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