MX2008004980A - Intranasal administration of rapid acting insulin - Google Patents

Abstract

What is described are pharmaceutical compositions, formulations, and uses thereof, for medicaments for intranasal delivery of insulin to a patient, comprising an aqueous mixture of human insulin, a solubilizing agent, and a surface active agent, wherein the human insulin may be rapid actin insulin.

Description

INTRANASAL ADMINISTRATION OF RAPID ACTION INSULIN BACKGROUND Insulin is an important polypeptide hormone that regulates glucose. It is a hormone of natural origin secreted by the pancreas. The cells of the body require insulin to remove and use the blood glucose. Glucose allows cells to produce the energy necessary to carry out cellular functions. In addition to being the main effector in carbohydrate homeostasis, insulin has effects on fat metabolism. You can change the capacity of the liver to release accumulations of fat. Insulin has various pharmacodynamic effects throughout the body. The researchers first provided an active extract of the pancreas containing insulin to a young diabetic patient in 1922, and the FDA approved insulin for the first time in 1939. Currently, the insulin used for the treatment is derived from the pancreas of cattle and pig as well as recombinant technology (human). The first recombinant human insulin was approved by the FDA in 1982. Insulin was used medically in some forms of diabetes mellitus. Patients with diabetes mellitus have an inability to absorb and use glucose from the blood, and, as a result, the level of glucose in the blood rises. In type 1 diabetes, the pancreas can not make enough insulin. Therefore, insulin therapy is needed. In type 2 diabetes, patients produce insulin, but cells throughout the body do not respond normally to insulin. However, insulin can also be used in type 2 diabetes to overcome the cellular resistance to insulin. By increasing the absorption of glucose by the cells and reducing the concentration of glucose in the blood, insulin prevents or reduces the long-term complications of diabetes, including damage to blood vessels, eyes, kidneys, and nerves. Insulin is commonly administered by injection under the skin (subcutaneously). The subcutaneous tissue of the abdomen is pretreated because the absorption of insulin is more consistent from this location than in the subcutaneous tissues in other locations. When insulin was first discovered and made available to people with diabetes, there was only one type of short-acting insulin. This required several injections a day. With the passage of time, new insulins were developed that lasted longer, requiring fewer injections, but requiring strict attention in the synchronization of food. Now, there are different types of insulin available. This provides greater flexibility in the amount and timing of administration, making it easier to maintain target blood glucose levels, based on the patient's lifestyle. Insulin is available in several forms, for example, fast acting, medium and prolonged. Insulin is typically delivered by subcutaneous injections. Other options are available, such as pump delivery, and more recently pulmonary delivery. Several insulin analogs prepared with recombinant DNA technology are available for clinical use. Among these agents is insulin aspart (NovoLog ™, Novo Nordisk Pharmaceuticals), which is homologous to regular human insulin except for a single substitution of aspartic acid for proline at position B28. This unique substitution reduces the tendency of the molecules to form hexamers. Consequently, insulin aspart is absorbed more rapidly after subcutaneous injection and has both a faster onset of action and a shorter duration of action than short-acting insulin. Mixtures of insulin are also used, especially for people with type 2 diabetes. Mixtures of insulin allow treatment with different types of insulin in a combined administration. Injectable insulin comes in three different forms, of vials, pre-filled syringes, and cartridges. The cartridges are used in a pen type device that simplifies the injection. The commonly used insulins are recombinant human insulin, insulin lispro, insulin aspart, and insulin glargine. The insulins of cattle and pigs are used infrequently. Regular human insulin (Novolin R, Humulin R) is available in vials, cartridges, and pre-filled syringes. NPH human insulin (Novolin N, Humulin N) is available in vials, cartridges, and pre-filled syringes. A mixture of 70% NPH human insulin and 30% regular human insulin (Novolin 70/30, Humulin 70/30) is available in vials, cartridges, and pre-filled syringes. A mixture of 50% NPH human insulin and 50% human regular insulin (Humulin 50/50) is available in vials. Human insulin Lens (Novolin L, Humulin L) is available in vials. Ultralente human insulin (Humulin U) is available in vials. Insulin lispro (Humalog) is available in vials and cartridges. Insulin aspart (Novolog) is available in vials and cartridges. Insulin glargine (Lantus) is available in vials and cartridges. Monomeric forms of insulin include insulin homologs and are known to be fast acting, eg, insulin glulisine (LysB3, GluB29), HMR-1153 (LysB3, IleB28), HMR-1423 (GlyA21, HisB31, HisB32), insulin aspart (AspB28) or (AspBlO), lispro (LysB28, ProB29). In each of the above cases, the nomenclature of the analogs is based on a description of the substitution of amino acids at specific positions in the A or B chain of insulin, numbered from the N-terminus of the chain, in which the The rest of the sequence is that of natural human insulin. A dry powder formulation of a rapid-acting insulin for pulmonary delivery comprising an insulin having the amino acid sequence of natural human insulin (US Patent No. 6,737,045) has been described. There is a need to develop additional pharmaceutical formulations comprising fast acting insulins, i.e., those which are capable of providing peak serum levels within 60 minutes and glucose depressions within 90 minutes. There are several options available for people who inject insulin. Insulin can be injected manually, or it can be infused into the body with the help of a small electronic infusion device called an insulin pump. Syringes are probably the most common and most cost-effective choice, and they are useful for patients who take two types of insulin mixed together. An alternative to syringes is an insulin pen, which is pre-filled with insulin and can be both disposable and reusable (with disposable insulin cartridges). The device resembles a large ballpoint pen, with a thin needle under the cap and a plunger at the other end. An indicator allows the user to regulate the dose. Insulin pens are also available in the most frequently prescribed types of insulin mixtures, such as 70/30 (NPH and regular insulin). Some people prefer pens to syringes because they are easier to transport and use. Another device known as an insulin jet injector works by using a burst of high-pressure air to send a fine spray of insulin through the skin. This may be a good option for patients who elude needles. However, jet injectors require a significant financial investment and are not always covered by insurance. An insulin pump may be a more effective way to control type 1 diabetes for some people because it more closely mimics the production of insulin from a pancreas. An insulin pump is a compact electronic device with a connected infusion set (or tube) that delivers a small flow, insulin constant to a patient throughout the day, known as a "basal rate." Before eating, the pump user schedules the pump to deliver a fast-acting "bolus" of insulin to cover the corresponding rise. In the blood glucose levels from the food, the flow of the pump can also be adjusted manually by a user throughout the day as needed.The peptides that regulate glucose are a class of peptides that have been shown to have a therapeutic potential in the treatment of insulin-dependent diabetes mellitus (IDDM), gestational diabetes or non-insulin dependent diabetes mellitus (NIDDM), the treatment of obesity and the treatment of dyslipidemia, see U.S. Patent No. 6,506,724; the Publication of the Patent Application of E.U. No. 20030036504A1; European Patent No. EP1083924B1; Publication of International Patent Application No. WO 98 / 30231A1; and International Patent Application No. WO 00 / 73331A2. In addition to insulin and insulin analogues, peptides that regulate glucose include glucagon-like peptide, GLP, eg, GLP-1, exendins, especially exendin-4, also known as exenatide, and amylin peptides and analogs of amylin such as pramlintide. To date these peptides have been administered to humans by injection. Therefore, there is a need to develop pharmaceutical formulations for the administration of glucose-regulating peptides, especially fast-acting insulins, other than injection. DESCRIPTION OF THE DRAWINGS Figure 1: PK Results for the Rabbit Study 1 of PDF Comparison only for PDF with Tween formulations; Figure 2: PK Results for the Study of Rabbit 2 of Comparison of Administration IN of PDF with Formulations Tween with Administration S of Q of Novolog; Figure 3: PD Results for the Rabbit Study 1, Glucose% (Linear Record Scale) of Control Comparison IN, IN PDF, IN PDF with Tween, IV and SC Formulations; Figure 4: PD Results of Comparison of Data from Study 1 and Study 2,% of Glucose from the Start (Linear Graph); Figure 5: PD data for groups dosed in the Preclinical Study 3, Glucose% from the Start (Linear Graph); Figure 6: PK data for groups dosed in Study 3 Preclinical Comparison of IN PDF with Tween (with and without DDPC), SC PDF, and Control Formulations SC; Figure 7: PD data for groups dosed in Study 4 Preclinical Comparison of IN PDF with Tween containing 0.2% Gelatin or PG (with and without DDPC); Figure 8: PK data for groups dosed in Study 4 Preclinical Comparison of IN PDF with Tween that Contains 0.2% Gelatin or PG (with and without DDPC), TDMthotonic, TDMIsotonic, PDF Oral (with and without PG and / or DDPC), and Control SC; Figure 9:% Glucose from Initial PD data for all groups dosed with viscosity enhancing formulations (Gelatin, HPMC, MC, Carcomer, and CMC); and Figure 10: PK data for all groups dosed with viscosity enhancing formulations (Gelatin, HPMC, MC, Carbomer, and CMC). DESCRIPTION OF THE INVENTION In order to provide a better understanding of the present invention, the following definitions are provided: Insulin and Insulin Analogs The present invention focuses primarily on the intranasal administration of fast acting insulins which are capable of providing peak levels of serum within 60 minutes and glucose depressions within 90 minutes. In accordance with the present invention, the glucose-regulating peptides also include free bases, acid addition salts or metal salts, such as potassium or sodium salts of the peptides, and peptides that have been modified by processes such as amidation, glycosylation, acylation, sulfation, phosphorylation, acetylation, cyclization and other well-known covalent modification methods. As used herein, the term "human insulin" includes recombinant human insulin. The pharmaceutically acceptable salts include inorganic acid salts, organic amine salts, organic acid salts, alkaline earth metal salts and mixtures thereof. Suitable examples of pharmaceutically acceptable salts include, but are not limited to, halide, glucosamine, alkyl glucosamine, sulfate, hydrochloride, carbonate, hydrobromide, N, N'-dibenzylethylene diamine, triethanolamine, diethanolamine, trimethylamine, triethylamine, pyridine, picoline, dicyclohexylamine, phosphate, sulfate, sulfonate, benzoate, acetate, salicylate, lactate, tartate, citrate, mesylate, gluconate, tosylate, maleate, fumarate, stearate and mixtures thereof. Therefore, according to the present invention, the peptides described above, and mixtures thereof, are incorporated into pharmaceutical formulations suitable for transmucosal delivery, especially intranasal delivery. Analogs and Mimetics of Peptides Included within the definition of biologically active peptides for use within the invention, are natural or synthetic peptides, therapeutically or prophylactically active (comprised of two or more covalently linked amino acids), proteins, peptide fragments or protein, peptide or protein analogs, and derivatives or chemically modified salts of active peptides or proteins. A wide variety of useful analogs and mimetics of glucose-regulating peptides are contemplated for use within the invention and can be produced and tested for their biological activity according to known methods. Often, the peptides or proteins of the glucose regulatory peptide or other biologically active peptides or proteins for use within the invention, are muteins easily obtained by partial substitution, addition, or deletion of amino acids within a peptide or protein sequence of natural or native origin (e.g., wild-type mutant, or of natural origin, or allelic variant). Additionally, biologically active fragments of native peptides or proteins are included. Such mutant derivatives and fragments retain substantially the desired biological activity of the native peptides and proteins. In the case of peptides and proteins having carbohydrate chains, biologically active variants marked by alterations in these carbohydrate species are also included within the invention. As used herein, the term "Conservative amino acid substitution" refers to the general exchange capacity of amino acid residues that have similar side chains. For example, a commonly interchangeable group of amino acids having aliphatic chains is alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic hydroxyl side chains is serine and threonine; A group of amino acids having side chains containing amide is asparagine and glutamine; A group of amino acids that have aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids that have basic side chains is lysine, arginine, and histidine; A group of amino acids that have side chains that contain sulfur is cysteine and methionine. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another. In the same way, the present invention contemplates the substitution of a polar (hydrophilic) residue such as between arginine and lysine, between glutamine and asparagine, and between threonine and serine. Additionally, replacement of a basic residue such as lysine, arginine or histidine by another or replacement of an acidic residue such as aspartic acid or glutamic acid by another is also contemplated. Exemplary conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. By aligning a peptide or protein analogue optimally with a corresponding native peptide or protein, and by using appropriate assays, eg, protein adhesion analysis or receptor binding, to determine a selected biological activity, analogs can be easily identified. of operable peptide and protein for use within the methods and compositions of the invention. The operable peptide and protein analogs are typically specifically immunoreactive with antibodies cultured for the corresponding native peptide or protein. Mucose Supply Improvement Agents "Mucous supply enhancing agents are defined as chemicals and other excipients that, when added to a formulation comprising water, common salts and / or buffers, and glucose regulatory peptide (the control formulation) produce a formulation that produces an effective increase in the transport of glucose regulating peptide through a mucosa, measured by the maximum concentration in blood, serum, or cerebral spinal fluid (Cmax) or by the area under the curve, AUC, In a concentration plot against time, a mucosa includes the nasal, oral, intestinal, buccal, bronchopulmonary, vaginal, and rectal surfaces and in fact includes all the mucous secretion membranes that cover all the cavities or body passages that communicate with The exterior mucosal supply enhancing agents are sometimes called "vehicles." "Endotoxin-free formulation" means to a formulation containing a glucose regulatory peptide and one or more mucosal delivery enhancing agents that is substantially free of endotoxins and / or related pyrogenic substances. Endotoxins include toxins that are confined within a microorganism and are released only when microorganisms break down or die. Pyrogenic substances include thermostable substances for the induction of fever (glycoproteins) of the outer membrane of bacteria and other microorganisms. Both substances can cause fever, hypotension and concussion if administered to humans. The production of endotoxin-free formulations may require special equipment, skilled technicians, and may be significantly more expensive than the preparation of formulations that are not free of endotoxin. Because the intravenous administration of GLP or amylin, simultaneously with the infusion of endotoxin in rodents, has been shown to prevent hypotension and even death associated with the administration of endotoxin alone (US Patent No. 4,839,343), the production of endotoxin-free formulations of these and other peptide therapeutic agents Glucose regulator is not expected to be necessary for a non-parental administration (not injected). Uninfused Administration "Uninfused Administration" means any method of delivery that does not involve an injection directly into an artery or vein, a method that forces or drives (typically a fluid) into something and in particular enters a part of the body by a needle, syringe or other invasive method. Uninfused administration includes subcutaneous injection, intramuscular injection, intraperitoneal injection, and delivery methods to a mucosa without injection. Methods and Delivery Compositions Improved methods and compositions for mucosal administration of the glucose regulatory peptide to mammalian subjects optimize glucose regulator peptide dosage schedules. The present invention provides the mucosal delivery of the glucose regulatory peptide formulated with one or more mucosal delivery enhancing agents wherein the dose release of the glucose regulatory peptide is substantially normalized and / or sustained during an effective delivery period of glucose ranges. Release of the glucose regulator peptide from about 0.1 to 2.0 hours; from 0.4 to 1.5 hours; from 0.7 to 1.5 hours; or from 0.8 to 1.0 hours; after mucosal administration. Sustained release of the achieved glucose regulatory peptide can be facilitated by repeated administration of the exogenous glucose regulatory peptide using the methods and compositions of the present invention. Improved compositions and methods for mucosal administration of the glucose-regulating peptide to mammalian subjects optimize the glucose regulator peptide dosage schedules. The present invention provides improved mucosal (e.g., nasal) delivery of a formulation comprising the glucose regulatory peptide in combination with one or more mucosal delivery enhancing agents and an optional sustained release enhancing agent (s). The mucosal delivery enhancing agents of the present invention produce an effective increase in delivery, e.g., an increase in the maximum plasma concentration (Cmax) to improve the therapeutic activity of the glucose-regulating peptide administered mucosally. A second factor that affects the therapeutic activity of the glucose regulating peptide in the blood plasma and the CNS is the residence time (RT).

Sustained-release enhancing agents, in combination with nasal delivery agents, increase the Cmax and increase the residence time (RT) of the glucose-regulating peptide. Polymeric delivery vehicles and other agents and methods of the present invention that produce sustained release enhancer formulations, for example, polyethylene glycol (PEG), are described herein. The present invention provides an improved glucose regulator peptide delivery method and a dosage form for the treatment of symptoms related to diseases and conditions including diabetes, hyperglycemia, dyslipidemia, satiety induced in an individual, promotion of weight loss in an individual, obesity, colon cancer, prostate cancer, or other cancer in a mammalian subject. Within the mucosal delivery formulations and methods of this invention, the glucose regulating peptide is frequently combined or administered in synchronization with a carrier or vehicle suitable for mucosal delivery. As used herein, the term "carrier" means a pharmaceutically acceptable solid, liquid or filler, filler, or encapsulation material. A liquid vehicle containing water may contain pharmaceutically acceptable additives such as acidifying agents, alkalizing agents, antimicrobial preservatives, antioxidants, buffering agents, chelating agents, complexing agents, solubilizing agents, humectants, solvents, suspending and / or increasing agents. viscosity, tonicity agents, wetting agents or other biocompatible materials. A tabulation of the ingredients listed by the above categories can be found in the U.S. National Pharmacopoeia Formulary, 1857-1859, 1990. Some examples of materials that can serve as pharmaceutically acceptable carriers are sugars, such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; jelly; talcum powder; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil; olive oil; corn oil and soybean oil; glycols, such as propylene glycol; polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen free water; isotonic saline; Ringer's solution, ethyl alcohol and phosphate buffer solutions, as well as other non-toxic compatible substances used in pharmaceutical formulations, and mixtures thereof. Thus, some examples of humectants include propylene glycol, glycerin, glyceryl triacetate, a polyol, a polymeric polyol, lactic acid, urea, and mixtures thereof. Some examples of buffer and buffer salt are based on glutamate, acetate, glycine, histidine, arginine, lysine, methionine, lactate, formate, glycolate, and mixtures thereof. Wetting agents, emulsifiers and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants may also be present in the compositions, according to the formulator's wishes. Examples of pharmaceutically acceptable antioxidants include water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol and the like; metal chelating agents such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and mixtures thereof. The amount of the active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending on the particular mode of administration. Within the mucosal delivery compositions and methods of the invention, various supply enhancing agents are employed which improve delivery of the glucose regulating peptide within or through a mucosal surface. In this regard, the delivery of the glucose regulating peptide through mucosal epithelium can occur "transcellularly" or "paracellularly". The degree to which these trajectories contribute to the total flow and bioavailability of the glucose-regulating peptide depends on the mucosal environment, the psycho-chemical properties of the active agent, and the properties of the mucosal epithelium. Paracellular transport involves only passive diffusion, while transcellular transport can occur through passive, facilitated or active processes. Generally, hydrophilic polar solutes, passively transported, diffuse through the paracellular pathway, while more lipophilic solutes use the transcellular pathway. Absorption and bioavailability (eg, as reflected by a permeability or physiological coefficient analysis), for various passive and active absorption solutes, can be easily evaluated, in terms of both paracellular and transcellular delivery components, for any regulatory peptide of glucose selected within the invention. For passive absorption drugs, the relative contribution of paracellular and transcellular trajectories to the transport of the drug depends on the pKa, the separation coefficient, the molecular radio and the charge of the drug, the pH of the luminal environment in which it is supplied the drug, and the area of the absorbent surface. The paracellular pathway represents a relatively small fraction of the accessible surface area of the nasal mucosal epithelium. In general terms, it has been reported that the membranes of the cell occupy a mucosal surface area that is a thousand times greater than the area occupied by the paracellular spaces. Therefore, the smallest accessible area and the discrimination based on the size and charge against the macromolecular permeation would suggest that the paracellular pathway would be a generally less favorable route than the transcellular supply for transporting the drug. Surprisingly, the methods and compositions of the invention provide significantly improved transport of biotherapeutics into and through the mucosal epithelium via the paracellular route. Therefore, the methods and compositions of the invention are successfully directed to both paracellular and transcellular pathways, alternatively or within a single method or composition. As used herein, "mucosal delivery enhancing agents" include agents that enhance release or solubility (eg, from a formulation delivery vehicle), rate of diffusion, capacity and temporality of penetration, absorption, residence time, stability, effective half-life, peak or sustained concentration levels, clarification and other desired mucosal delivery characteristics (eg, as measured at the site of delivery, or at a site of selected target activity such as the bloodstream or central nervous system) of glucose-regulating peptide or other biologically active compound (s). The improvement of the mucosal supply can therefore occur by any of a variety of mechanisms, for example, by increasing the diffusion, transport, persistence or stability of the glucose regulating peptide, increasing the membrane fluidity, modulating the availability or action of the calcium and other ions that regulate intracellular or paracellular permeability, by dissolving the components of the mucous membrane (eg, lipids), by changing the levels of non-protein sulfidyl and of protein in the mucosal tissues, increasing the flow of water through the mucosal surface, modulating the physiology of the epithelial junction, reducing the viscosity of the mucus that lines the mucous epithelium, reducing mucociliary clearance rates, and other mechanisms. As used herein, a "mucosively effective amount of the glucose regulatory peptide" contemplates the effective mucosal delivery of the glucose regulatory peptide to a target site for the drug's activity in the subject, which may involve a variety of pathways of supply or transfer. For example, a given active agent can find its way through the spaces between the mucosal cells and reach an adjacent vascular wall, whereas through another pathway the agent can, either passively or actively, be absorbed into the mucous cells. to act within the cells or to be discharged or transported out of the cells to reach a secondary target site, such as the systemic circulation. The methods and compositions of the invention can promote the translocation of active agents along one or more such alternate routes, or they can act directly on the mucosal tissue or nearby vascular tissue to promote absorption or penetration of the active agent (s). The promotion of absorption or penetration in this context is not limited to these mechanisms. As used herein the "peak concentration (Cmax) of the glucose regulating peptide in a blood plasma", the "concentration curve under the area vs. the time (AUC) of the glucose regulating peptide in a blood plasma. ", the" time for the maximum plasma concentration (tmax) of the glucose regulating peptide in a blood plasma "are pharmacokinetic parameters known to those skilled in the art. Laursen et al., Eur. J. Endocrinology 135: 309-315, 1996. The "concentration versus time curve" measures the concentration of the glucose-regulating peptide in a blood serum of a subject vs. the time following the administration of a dose of the glucose regulating peptide to a subject either by intranasal, intramuscular, subcutaneous, or other parenteral administration. The "Cmax" is the maximum concentration of the glucose-regulating peptide in the blood serum of a subject after a single dose of the glucose-regulating peptide to the subject. The "tmax" is the time to reach the maximum concentration of the glucose regulating peptide in the blood serum of a subject after the administration of a single dose of the glucose regulating peptide to a subject. As used herein, the "concentration curve under the area vs. the time (AUC) of the glucose regulating peptide in a blood plasma" is calculated according to a linear trapezoidal rule and with the addition of residual areas . A decrease of 23% or an increase of 30% between two doses will be detected with a probability of 90% (type p error ß = 10%). The "supply rate" or "absorption rate" is estimated by comparing the time (tmax) to reach the maximum concentration (Cmax). Both Cmax and tmax are analyzed using nonparametric methods. Comparisons of the pharmacokinetics of intramuscular, subcutaneous, intravenous and intranasal glucose regulatory peptide administrations were carried out by variation analysis (ANOVA). A Bonferroni-Holmes sequential procedure is used to evaluate the significance for pairwise comparisons. The dose-response relationship between the three nasal doses is estimated by regression analysis. P <0.05 is considered significant. The results are given as mean values +/- SEM. Although the absorption promotion mechanism can vary with the different mucosal supply enhancing agents of the invention, the reagents useful in this context will not substantially adversely affect the mucosal tissue and will be selected according to the psychochemical characteristics of the glucose regulating peptide. in particular or another active agent or supply enhancer. In this context, the supply enhancing agents that increase the penetration or permeability of the mucosal tissues will often result in some alteration of the mucosal permeability protection barrier. For such supply enhancing agents to be valuable within the invention, it is generally desired that any significant change in mucosal permeability be reversible within an appropriate time frame for the desired duration of delivery of the drug. In addition, there should be no substantial, cumulative toxicity, or any permanent damaging change induced in the properties of the mucosal barrier with long-term use. Within certain aspects of the invention, the absorption promotion agents for co-ordinated administration or the combinatorial formulation with the glucose-regulating peptide of the invention are selected from small hydrophilic molecules, including but not limited to, dimethyl sulfoxide ( DMSO), dimethylformamide, ethanol, propylene glycol, and the 2-pyrrolidones. Alternatively, long chain amphipathic molecules, for example, deacylmethyl sulfoxide, azone, sodium lauryl sulfate, oleic acid, and the bile salts, can be employed to improve mucosal penetration of the glucose regulating peptide. In additional aspects, surfactants (e.g., polysorbates) are used as adjunct components, processing agents, or formulation additives to improve the intranasal delivery of the glucose regulatory peptide. Agents such as DMSO, polyethylene glycol, and ethanol, if present in sufficiently high concentrations in the delivery environment (eg, by pre-administration or incorporation into a therapeutic formulation), can enter the aqueous phase of the mucosa. and altering its solubility properties, thereby improving the separation of the glucose regulating peptide from the vehicle within the mucosa. Therefore, some examples of solubility agents include cyclodextrins, hydroxypropyl-β-cyclodextrin, sulfobutylether-β-cyclodextrin, methyl-β-cyclodextrin, and mixtures thereof. Additional mucosal delivery enhancing agents useful within the co-ordinated administration and processing methods and combinatorial formulations of the invention include, but are not limited to, mixed micelles; enamines, nitric oxide donors (e.g., S-nitroso-N-acetyl-DL-penicillamine, NOR1, NOR4 which are preferably coadministered with a NO scavenger such as carboxy-PITO or sodium doclofenac); sodium salicylate; glycerol esters of acetoacetic acid (e.g., glyceryl-1,3-diacetoacetate or 1,2-isopropyldeneglycerin-3-acetoacetate); and other release-diffusion agents or transepithelial penetration promoters that are physiologically compatible for mucosal delivery. Other absorption promoting agents are selected from a variety of vehicles, bases and excipients that improve mucosal delivery, stability, activity or transepithelial penetration of the glucose regulatory peptide. These include, inter alia, cyclodextrins and beta-cyclodextrin derivatives (eg, 2-hydroxypropyl-beta-cyclodextrin and heptakis (2,6-di-0-methyl-beta-cyclodextrin) .These compounds, optionally conjugated to one or more of the active ingredients and also optionally formulated in an oily base, improve the bioavailability in the mucous formulations of the invention Still other additional absorption enhancers for the mucosal delivery include medium chain fatty acids, including mono and diglycerides (e.g., sodium caprate-coconut oil extracts, Capmul), and triglycerides (e.g., amylodextrin, Estaram 299, Miglyol 810). The therapeutic and prophylactic mucosal compositions of the present invention can be supplemented with any suitable penetration promoting agent that facilitates the absorption, diffusion or penetration of the glucose regulating peptide through the mucosal barriers. The penetration promoter can be any promoter that is pharmaceutically acceptable. Therefore, in more detailed aspects of the invention, compositions incorporating one or more penetration promoter agents selected from sodium salicylate and salicylic acid derivatives (acetyl salicylate, choline salicylate, salicylamide, etc.) are provided.; amino acids and salts thereof (eg, monoaminocarboxylic acids such as glycine, alanine, phenylalanine, proline, hydroxyproline, etc., hydroxyamino acids such as serine, amino acid acids such as aspartic acid, glutamic acid, etc .; basic amino acids such as lysine, etc., including their alkali metal or alkaline earth metal salts); and N-acetylamino acids (N-acetylalanine, N-acetylphenylalanine, N-acetylserine, N-acetylglycine, N-acetylisine, N-acetylglutamic acid, N-acetylproline, N-acetylhydroxyproline, etc.) and their salts (metal salts) alkali and alkaline earth metal salts). Penetration promoting agents within the methods and compositions of the invention are also provided as substances which are generally used as emulsifiers (eg, sodium oleyl phosphate, lauryl sodium phosphate, lauryl sodium sulfate, myristyl sodium sulfate, polyoxyethylene alkyl, polyoxyethylene alkyl esters, etc.), caproic acid, lactic acid, malic acid, and citric acid and its alkali metal salts, pyrrolidonecarboxylic acids, esters of alkylpyrrolidonecarboxylic acid, N-alkylpyrrolidones, acyl proline esters and the like . Within various aspects of the invention, improved nasal mucosal delivery formulations and methods are provided which allow the delivery of glucose regulatory peptides and other therapeutic agents within the invention, through mucosal barriers between administration and the selected target sites . Certain formulations are specifically adapted for a selected target cell, tissue or organ or even a particular disease state. In other aspects, the formulations and methods provide efficient selective endo or transcytosis of the glucose targeting peptide specifically directed along a defined intracellular or intercellular pathway. Typically, the glucose-regulating peptide is efficiently loaded at effective concentration levels in a vehicle or other delivery vehicle, and is supplied and maintained in a stabilized form, eg, in the nasal mucosa and / or during passage through the nasal mucosa. the compartments and intracellular membranes to a remote target site for the action of the drug (eg, the bloodstream or a defined extracellular tissue, organ or compartment). The glucose regulatory peptide can be provided in a delivery vehicle or otherwise modified (eg, in the form of a prodrug), wherein the release or activation of the glucose regulatory peptide is triggered by a physiological stimulus (eg, change in pH, lysosomal enzymes, etc.). Frequently, the glucose regulating peptide is pharmacologically inactive until it reaches its target site for its activity. In most cases, the glucose regulatory peptide and other components of the formulation are non-toxic and non-immunogenic. In this context, vehicles and other components of the formulation are generally selected for their ability to rapidly degrade and excrete under physiological conditions. At the same time, the formulations are chemically and physically stable in the dosage form for effective storage. A variety of additives, diluents, bases and delivery vehicles are provided within the invention, which effectively control the water content to improve the stability of the protein. These effective reagents and vehicle materials as anti-aggregation agents in this regard include, for example, polymers of various functionalities, such as polyethylene glycol, dextran, diethylaminoethyl dextran, and carboxymethyl cellulose, which significantly increase stability and reduce solid phase aggregation of peptides and proteins mixed with them or linked thereto. In some examples, the activity or physical stability of the proteins can also be improved by various additives to aqueous solutions of peptide or protein drugs. For example, additives such as polyols (including sugars), amino acids, proteins such as collagen and gelatin, and various salts can be used.

Certain additives, in particular sugars and other polyols, also impart significant physical stability to dry, e.g., lyophilized proteins. These additives can also be used within the invention to protect the proteins against aggregation, not only during lyophilization but also during storage is the dry state. For example, sucrose and Ficoll 70 (a polymer with sucrose units) exhibit significant protection against aggregation of the peptide or protein during solid phase incubation under various conditions. These additives can also improve the stability of solid proteins embedded within polymer matrices. Still other additional additives, for example sucrose, stabilize the proteins against aggregation in the solid state in humid atmospheres at elevated temperatures, such as may occur in certain sustained release formulations of the invention. Proteins such as gelatin and collagen also serve as stabilizing or bulking agents to reduce the denaturation and aggregation of unstable proteins in this context. These additives can be incorporated in polymer melt processes and in the compositions within the invention. For example, polypeptide microparticles can be prepared by simply lyophilizing or spray drying a solution containing various stabilization additives described above. the non-aggregated sustained-release peptides and proteins can, therefore, be obtained over an extended period of time. Various components and additional preparation methods, as well as specific formulation additives, are provided herein that produce formulations for the mucosal delivery of peptides and proteins prone to aggregation, wherein the peptide or protein is stabilized in a substantially pure form using a solubilizing agent. A range of components and additives are contemplated for use within these methods and formulations. Exemplary of these solubilizing agents are the cyclodextrins (CDs), which selectively bind to the hydrophobic side chains of the polypeptides. It has been found that these CDs bind to hydrophobic patches of proteins in a manner that significantly inhibits aggregation. This inhibition is selective with respect to both the CD and the protein involved. Such selective inhibition of protein aggregation provides additional advantages within the methods and compositions of intranasal delivery of the invention. Additional agents for use in this context include CD dimers, trimers and tetramers with variable geometries controlled by the linkages that specifically block the aggregation of peptides and proteins. In addition, the solubilization agents and methods for their incorporation into the invention involve the use of peptides and peptide mimetics to selectively block protein-protein interactions. In one aspect, the specific binding of hydrophobic side chains reported for CD multimers is extended to the proteins by the use of peptides and peptide mimetics that similarly block protein aggregation. A wide range of suitable anti-aggregation methods and agents are available for incorporation into the compositions and methods of the invention. Charging and pH Control Modifying Agents and Methods To improve the transport characteristics of biologically active agents (including glucose-regulating peptides, other peptides and active proteins and macromolecular and small-molecule drugs) for enhanced delivery through the hydrophobic barriers of the mucous membrane, the invention also provides techniques and reagents for charge modification of biologically active agents or selected delivery enhancing agents described herein. In this regard, the relative permeabilities of macromolecules are generally related to their separation coefficients. The degree of ionization of the molecules, which depends on the pKa of the molecule and the pH on the mucous membrane surface, also affects the permeability of the molecules. Permeation and separation of the biologically active agents, including glucose-regulating peptides and analogs of the invention, for mucosal delivery, can be facilitated by altering the charge or expanding the charge of the active agent or the permeabilizing agent, which is achieved, for example, by altering the charged functional groups, by modifying the pH of the vehicle or delivery solution in which the active agent is delivered, or by the coordinated administration of a charge alteration reagent or pH with the active agent. Consistent with these general teachings, the mucosal delivery of charged macromolecular species, including the glucose regulatory peptide and other biologically active peptides and proteins, within the methods and compositions of the invention, substantially improves when the active agent is delivered to the mucosal surface in a substantially un-ionized or neutral electrical charge state. Certain glucose regulating peptides and other biologically active peptide and protein components of mucosal formulations for use within the invention will be modified in their charge to produce an increase in the positive charge density of the peptide or protein. These modifications also extend to the cationization of peptide and protein conjugates, carriers and other delivery forms described herein. Cationization offers a convenient means for altering the biodistribution and transport properties of proteins and macromolecules within the invention. The cationization is carried out in a manner that substantially preserves the biological activity of the active agent and potentially limits adverse side effects, including tissue damage and toxicity. A "buffer" is generally used to maintain the pH of a solution at an almost constant value. A buffer maintains the pH of a solution, even when small amounts of a strong acid or a strong base are added to the solution, avoiding or neutralizing large changes in the concentrations of hydrogen and hydroxide ions. A buffer usually consists of a weak acid and its appropriate salt (or a weak base and its proper salt). The appropriate salt for a weak acid contains the same negative ion present in the weak acid (see Lagowski, Macmillan Encyclopedia of Chemistry, Vol. 1, Simon &; Schuster, New York, 1997 on p. 273-4). The Henderson-Hasselbach equation, pH = Pka + log10 [A] / [HA], is used to describe a buffer and is based on the standard equation for weak acid dissociation, HA = H + + A ".

Examples of commonly used buffer sources include the following: acetate, tartrate, citrate, phosphate, lactate, glycine, lysine, arginine, histidine, glutamate, methionine, formate, and glycolate. "Buffering capacity" means the amount of acid or base that can be added to a buffer solution before a significant change in pH occurs. If the pH falls within the range of pK-1 and pK + 1 of the weak acid, the buffer capacity is appreciated, but outside this range it falls to such a degree that it is of little value. Consequently, a given system has only a buffering action in a range of one pH unit on either side of the pK of the weak acid (or weak base) (see Dawson, Data for Biochemical Research, third edition, Oxford Science Publications, 1986, p.419). Generally, adequate concentrations are selected so that the pH of the solution is close to the pKa of the weak acid (or weak base) (see Lide, CRC Handbook of Chemistry and Physics, 86th edition, Taylor & Francis Group, 2005-2006, pp. 2-41). In addition, solutions of strong acids and bases are not normally classified as buffer solutions and do not display a buffering capacity between pH values of 2.4 to 11.6. Inhibitory Agents and Methods of Degradative Enzyme Another excipient that can be included in a transmucosal preparation is a degradative enzyme inhibitor. Exemplary mucoadhesive-enzyme inhibitor polymer complexes that are useful within the mucosal delivery formulations and methods of the invention, include, but are not limited to: carboxymethylcellulose-pepstatin (with anti-pepsin activity); poly (acrylic acid) -Bowman-Birk inhibitor (anti-chymotrypsin); poly (acrylic acid) -chimiostatin (anti-chymotrypsin); poly (acrylic acid) -elastatinal (anti-elastase); carboxymethylcellulose-elastatinal (anti-elastase); polycarbophil-elastatinal (anti-elastase); cytosan-antidolor (anti-trypsin); poly (acrylic acid) -bacitracin (anti-aminopeptidase N); cytosan-EDTA (anti-aminopeptidase N, anti-carboxypeptidase A); cytosan-EDTA-antidolor (anti-trypsin, anti-chymotrypsin, anti-elastase). As described in greater detail below, certain embodiments of the invention will optionally incorporate a new cytosan derivative or a chemically modified form of cytosan. A new derivative such for use within the invention is denoted as the polymer beta- [1-4] -2-guanidino-2-deoxy-D-glucose (poly-GuD). Any inhibitor that inhibits the activity of an enzyme to protect the biologically active agent (s) may be commonly employed in the compositions and methods of the invention. The enzyme inhibitors useful for the protection of biologically active proteins and peptides include, for example, soybean trypsin inhibitor, exendin trypsin inhibitor, chymotrypsin inhibitor and trypsin inhibitor, and chymotrypsin isolated from potato tubers (Solanum tuberosum L). A combination or mixtures of inhibitors may be used. Additional inhibitors of proteolytic enzymes for use within the invention include ovomucoid-enzyme, gabaxate mesylate, alpha-1-antitrypsin, aprotinin, amastatin, bestatin, puromycin, bacitracin, leupepsin, alpha2-macroglobulin, pepstatin and clear trypsin inhibitor. of egg or soy. These and other inhibitors can be used alone or in combination. The inhibitor (s) can (are) incorporated into or attached to a vehicle, eg, a hydrophilic polymer, coated on the surface of the dosage form that is to be contacted with the nasal mucosa, or incorporated into the surface phase of the surface, in combination with the biologically active agent or in a separately administered formulation (eg, pre-administered). The amount of the inhibitor, e.g., of a proteolytic enzyme inhibitor, which is optionally incorporated in the compositions of the invention, will vary depending on (a) the properties of the specific inhibitor, (b) the number of functional groups present in the molecule (which can be reactivated to introduce the ethylenic unsaturation necessary for the copolymerization with hydrogel-forming monomers), and (c) the number of lectin groups, such as glycosides, which are present in the inhibitor molecule. It may also depend on the specific therapeutic agent that is intended to be administered. Generally speaking, a useful amount of an enzyme inhibitor is from about 0.1 mg / ml to about 50 mg / ml, frequently from about 0.2 mg / ml to about 25 mg / ml, and more commonly from about 0.5 mg / ml to 5 mg / ml. mg / ml of the formulation (ie, a separate protease inhibitor formulation or a formulation combined with the inhibitor and the biologically active agent). In the case of trypsin inhibition, suitable inhibitors can be selected from eg, aprotinin, BBI, soybean trypsin inhibitor, chicken ovomucoid, chicken ovoinhibitor, human exendin trypsin inhibitor, camostat mesylate, flavonoid inhibitors, anti-pain , leupeptin, p-aminobenzamidine, AEBSF, TLCK (tosyllysine chloromethyl ketone), APMSF, DFP, PMSF and poly (acrylate) derivatives. In the case of inhibition of chymotrypsin, suitable inhibitors can be selected from eg, aprotinin, BBI, soybean trypsin inhibitor, chemostatin, benzoyloxycarbonyl-Pro-Phe-CHO, FK-448, chicken ovoinhibitor, sugar acid complexes biphenylboronic acid, DFP, PMSF, beta-phenylpropionate and poly (acrylate) derivatives. In the case of elastase inhibition, suitable inhibitors can be selected from, eg, elastatinal, methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone (MPCMK), BBI, soybean trypsin inhibitor, chicken ovoinhibitor, DFP, and PMSF. The additional enzyme inhibitors for use within the invention are selected from a wide range of non-protein inhibitors that vary in their degree of potency and toxicity. As described in more detail below, the immobilization of these adjunct agents to matrices or other delivery vehicles, or the development of chemically modified analogs, can be easily implemented to reduce or even eliminate the toxic effects, when they are found. Among this broad group of candidate enzyme inhibitors for use within the invention, are organophosphorus inhibitors, such as diisopropyl fluorophosphate (DFP) and phenylmethylsulfonyl fluoride (PMSF), which are potent irreversible inhibitors of serine proteases (eg, trypsin and chymotrypsin). The additional inhibition of acetylcholinesterase by these compounds makes them highly toxic in uncontrolled supply adaptations. Another candidate inhibitor, 4- (2-aminoethyl) -benzenesulfonyl fluoride (AEBSF), has an inhibitory activity comparable to DFP and PMSF, but is markedly less toxic. (4-Aminophenyl) -methanesulfonyl fluoride hydrochloride (APMSF) is another potent trypsin inhibitor, but it is toxic in uncontrolled adaptations. In contrast to these inhibitors, the 4- (4-isopropylpiperadinocarbonyl) phenyl 1,2-, 3,4-tetrahydro-1-naphthoate methanesulfonate (FL-448) is a low toxic substance, which represents a potent and specific inhibitor of chymotrypsin Additional representatives of this non-protein group of inhibitor candidates, and which also exhibit low toxic risk, are camostat mesylate (N, N 'dimethyl carbamoylmethyl-p- (p'-guanidino-benzoyloxy) phenylacetate methanesulfonate). Still another type of enzyme inhibiting agent for use within the methods and compositions of the invention are the amino acids and modified amino acids that interfere with the enzymatic degradation of specific therapeutic compounds. For use in this context, amino acids and modified amino acids are substantially non-toxic and can be produced at low cost. NeverthelessDue to their low molecular size and good solubility, they are easily diluted and absorbed in mucous environments. However, under the appropriate conditions, amino acids can act as reversible competitive inhibitors of protease enzymes. Certain modified amino acids may display a much stronger inhibitory activity. A modified amino acid desired in this context is known as a "transition state" inhibitor. The strong inhibitory activity of these compounds is based on their structural similarity to a substrate in their transition state geometry, while they are generally selected to have a much greater activity for the active site of an enzyme than for the substrate itself. Inhibitors in the transition state are reversible competitive inhibitors. Examples of this type of inhibitor are the alpha-aminoboronic acid derivatives, such as boro-leucine, boron-valine and boron-alanine. The boron atom in these derivatives can form a tetrahedral boronate ion which is believed to mimic the transition state of the peptides during their hydrolysis by aminopeptidases. These amino acid derivatives are potent reversible inhibitors of aminopeptidases and it is reported that boro-leucine is more than 100 times more effective at inhibiting enzyme than bestatin and more than 1000 times more effective than puromycin. Another modified amino acid for which a strong protease inhibitory activity has been reported is N-acetylcysteine, which inhibits the aminopeptidase N enzymatic activity. This adjunct agent also displays mucolytic properties that can be employed within the methods and compositions of the invention for reduce the effects of the mucus diffusion barrier. Still other enzyme inhibitors useful for use within the methods of coordinated administration and combinatorial formulations of the invention may be selected from peptides and modified peptide enzyme inhibitors. An important representative of this class of inhibitors is the cyclic dodecapeptide, bacitracin, obtained from Bacillus licheniformis. In addition to these types of peptides, certain dipeptides and tripeptides display a weak inhibitory activity that does not specify any protease. By its analogy with amino acids, its inhibitory activity can be improved by chemical modifications. For example, dipeptide analogs of phosphinic acid are also inhibitors in the transition state with strong inhibitory activity towards aminopeptidases. They have been used reportedly to stabilize encephalin leucine administered nasally. Another example of an analogue in the transition state is the modified pentapeptide pepstatin, which is a very potent pepsin inhibitor. The structural analysis of pepstatin, testing the inhibitory activity of several synthetic analogues, demonstrated the main structure-function characteristics of the molecule responsible for the inhibitory activity. Another special type of modified peptide includes inhibitors with an aldehyde function terminally located in its structure.

For example, it has been found that the benzyloxycarbonyl-Pro-Phe-CHO sequence, which meets the known primary and secondary specificity requirements of chymotrypsin, is a potent reversible inhibitor of this target proteinase. Chemical structures of additional inhibitors with a terminally located aldehyde function, eg, antidolor, leupeptin, chemostatin and elastatin, are also known in the art, as are the structures of other known reversible modified peptide inhibitors such as phosphoramidon, bestatin, puromycin and amastatin. Due to their comparably high molecular mass, the polypeptide protease inhibitors are more docile than the smaller compounds to the concentrated supply in a drug-vehicle matrix. Additional agents for protease inhibition within the formulations and methods of the invention involve the use of complexing agents. These agents mediate the inhibition of enzymes by depriving the intranasal environment (or the preparation or therapeutic composition) of bivalent cations, which are co-factors for many proteases. For example, the EDTA and DTPA complexing agents, as coordinated or combinatorially formulated adjunctive agents, at the appropriate concentration, will be sufficient to inhibit the selected proteases to thereby improve the intranasal delivery of biologically active agents according to the invention. Additional representatives of this class of inhibitory agents are EGTA, 1, 10-phenanthroline and hydroxyquinoline. Additionally, due to their propensity to chelate divalent cations, these and other complexing agents are useful within the invention as direct absorption promoting agents. As noted in more detail elsewhere herein, the use of various polymers, particularly mucoadhesive polymers, as enzyme inhibiting agents within the methods and compositions of co-ordinated administration, multi-processing formulation and / or combinatorial of the invention. For example, poly (acrylate) derivatives, such as poly (acrylic acid) and polycarbonyl, can affect the activity of various proteases, including trypsin and chymotrypsin. The inhibitory effect of these polymers can also be based on the complexation of bivalent cations such as Ca2 + and Zn2 +. It is further contemplated that these polymers may serve as partners or conjugate vehicles for the additional enzyme inhibiting agents, as described above. For example, a cytosan-EDTA conjugate has been developed, useful within the invention, which exhibits a strong inhibitory effect towards the enzymatic activity of zinc-dependent proteases. The mucoadhesive properties of the polymers after the covalent attachment of other enzyme inhibitors in this context are not expected to be substantially compromised, nor is the general utility of such polymers expected as a delivery vehicle for the biologically active agents within of the invention, be diminished. In contrast, the reduced distance between the delivery vehicle and the mucosal surface provided by the mucoadhesive mechanism will minimize the presystemic metabolism of the active agent, while the covalently bound enzyme inhibitors remain concentrated at the drug delivery site. , minimizing the unwanted dilution effects of the inhibitors as well as the toxic and other side effects caused by them. In this way, the effective amount of an enzyme inhibitor administered in a coordinated manner can be reduced due to the exclusion of the effects of dilution. Exemplary mucoadhesive-enzyme inhibitor polymer complexes that are useful within the mucosal formulations and methods of the invention, include, but are not limited to: carboxymethylcellulose-pepstatin (with anti-pepsin activity); poly (acrylic acid) -Bowman-Birk inhibitor (anti-chymotrypsin); poly (acrylic acid) -chimiostatin (anti-chymotrypsin); poly (acrylic acid) -elastatinal (anti-elastase); carboxymethylcellulose-elastatinal (anti-elastase); polycarbophil-elastatinal (anti-elastase); cytosan-antidolor (anti-trypsin); poly (acrylic acid) -bacitracin (anti-aminopeptidase N); cytosan-EDTA (anti-aminopeptidase N, anti-carboxypeptidase A); cytosan-EDTA-antidolor (anti-trypsin, anti-chymotrypsin, anti-elastase). Mucolytic and Mucus Cleaning Agents and Methods Effective delivery of biotherapeutic agents by intranasal administration should take into account the rate of drug transport decreased through the protective mucus covering of the nasal mucosa, in addition to the loss of drug due to the binding to the glycoproteins of the mucus layer. Normal mucus is a viscoelastic, gel-like substance that consists of water, electrolytes, mucins, macromolecules and recessed epithelial cells. This mainly serves as a cryoprotective and lubricating cover for the underlying mucosal tissues. The mucus is secreted by the randomly distributed secretory cells located in the nasal epithelium and in another mucosal epithelium. The structural unit of mucus is mucin. This glycoprotein is mainly responsible for the viscoelastic nature of the mucus, although other macromolecules may also contribute to this property. In airborne mucus, such macromolecules include locally produced secretory IgA, IgM, IgE, lysozyme and broncotransferrin, which also play an important role in host defense mechanisms. The methods of co-ordinated administration of the present invention optionally incorporate mucolytic or mucus cleansing agents, which serve to degrade thin or clear mucus from intranasal mucosal surfaces to facilitate the absorption of biotherapeutic agents administered intranasally. Within these methods, a mucolytic or mucus cleansing agent is coadministered as an adjunct to improve the intranasal delivery of the biologically active agent. Alternatively, an effective amount of a mucolytic agent or mucus cleaning agent is incorporated as a processing agent within a multi-processing method of the invention, or as an additive within a combinatorial formulation of the invention, to provide an improved formulation that improves the intranasal supply of biotherapeutic compounds by reducing the barrier effects of intranasal mucus. A variety of mucolytic or mucus cleaning agents are available for incorporation into the methods and compositions of the invention. Based on their mechanisms of action, mucolytic and mucus-cleansing agents can frequently be classified into the following groups: proteases (e.g., pronase, papain) that unfold the protein nucleus of mucin glycoproteins; sulfhydryl compounds that separate the disulfide bonds from mucoproteins; and detergents (e.g., Triton X-100, Tween 20) that break the non-covalent bonds within the mucus. Additional compounds in this context include, but are not limited to, bile salts and surfactants, for example, sodium deoxycholate, sodium taurodeoxycholate, sodium glycocholate and lysophosphatidylcholine. The effectiveness of bile salts to cause structural breakdown of mucus is in the order of deoxycholate > taurocholate > glycolate. Other effective agents that reduce mucus viscosity or adhesion to improve intranasal delivery according to the methods of the invention include, eg, short chain fatty acids, and mucolytic agents that function by chelation, such as N-acyl collagen peptides. , bile acids and saponins (the latter functions in part by chelating Ca2 + and / or Mg2 + that play an important role in maintaining the structure of the mucous layer). Additional mucolytic agents for use within the methods and compositions of the invention include N-acetyl-L-cysteine (ACS), a potent mucolytic agent that reduces both the viscosity and adherence of bronchopulmonary mucus and reports modest increase in the nasal bioavailability of human growth hormone in anesthetized rats (from 7.5 to 12.2%). This and other mucolytic or mucus-cleansing agents are contacted with the nasal mucosa, typically in a concentration range of about 0.2 to 20 mM, in coordination with the administration of the biologically active agent, to reduce the polar viscosity and / or the elasticity of intranasal mucus. Still other mucus cleansing agents can be selected from a range of glycosidase enzymes, which are capable of cleaving the glycosidic linkages within the mucosal glycoprotein. Alpha-amylase and beta-amylase are representative of this class of enzymes, although their mucolytic effect may be limited. In contrast, bacterial glycosidases allow these microorganisms to permeate the mucous layers of their hosts. For combinatorial use with most of the biologically active agents within the invention, including peptide and protein therapeutics, non-ionogenic detergents such as mucolytic or mucus cleaning agents are also useful. These agents will typically not modify or substantially damage the activity of the therapeutic polypeptides. Cilostatic Agents and Methods Due to the self-cleansing capacity of certain mucosal tissues (eg, nasal mucosal tissues) through the necessary mucociliary clearance as a protective function (eg, to remove dust, allergens, and bacteria), it has generally been considered that this function should not be substantially damaged by mucous medications. Mucociliary transport in the respiratory tract is a particularly important defense mechanism against infections. To achieve this function, the ciliary blow in the nasal and air passages moves a layer of mucus along the mucosa to remove the inhaled particles and microorganisms. Ciliastic agents find their use within the methods and compositions of the invention, to increase the residence time of glucose-regulating peptides, analogs and glucose-regulating mimetics (eg, intranasally) and other biologically active agents described in I presented. In particular, the delivery of these agents within the methods and compositions of the invention, significantly improves in certain aspects by means of coordinated administration or combined formulation of one or more ciliatatic agents that function to reversibly inhibit the ciliary activity of mucosal cells, to provide a temporary, reversible increase in the residence time of the active agent (s) administered mucosally. For use within these aspects of the invention, the above ciliatic factors, either specific or indirect in their activity, are all candidates for their successful use as ciliates in the appropriate amounts (depending on the concentration, duration and mode of delivery). ) so as to produce a transient (ie, reversible) reduction or cessation of mucociliary clearance at the mucosal site of administration to improve the delivery of glucose regulating peptides, analogs and mimetics, and other biologically active agents described herein, without Unacceptable side effects Within more detailed aspects, a specific ciliostatic factor is employed in a combined formulation or in a coordinated administration protocol, with one or more peptides, proteins, analogs and glucose regulating mimetics and / or other biologically active agents described herein. Various bacterial ciliates isolated and characterized in the literature can be used within these embodiments of the invention. Cystoic factors of the bacterium Pseudomonas aeruginosa include a phenazine derivative, a pyo compound (2-alkyl-4-hydroxyquinolines), and a rhamnolipid (also known as hemolysin). The compound pyo produced ciliostasis at concentrations of 50 ug / ml and without obvious ultrastructural lesions. The phenazine derivative also inhibited ciliary mobility, but caused some membrane ruptures, although at concentrations substantially higher than 400 ug / ml. Limited exposure of tracheal explants to the rhamnolipid resulted in ciliostasis, which is associated with altered ciliary membranes. The most extensive exposure to rhamnolipid is associated with the removal of dynein arms from axonemes. Surface-active Agents and Methods Within more detailed aspects of the invention, one or more membrane penetration enhancing agents may be employed within a mucosal delivery method or formulation of the invention to improve the mucosal delivery of peptides, proteins, analogs and regulatory mimics. of glucose, and other biologically active agents described herein. Membrane penetration enhancing agents in this context can be selected from: (i) a surfactant; (ii) a bile salt; (iii) a phospholipid additive, mixed micelle, liposome or vehicle; (iv) an alcohol; (v) an enamine; (vi) a compound NOT of donor; (vii) a long-chain amphipathic molecule; (viii) a small hydrophobic penetration enhancer; (ix) sodium or a salicylic acid derivative; (x) a glycerol ester of acetoacetic acid; (xi) a cyclodextrin or beta-cyclodextrin derivative; (xii) a medium chain fatty acid; (xiii) a chelating agent; (xiv) an amino acid or salt thereof; (xv) an N-acetylamino acid or salt thereof; (xvi) a degrading enzyme for a selected membrane component; (xvii) a fatty acid synthesis inhibitor; (xviii) a cholesterol synthesis inhibitor; or (xix) any combination of the membrane penetration enhancing agents mentioned in (i) - (xviii). Certain surfactants are easily incorporated into the mucous delivery formulations and methods of the invention as mucosal absorption enhancing agents. These agents, which can be co-administered or formulated in combination with peptides, proteins, analogs and glucose-regulating mimetics, and other biologically active agents described herein, can be selected from a broad assemblage of known surfactants. Surfactants, which generally fall into three classes: (1) nonionic polyoxyethylene ethers; (2) bile salts such as sodium glycollate (SGC) and deoxycholate (DOC); and (3) fusidic acid derivatives such as sodium taurodihydrofusidate (STDHF). The mechanisms of action of these various classes of surfactants typically include the solubilization of the biologically active agent. For proteins and peptides that frequently form aggregates, the surfactant properties of these absorption promoters can allow interactions with proteins in such a way that smaller units, such as surfactant-coated monomers, can be more easily maintained in solution. Examples of other surfactants are L-alpha-phosphatidylcholine didecanoyl (DDPC), polysorbate 80 and polysorbate 20. These monomers are presumably more transportable units than the aggregates. A second potential mechanism is the protection of the peptide or protein from proteolytic degradation by proteases. in the mucous environment. Both bile salts and some derivatives of fusidic acid report inhibiting proteolytic degradation of proteins by nasal homogenates at concentrations lower than or equivalent to those required to improve protein absorption. This inhibition of protease may be especially important for peptides with short biological half-lives. Thus, some examples of surfactants include nonionic polyoxyethylene ether, fusidic acid and its derivatives, sodium taurodihydrofusidate, L-alpha-phosphatidylcholine didecanoyl, polysorbate 80, polysorbate 20, polyethylene glycol, cetyl alcohol, polyvinylpyrrolidone, polyvinyl alcohol, alcohol lanolinic, sorbitan monooleate, and mixtures thereof. Viscosity Enhancing Agents Viscosity improving agents or suspending agents can affect the release rate of a drug from the dose formulation and absorption. Some examples of the materials that can serve as pharmaceutically acceptable viscosity improving agents are gelatin; methylcellulose (MC); hydroxypropylmethylcellulose (HPMC); carboxymethylcellulose (CMC); cellulose; starch; starch; polyoxamers; pluronics; Sodium CMC; sorbitol; acacia; povidone; carbomer; polycarbophil; cytosan; microspheres of cytosan; alginate microspheres; cytosan glutamate; amberlite resin; hyaluronan; ethyl cellulose; OF maltodextrin; dried barley corn starch (DDWM); degradable starch microspheres (DSM); deoxyglycolate (GDC); hydroxyethyl cellulose (HEC); hydroxypropyl cellulose (HPC); microcrystalline cellulose (MCC); polymethacrylic acid and polyethylene glycol; sulfobutyl ether B cyclodextrin; biospheres of cross-linked eldexomer starch; sodium taurodihydrofusidate (STDHF); N-trimethyl cytosan chloride (TMC); microspheres of degraded starch; amberlite resin; nanoparticles of cytosan, crospovidone spray dried; spray dried dextran microspheres; spray-dried microcrystalline cellulose; and starch microspheres of the cross-linked eldexomer. Other viscosity improving agents in Ugwoke et al., Adv. Drug. Deliv. Rev., 29: 1656-57, 1998, are incorporated by reference. Degradation Enzymes and Fatty Acid Synthesis Inhibitors and Cholesterol In related aspects of the invention, the peptides, proteins, analogs and glucose regulating mimetics, and other biologically active agents for mucosal administration, are formulated or administered in coordination with an improving agent. of selected penetration of a degradation enzyme, or a metabolic stimulating agent or inhibitor of the synthesis of selected fatty acids, sterols or other epithelial barrier components, US Pat. No. 6,190,894. For example, degrading enzymes such as phospholipase, hyaluronidase, neuraminidase and chondroitinase can be used to improve mucosal penetration of peptides, proteins, analogs and glucose-regulating mimetics and other biologically active agents without causing irreversible damage to the mucosal barrier. In one embodiment, chondroitinase is used within a method or composition as provided herein, to alter the constituents of the glycoprotein or glycolipid of the mucosal permeability barrier, thereby improving mucosal absorption of peptides, proteins, analogs and glucose regulator mimetics and other biologically active agents described herein. With respect to inhibitors of the synthesis of mucosal barrier constituents, it is noted that free fatty acids account for 20-25% of epithelial lipids by weight. Two rate-limiting enzymes in the biosynthesis of free fatty acids are acetyl CoA carboxylase and fatty acid synthetase. Through a series of stages, free fatty acids are metabolized into phospholipids. Therefore, inhibitors of the synthesis and metabolism of free fatty acids for use within the methods and compositions of the invention, include, but are not limited to, acetyl CoA carboxylase inhibitors such as 5-tetradecyloxy-2 acid. -furancarboxilico (TOFA); fatty acid synthetase inhibitors; phospholipase A inhibitors such as gomisin A, 2- (p-amilcinnamyl) amino-4-chlorobenzoic acid, bromofenacrylic bromide, monoalide, 7,7-dimethyl-5,8-eicosadienoic acid, niceroglin, cepharanthine, nicardipine, quercetin, Dibutyryl-cyclic AMP, R-24571, N-oleoylethanolamine, N- (7-nitro-2, 1,3-benzoxadiazol-4-yl) phosphoestidyl serine, cyclosporin A, topical anesthetics, including dibucaine, prenylamine, retinoids, such as all-trans and 13-cis-retinoic acid, W-7, trifluoperazine, R-24571 (calmidazolium), l-hexadocyl-3-trifluoroethyl glycero-sn-2-phosphomentol (MJ33); calcium channel blockers including nicardipine, verapamil, diltiazem, nifedipine and nimodipine; antimalarials including quinacrine, mepacrine, chloroquine and hydroxychloroquine; beta-blockers including propanalol and labetalol; calmodulin antagonists; EGTA; timersol; glucocorticosteroids including dexamethasone and prednisolone; and non-spheroidal anti-inflammatory agents including indomethacin and naproxen. Free sterols, mainly cholesterol, account for 20-25% of epithelial lipids by weight. The rate-limiting enzyme in cholesterol biosynthesis is 3-hydroxy-3-methylglutaryl (HGM) CoA reductase. Inhibitors of cholesterol synthesis for use within the methods and compositions of the invention include, but are not limited to, competitive inhibitors of (HGM) CoA reductase, such as simvastatin, lovastatin, fluindostatin, (fluvastatin), pravastatin, mevastatin, as well as other HMG CoA reductase inhibitors, such as cholesterol oleate, cholesterol sulfate and phosphate, and oxygenated sterols, such as 25-OH- and 26-OH-cholesterol; squalene synthetase inhibitors; squalene epoxidase inhibitors; inhibitors of DELTA7 or DELTA24 reductases such as 22, 25-diazacholesterol, 20, 25-diazacholesterol, AY9944 and triparanol. Each of the inhibitors of fatty acid synthesis or sterol synthesis inhibitors can be co-administered or formulated in combination with one or more peptides, proteins, analogs and glucose-regulating mimics, and other biologically active agents described herein to achieve improved epithelial penetration of the active agent (s). An effective concentration range for the sterol inhibitor in a therapeutic formulation or adjunct for mucosal delivery is generally from about 0.0001% to about 20% by weight of the total, more typically from about 0.01% to about 5%. Nitric Oxide Donors and Methods Within other related aspects of the invention, a nitric oxide (NO) donor is selected as a membrane penetration enhancing agent to improve the mucosal delivery of one or more peptides, proteins, analogues and mimetics glucose regulators, and other biologically active agents described herein. Several NO donors are known in the art and are useful in effective concentrations within the methods and formulations of the invention. Non-exemplary donors include, but are not limited to, nitroglycerin, nitroprusside, NOC5 [3- (2-hydroxy-1- (methyl-ethyl) -2-nitrosohydrazino) -1-propanamine], NOC12 [N-ethyl-2 - (1-ethyl-hydroxy-2-nitrosohydrazino) -etanamine], SNAP [S-nitroso-N-acetyl-DL-penicillamine], NOR1 and NOR4. Within the methods and compositions of the invention, an effective amount of a NO donor with one or more peptides, proteins, analogs, and glucose-regulating mimetics and / or other biologically active agents described herein is co-administered or formulated in combination. present inside or through the mucosal epithelium. Agents to Modulate the Structure and / or Physiology of the Epithelial Junction The present invention provides a pharmaceutical composition containing one or more glucose regulating peptides, proteins, analogs or mimetics and / or other biologically active agents in combination with the mucosal delivery enhancing agents described herein, formulated in a pharmaceutical preparation. for the mucosal supply. The permeabilization agent reversibly improves epithelial paracellular transport, typically by modulating the structure and / or epithelial binding physiology at the mucosal epithelial surface in the subject. This effect typically involves the inhibition by the permeabilization agent of the homotypic or heterotypic binding between the epithelial membrane adhesive proteins of the neighboring epithelial cells. The target proteins for this homotypic or heterotypic binding block can be selected from several related binding adhesion molecules (JAMs), occludins or claudins. Examples of these are antibodies, antibody fragments or single-chain antibodies that bind to the extracellular domains of these proteins. Still in detailed further embodiments, the invention provides permeabilization peptides and peptide analogs and mimetics to enhance paracellular epithelial mucosal transport. Peptides and peptide analogs and mimetics of subject peptide typically function within the compositions and methods of the invention by modulating the structure and / or epithelial binding physiology in a mammalian subject. In certain embodiments, the peptides and peptide analogs and mimetics effectively inhibit the homotypic and / or heterotypic binding of an epithelial membrane adhesive protein selected from a binding adhesion molecule (JAM), occludin or claudin. One such agent that has been extensively studied is the bacterial toxin of Vibrio cholerae known as the "zonula occludens toxin" (ZOT). This toxin mediates increased intestinal mucosal permeability and causes symptoms of disease including diarrhea in infected subjects. Fasano, et al., Proc. Natl. Acad. Sci., E.U.A., 8: 5242-5246, 1991. When tested in rabbit ileal mucosa, ZOT increased intestinal permeability by modulating the structure of the tight intercellular junctions. More recently, it has been found that ZOT is capable of reversibly opening the tight junctions in the intestinal mucosa. It has also been reported that ZOT is capable of reversibly opening the tight junctions in the nasal mucosa. Patent of E.U. No. 5,908,825.

Within the methods and compositions of the invention, ZOT, as well as various analogs and mimetics of ZOT that function as agonists or antagonists of ZOT activity, are useful for improving the intranasal delivery of biologically active agents, increasing paracellular absorption within and through the nasal mucosa. In this context, ZOT typically acts by causing a structural rearrangement of the tight junctions marked by the altered location of the ZO1 binding protein. Within these aspects of the invention, ZOT is administered in a coordinated manner or formulated in combination with the biologically active agent in an effective amount to produce a significantly improved absorption of the active agent, reversibly increasing nasal mucosal permeability without side effects. substantial adverse Vasodilating Agents and Methods Still another class of absorption promoting agents that show a beneficial utility within the methods and compositions of coordinated administration and combined formulation of the invention, are the vasoactive compounds, more specifically vasodilators. These compounds function within the invention to modulate the structure and physiology of the submucosal vasculature, to increase the rate of transport of glucose regulating peptides, analogs and mimetics, and other biologically active agents within or through the mucosal epithelium and / or to target specifically target tissues or compartments (eg, the systemic circulation or the central nervous system). Vasodilating agents for use within the invention typically cause the relaxation of submucosal blood vessels either by decreasing cytoplasmic calcium, increasing nitric oxide (NO), or inhibiting light chain myosin kinase. They are generally divided into 9 classes: calcium channel blockers, potassium channel openers, ACE inhibitors, angiotensin-II receptor antagonists, alpha-adrenergic antagonists and imidazole receptors, beta-adrenergic agonists, phosphodiesterase inhibitors, eicosanoids and DO NOT. Despite the chemical differences, the pharmacokinetic properties of calcium antagonists are similar. Absorption within the systemic circulation is high, and these agents, therefore, experience a considerable first-pass metabolism in the liver, resulting in individual variation in pharmacokinetics. Except for the newer drugs of the dihydropyridine type (amlodipine, felodipine, isradipine, nilvadipine, nisoldipine and nitrendipine), the half-life of calcium antagonists is short. Accordingly, in order to maintain an effective drug concentration for many of these, multiple dose or controlled release formulations may be required, as described elsewhere herein. Treatment with the potassium channel opener minoxidil may also be limited in a manner and level of administration due to potential adverse side effects. ACE inhibitors prevent the conversion of angiotensin-I to angiotensin-II, and are more effective when renin production is increased. Since ACE is identical to kininase-II, which deactivates the potent endogenous vasodilator bradykinin, inhibition of ACE causes a reduction in bradykinin degradation. ACE inhibitors provide the additional advantage of cardioprotective and cardioreparative effects, preventing and reversing cardiac fibrosis and ventricular hypertrophy in animal models. The predominant elimination pathway of most ACE inhibitors is by renal excretion. Consequently, kidney damage is associated with reduced elimination and dose reduction of 25 to 50% is recommended in patients with moderate to severe kidney damage. With respect to NO donors, these compounds are particularly useful within the invention for their additional effects on mucosal permeability. In addition to the NO donors noted above, the NO complexes with nucleophiles called NO / nucleophiles, or NONOates, spontaneously and non-enzymatically release the NO when an aqueous solution is dissolved at a physiological pH. In contrast, nitro vasodilators such as nitroglycerin require a specific enzyme activity for the release of NO. NONOates release NO with a defined stoichiometry and at predictable rates that vary from <; 3 minutes for diethylamine / NO to approximately 20 hours for diethylenetriamine / NO (DETANE). Within certain methods and compositions of the invention, a selected vasodilator agent is administered in a coordinated manner (eg, systemically or intranasally, simultaneously or in combinatorially effective temporal association) or formulated in combination with one or more peptides, analogues and glucose regulating mimetics, and other biologically active agent (s) in an amount effective to improve mucosal absorption of the active agent (s) to reach a target tissue or compartment in the subject (eg, liver, hepatic portal vein, CNS tissue or fluid, or blood plasma). Agents and Methods of Selective Transport Improvement The compositions and delivery methods of the invention optionally incorporate a selective transport enhancing agent that facilitates the transport of one or more biologically active agents. The transport enhancing agents can be employed in a combinatorial formulation or in a coordinated delivery protocol with one or more of the glucose regulatory peptides, proteins, analogs and mimetics described herein, to coordinately improve the delivery of one or more agents biologically additional active through the mucosal transport barriers, to improve the mucosal delivery of the active agents to reach a target tissue or compartment in the subject (eg, in the mucosal epithelium, the liver the tissue or fluid of the CNS or the blood plasma ). Alternatively, the transport enhancing agents can be employed in a combinatorial formulation or in a coordinated administration protocol to directly improve the mucosal delivery of one or more of the glucose regulating peptides, proteins, analogs and mimetics with or without improved delivery of a additional biologically active agent. Exemplary selective transport enhancing agents for use within this aspect of the invention include, but are not limited to, glycosides, sugar-containing molecules, and binding agents such as lectin binding agents, which are known to interact specifically with the components of the epithelial transport barrier. For example, specific "bioadhesive" ligands, including various plant and bacterial lectins, that bind to cell surface sugar residues through receptor mediated interactions, can be employed as vehicles or conjugated transport mediators to improve mucosal delivery, eg , nasal, of the biologically active agents within the invention. Certain bioadhesive ligands for use within the invention will mediate the transmission of biological signals to target epithelial cells that trigger selective absorption of the adhesive ligand by specialized cellular transport processes (endocytosis or transcytosis). These transport mediators, therefore, can then be used as a "transport system" to stimulate or direct the selective uptake of one or more peptides, proteins, analogs and glucose regulating mimetics and other biologically active agents into and / or through the mucosal epithelium. These and other selective transport enhancing agents significantly improve the mucosal delivery of macromolecular biopharmaceuticals (particularly peptide, protein, oligonucleotide and polynucleotide vectors) within the invention, lectins are plant proteins that bind to the specific sugars found on the surface of Glycoproteins and glycolipids of eukaryotic cells. Concentrated solutions of lectins have a "mucotractive" effect and several studies have shown rapid receptor-mediated endocytosis (EMR) of lectins and lectin conjugates (eg, concanavalin A conjugated to colloidal gold particles) through mucosal surfaces. Additional studies have reported that the absorption mechanisms for lectins can be used to direct intestinal drugs in vivo. In certain of these studies, the polystyrene nanoparticles (500 nm) were covalently coupled to tomato lectin and reported to produce an improved systemic absorption after oral administration to rats. In addition to the plant lectins, the microbial adhesion and invasion factors provide a rich source of candidates for use as adhesive / selective transport vehicles within the methods and mucosal delivery compositions of the invention. Two components are necessary for bacterial adhesion processes, a bacterial "adhesin" (adherence factor or colonization) and a receptor on the host cell surface. Bacteria that cause mucosal infections need to penetrate the mucosal layer before they attach themselves to the epithelial surface. This union is commonly mediated by the bacterial structures of fimbrae or pilus, although other components of the cell surface can also take part in the process. Adherent bacteria colonize the mucosal epithelium by multiplication and initiation of a series of biochemical reactions within the target cell through signal transduction mechanisms (with or without the help of toxins). Associated with these invasive mechanisms, a wide variety of bioadhesive proteins (e.g., invasin, internalin) originally produced by various bacteria and viruses are known. These allow the extracellular binding of such microorganisms with an impressive selectivity for host species and even with particular target tissues. The signals transmitted by such receptor-ligand interactions trigger the transport of intact living microorganisms within, and eventually through the epithelial cells by endo or transcytotic processes. Such a phenomenon of natural origin can be sustained (eg, by complexing biologically active agents such as the glucose regulatory peptide with an adhesin) according to the teachings herein for the improved delivery of biologically active compounds into or through the mucosal epithelium and / or Other designated target sites for drug action. Various bacterial and plant toxins that bind to epithelial surfaces in a lectin-specific manner are also useful within the methods and compositions of the invention. For example, diphtheria toxin (DT) is rapidly introduced into host cells by RME. Similarly, subunit B of heat labile toxin from E. coli binds to the brush border of intestinal epithelial cells in a highly specific lectin-like manner. Absorption of this toxin and transcytosis to the basolateral side of the enterocytes has been reported in vivo and in vitro. Other investigations have expressed the transmembrane domain of diphtheria toxin in E. coli such as a maltose binding fusion protein and chemically coupled with high Mw poly-L-lysine. The resulting complex is successfully used to mediate the internalization of an in vitro reporter gene. In addition to these examples, Staphylococcus aureus produces a set of proteins (eg, staphylococcal enterotoxin A (SEA), SEB, toxic shock syndrome toxin 1 (TSST-1) that act as super-antigens and as toxins. They have reported facilitated transcitosis dependent on the dose of SEB and TSST-1 in Caco-2 cells.The viral hemagglutinins comprise another type of transport agent to facilitate the mucosal delivery of biologically active agents within the methods and compositions of the invention. Initial stage in many viral infections is the binding of surface proteins (hemagglutinins) to mucous cells.These binding proteins have been identified for most viruses, including, rotavirus, varicella zoster virus, semliki forest virus, adenovirus, potato leaf virus, and reovirus These and other exemplary viral hemagglutinins can be used in a combinatorial formulation (eg, a conjugate mixture or formulation) or in a coordinated administration protocol with one or more of the glucose regulating peptides, analogs and mimetics described herein to coordinately improve the mucosal delivery of one or more additional biologically active agents. Alternatively, the viral hemagglutinins may be employed in a combinatorial formulation or in a co-ordinated administration protocol to directly improve the mucosal delivery of one or more of the glucose regulating peptides, proteins, analogs and mimetics, with or without improved delivery of an agent additional biologically active A variety of endogenous factors mediating selective transport are also available for use within the invention. Mammalian cells have developed a selection of mechanisms to facilitate the internalization of specific substrates and direct them to defined compartments. Collectively, these membrane deformation processes are termed "endocytosis" and comprise phagocytosis, pinocytosis, receptor-mediated endocytosis (clathrin-mediated RME) and potocitosis (RME not mediated by clathrin). RME is a highly specific cellular biological process by which, as the name implies, several ligands bind to cell surface receptors and are internalized and subsequently transported within the cell. In many cells, the endocytosis process is so active that the entire membrane surface is internalized and replaced in less than half an hour. Two classes of receptors are proposed based on their orientation in the cell membrane; the amino terminal of Type I receptors is located on the extracellular side of the membrane, while Type II receptors have this same end of the protein in the intracellular milieu. Still other embodiments of the invention utilize transferrin as a vehicle or stimulator of EMR of biologically active agents mucosally delivered. Transferrin, an 80 kDa iron transport glycoprotein, is efficiently absorbed into cells by RME. Transferrin receptors are found on the surface of most proliferating cells, in high numbers in erythroblasts and in many types of tumors. Transcytosis of transferrin (Tf) and transferrin conjugates are reportedly improved in the presence of Brefeldin A (BFA), a fungal metabolite. In other studies, it has been reported that treatment with BFA rapidly increases the apical endocytosis of both ricin and HRP in MDCK cells. Therefore, BFA and other agents that stimulate transport mediated by the receptor, can be employed within the methods of the invention as agents formulated in a combinatorial manner (eg, conjugates) and / or administered in coordination to improve transport mediated by the patient. receptor for biologically active agents, including peptides, proteins, analogs and glucose regulating mimetics. Vehicles and Methods of Polymeric Delivery Within certain aspects of the invention, the glucose regulating peptides, proteins, analogs and mimetics, other biologically active agents described herein and the supply enhancing agents as described above, individually or in combination, they are incorporated into a mucosally administered formulation (eg, nasally) that includes a biocompatible polymer that functions as a carrier or base. Such polymer carriers include polymer powders, matrices or microparticle delivery vehicles, among other forms of the polymer. The polymer may be of vegetable, animal or synthetic origin. Frequently the polymer is crosslinked. Additionally, in these delivery systems, the glucose-regulating peptide, analog or mimetic can be functionalized in a manner that can be covalently bound to the polymer and made inseparable from the polymer. In other embodiments, the polymer is chemically modified with an inhibitor of enzymes or other agents that can degrade or deactivate the biologically active agent (s) and / or improve the delivery of the agent (s) . in certain formulations, the polymer is partially or completely insoluble in water but is a polymer that swells with water, e.g., a hydrogel. The polymers useful in this aspect of the invention are desirably inactive and / or hydrophilic in nature to absorb significant amounts of water, and often form hydrogels upon contact with water or the aqueous medium for a sufficient period of time to achieve equilibrium. with water In more detailed embodiments, the polymer is a hydrogel which, upon contact with an excess of water, absorbs at least twice its weight of water in equilibrium upon exposure to water at room temperature, US Pat. No. 6,004,583. Drug delivery systems based on biodegradable polymers are preferred in many biomedical applications because such systems are broken either by hydrolysis or by enzymatic reaction in non-toxic molecules. The rate of degradation is controlled by manipulating the composition of the biodegradable polymer matrix. These types of systems, therefore, can be used in certain adaptations for the long-term release of biologically active agents. Biodegradable polymers such as poly (glycolic acid) (PGA), poly (lactic acid) (PLA) and poly (D, L-lactic-co-glycolic acid) (PLGA), have received considerable attention as potential drug delivery vehicles, since it has been found that the degradation products of these polymers have low toxicity. During the normal metabolic function of the body, these polymers are degraded into carbon dioxide, and water. These polymers have also exhibited excellent biocompatibility. To prolong the biological activity of glucose regulating peptides, analogs and mimetics, and other biologically active agents described herein, as well as optional supply enhancing agents, these agents can be incorporated into polymer matrices, eg, polyorthoesters, polyanhydrides or polyesters . This produces an activity and a sustained release of the active agent (s), e.g., as determined by the degradation of the polymer matrix. Although the encapsulation of biotherapeutic molecules within synthetic polymers can stabilize them during storage and delivery, the biggest obstacle to polymer-based release technology is the loss of activity of therapeutic molecules during formulation processes that often involve heat, sonication or organic solvents. The absorption promoter polymers contemplated for use within the invention may include derivatives and chemically or physically modified versions of the above polymer types, in addition to other polymers, gums, resins and other agents of natural or synthetic origin, as well as mixtures of these materials with each other or with other polymers, provided that the alterations, modifications or mixtures do not adversely affect the desired properties, such as water absorption, hydrogel formation, and / or chemical stability for their useful application. In more detailed aspects of the invention, polymers such as nylon, acrylon, and other synthetic polymers normally hydrophobic, can be sufficiently modified by reaction to become swellable in water and / or to form stable gels in the aqueous medium. The absorption promoter polymers of the invention may include polymers of the group of homo and copolymers based on various combinations of the following vinyl monomers: acrylic and methacrylic acids, acrylamide, methacrylamide, hydroxyethylacrylate or methacrylate, vinylpyrrolidones, as well as polyvinyl alcohol and its co and terpolymers, polyvinylacetate, their co and terpolymers with the monomers listed above and 2-acrylamido-2-methyl-propanesulfonic acid (AMPS®). The copolymers of the monomers listed above with copolymerizable functional monomers such as acrylate or methacrylamide acrylate or methacrylate esters in which the ester groups are derived from straight or branched chain alkyl, aryl having up to four aromatic rings which may contain alkyl substituents of 1 to 6 carbons; spheroidal, sulphates, phosphates or cationic monomers such as N, N-dimethylaminoalkyl (meth) acrylamide, dimethylaminoalkyl (meth) acrylate, (meth) acryloxyalkyltrimethylammonium chloride (meth) acryloxyalkyldimethylbenzylammonium. The additional absorption promoter polymers for use within the invention are those classified as dextrans, dextrins, and from the class of materials classified as natural gums and resins, or from the class of natural polymers such as processed collagen, citin, cytosine, pulalan, zooglan, alginates and modified alginates such as "Kelcoloid" (an alginate modified with polyethylene glycol) gellan gums such as "Kelocogel", xanthan gums such as "Keltrol", estastine, alpha hydroxybutyrate and their copolymers, hyaluronic acid and their derivatives, polylactic and glycolic acids.

A very useful class of polymers applicable within the present invention are the olefinically unsaturated carboxylic acids containing at least one activated carbon-to-carbon double olefinic bond and at least one carboxyl group, ie, an acid or functional group easily converted to a acid containing a double olefinic bond that functions easily in polymerization due to its presence in the monomer molecule, either in the alpha-beta position with respect to a carboxyl group, or as part of a terminal methylene group. Olefinically unsaturated acids of this class include materials such as acrylic acids typified by acrylic acid itself, alpha-cyanoacrylic acid, beta methacrylic acid (crotonic acid), alpha-phenyl acrylic acid, beta-acryloxy propionic acid, cinnamic acid , p-chloro cinnamic acid, l-carboxy-4-phenylbutadiene-1,3, itaconic acid, citraconic acid, mesaconic acid, glutaconic acid, aconitric acid, maleic acid, fumaric acid, and tricarboxy ethylene. As used herein, the term "carboxylic acid" includes polycarboxylic acids and those acid anhydrides such as maleic anhydride, wherein the anhydride group is formed by the removal of one molecule of water from two carboxyl groups located on the same carboxylic acid molecule. Representative acrylates useful as absorption promoting agents within the invention include methyl acrylate, ethyl acrylate, propyl acrylate, isopropyl acrylate, butyl acrylate, isobutyl acrylate, methyl methacrylate, methyl ethacrylate, ethyl methacrylate, octyl acrylate, heptyl acrylate, octyl methacrylate, isopropyl methacrylate, 2-ethylhexyl methacrylate, nonyl acrylate, hexyl acrylate, n-hexyl methacrylate, and the like. The highest alkyl acrylic esters are decyl acrylate, isodecyl methacrylate, lauryl acrylate, stearyl acrylate, behenyl acrylate, and mesylsily acrylate, and their methacrylate versions. Mixtures of two or three or more long chain acrylic esters can be successfully polymerized with one of the carboxylic monomers. Other comonomers include olefins, including alpha olefins, vinyl ethers, vinyl esters and mixtures thereof. Other vinylidene monomers, including acrylic nitriles, can also be used as absorption promoter agents within the methods and compositions of the invention to improve the delivery and absorption of one or more peptides, proteins, analogs and glucose-regulating mimetics and others. biologically active agents, including for improving the delivery of the active agent (s) to a target tissue or compartment in the subject (eg, liver, hepatic portal vein, tissue or CNS fluid, or blood plasma) ). The alpha, beta-olefinically unsaturated nitriles are preferably monoolefinically unsaturated nitriles having from 3 to 10 carbon atoms such as acrylonitrile, methacrylonitrile and the like. More preferred are acrylonitrile and methacrylonitrile. Acrylic amides containing from 3 to 35 carbon atoms including monoolefinically unsaturated amides can also be used. Representative amides include acrylamide, methacrylamide, N-t-butyl-acrylamide, N-cyclohexyl acrylamide, higher alkyl amides, en.-where the alkyl group in the nitrogen contains from 8 to 32 carbon atoms, the acrylic amides including N-alkylol amides of alpha-beta-olefinically unsaturated acids including those having from 4 to 10 carbon atoms such as N-methylol acrylamide, N-propanol acrylamide, N-methylol methacrylamide, N-methylol maleimide, N-methylol maleamic acid esters, N-methylol-p-vinyl benzamide and the like . Further still useful absorption promoter materials are alpha-olefins containing from 2 to 18 carbon atoms, more preferably from 2 to 8 carbon atoms.; the dienes containing from 4 to 10 carbon atoms; vinyl esters and allyl esters such as vinyl acetate; vinyl aromatics such as styrene, methyl styrene and chloro styrene; vinyl and allyl ethers and ketones such as vinyl methyl ether and vinyl methyl ketone; chloroacrylates; cyanoalkyl acrylates such as alpha-cyanomethyl acrylate, and the alpha, beta and gamma-cyanopropyl acrylates; alkoxy acylates such as methoxy ethyl acrylate; haloacrylates such as chloroethyl acrylate; vinyl halides and vinyl chloride; vinylidene chloride and the like; divinyl, diacrylates and other polyfunctional monomers such as divinyl ether, diethylene glycol diacrylate, ethylene glycol dimethacrylate, methylene bis acrylamide, allyl pentaerythritol, and the like; and bis (beta-haloalkyl) alkenyl phosphonates such as bis (beta-chloroethyl) vinyl phosphonate and the like as known to those skilled in the art. Copolymers wherein the carboxy-containing monomer is a minor constituent, and the other vinylidene monomers present as major components, are readily prepared according to the methods described herein. When hydrogels are used as absorption promoting agents within the invention, they may be composed of synthetic copolymers of the group of acrylic and methacrylic acids, acrylamide, methacrylamide, hydroxyethyl acrylate (HEA) or methacrylate (HEMA) and vinylpyrrolidones which are interactive and swellable in Water. Illustrative specific examples of the polymers useful, especially for the delivery of peptides or proteins, are the following types of polymers: (meth) acrylamide and from 0.1 to 99% by weight of (meth) acrylic acid; (meth) acrylamides and 0.1-75% by weight of (meth) acryloxyethyl trimethylammonium chloride; (meth) acrylamide and 0.1-75% by weight of (meth) acrylamide; acrylic acid and 0.1-75% by weight of alkyl (meth) acrylates; (meth) acrylamide and 0.1-75% by weight of AMPS. RTM. (trademark of Lubrizol Corp.); (meth) acrylamide and from 0 to 30% by weight of alkyl (meth) acrylamides and from 0.1-75% by weight of AMPS. RTM; (meth) acrylamide and 0.1-99% by weight of HEMA; (meth) acrylamide and from 0.1 to 75% by weight of HEMA and from 0.1 to 99% of (meth) acrylic acid; (meth) acrylic acid and from 0.1 to 99% by weight of HEMA; 50 mol% vinyl ether and 50 mol% maleic anhydride; (meth) acrylamide and from 0.1 to 75% by weight of (meth) acryloxyalkyl dimethyl benzylammonium chloride; (meth) acrylamide and from 0.1 to 99% by weight of vinyl pyrrolidone; (meth) acrylamide and 50% by weight of vinyl pyrrolidone and from 0.1 to 99.9% by weight of (meth) acrylic acid; (meth) acrylic acid and from 0.1 to 75% by weight of AMPS, RTM and from 0.1 to 75% by weight of alkyl (meth) acrylamide. In the above examples, alkyl means from Ci to C3o, preferably from Ci to C22, linear and branched and from C4 to Cie cyclic; when (met) is used, it means that the monomers with and without the methyl group are included. Other very useful hydrogel polymers are swellable but insoluble versions of poly (vinyl pyrrolidone) starch, carboxymethyl cellulose and polyvinyl alcohol. Additional polymeric hydrogel materials useful within the invention include (poly) hydroxyalkyl (meth) acrylate; anionic and cationic hydrogels; poly complexes (electrolyte); poly (vinyl alcohols) having a low residual acetate; an inflatable mixture of cross-linked agar and cross-linked carboxymethyl cellulose; an inflatable composition comprising methyl cellulose mixed with a lightly cross-linked agar; a water-swellable copolymer produced by a finely divided copolymer dispersion of maleic anhydride with styrene, ethylene, propylene or isobutylene; a water-swellable polymer of N-vinyl lactams; inflatable sodium salts of carboxymethyl cellulose; and the similar. Other gellable, absorbent and water retaining polymers useful for forming the hydrophilic hydrogel for mucosal delivery of the biologically active agents within the invention include pectin; polysaccharides such as agar, acacia, karaya, tragacanth, alginos and guar and their reticulated versions; polymers of acrylic acid, copolymers and salt derivatives, polyacrylamides; inflatable indene maleic anhydride polymers; copolymers of starch graft; acrylate-type polymers and copolymers with an absorption capacity of approximately 2 to 400 times by original weight; polyglucan diesters; a mixture of cross-linked 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 indene-maleic anhydride acrylate polymers; and polysaccharides. Synthetic hydrogel polymers for use within the invention can be produced by an infinite combination of several monomers in various ratios. The hydrogel can be crosslinked and generally has the ability to imbibe and absorb the fluid and to swell or expand to an enlarged state of equilibrium. The hydrogel typically swells or expands to the supply to the surface of the nasal mucosa, absorbing about 2-5, 5-10, 10-50, up to 50-100 or more times its weight of water. The optimum degree of filling capacity for a given hydrogel will be determined for the different biologically active agents depending on factors such as molecular weight, size, solubility and diffusion characteristics of the active agent transported by or trapped or encapsulated within the polymer. , and of the specific separation and cooperative chain movement associated with each individual polymer.

The hydrophilic polymers useful within the invention are insoluble in water but swellable in water. Such polymers swollen in water are typically referred to as hydrogels or gels. Such gels can be conveniently produced from the water-soluble polymer by the process of cross-linking the polymers by a suitable cross-linking agent. However, stable hydrogels can also be formed from specific polymers under conditions of pH, temperature and / or ionic concentration defined according to methods known in the art. Typically, polymers that are crosslinked, i.e., crosslinked to the extent that the polymer possesses good hydrophilic properties, have an improved physical integrity (as compared to uncrosslinked polymers of the same or similar type) and exhibit an improved ability to retain within of the gel network both the biologically active agent of interest and the additional compounds for coadministration with them such as a cytosine or enzyme inhibitor, while retaining the ability to release the active agent (s) (s) at the appropriate location and time. Generally, hydrogel polymers for use within the invention are crosslinked with a dysfunctional crosslinking in the amount of from 0.01 to 25 percent by weight, based on the weight of the monomers form the copolymer, and more preferably 0.1. at 20 percent by weight and more frequently from 0.1 to 15 percent by weight of the crosslinked agent. Another useful amount of a crosslinking agent is 0.1 to 10 percent by weight. It is also possible to use tetra or more multifunctional crosslinking agents. When such reagents are used, smaller amounts may be required to achieve an equivalent crosslink density, i.e., the degree of crosslinking, or sufficient network properties to effectively contain the biologically active agent (s). The crosslinks can be covalent, ionic or hydrogen bonds, the polymer having the ability to swell in the presence of fluids containing water. Such crosslinkers and crosslinking reactions are known to those skilled in the art and in many cases depend on the polymer system. Therefore, a crosslinked network can be formed by free radical copolymerization of unsaturated monomers. Polymeric hydrogels can also be formed by cross-linked preformed polymers reactivating the functional groups found in polymers such as alcohols, acids, amines with groups such as glyoxal, formaldehyde or glutaraldehyde, bis anhydrides and the like. The polymers can also be crosslinked with any polyene, e.g., decadiene or trivinyl cyclohexane; acrylamides such as N, N-methylene-bis (acrylamide); polyfunctional acrylates, such as trimethylol propane triacrylate; or polyfunctional vinylidene monomers containing at least 2 terminal groups CH2 < , including, for example, divinyl benzene, divinyl naphthylene, allyl acrylates and the like. In certain embodiments, the crosslinking monomers for use in the preparation of the copolymers are polyalkenyl polyethers having more than one alkenyl ether group per molecule, which may optionally possess alkenyl groups in which there is a double olefinic bond attached. to a methylene terminal group (eg, produced by the etherification of a polyhydric alcohol containing at least 2 carbon atoms and at least 2 hydroxyl groups). Compounds of this class can be produced by reactivating an alkenyl halide such as allyl chloride or allyl bromide, with a strongly alkaline aqueous solution of one or more polyhydric alcohols. The product can be a complex mixture of polyethers with varying numbers of ether groups. The efficiency of the polyether crosslinking agent increases with the number of potentially polymerizable groups in the molecule. Typically, polyethers containing an average of two or more alkenyl ether groupings per molecule are used. Other crosslinking monomers include, for example, diallyl esters, dimetalyl ethers, allyl or methallyl acrylates and acrylamides, tetravinyl silane, polyalkenyl methanes, diacrylates and dimethacrylates, divinyl compounds such as benzene divinyl, polyallyl phosphate, diallyloxy and phosphate esters and the like. Typical agents are allyl pentaerythritol, allyl sucrose, trimethylolpropane triacrylate, 1,6-hexanediol diacrylate, diallyl ether of trimethylolpropane, pentaerythritol triacrylate, tetramethylene dimethacrylate, ethylene diacrylate, ethylene dimethacrylate, triethylene glycol dimethacrylate, and the similar. Allyl pentaerythritol, trimethylolpropane diallyl ether and allyl sucrose provide suitable polymers. When the crosslinking agent is present, the polymer blends commonly contain between about 0.01 and 20 percent by weight, eg, 1%, 5% or 10% or more by weight of the crosslinking monomer, based on the total monomer of the crosslinking agent. carboxylic acid, plus other monomers. In more detailed aspects of the invention, the mucosal delivery of glucose regulating peptides, analogs and mimetics and other biologically active agents described herein is improved by retaining the active agent (s) in a carrier or vehicle. enzymatically or physiologically protective slow release, for example, a hydrogel that protects the active agent from the action of degradative enzymes. In certain embodiments, the active agent is attached by chemical means to the carrier or vehicle, to which other additional agents such as enzyme inhibitors, cytokines, etc. can also be mixed or bound. The active agent can alternatively be immobilized through sufficient physical entrapment within the carrier or vehicle, e.g., a polymeric matrix. Polymers such as hydrogels useful within the invention may incorporate functional linked agents such as glycosides chemically incorporated within the polymer to improve the intranasal bioavailability of the active agents formulated therewith. Examples of such glycosides are glycosides, fructosides, galactosides, arabinosides, mannosides and their substituted alkyl derivatives and natural glycosides such as arbutin, phlorizin, amigdalin, digitonin, saponin, and indicate. There are several ways in which a typical glycoside can bind to a polymer. For example, the hydrogen of the hydroxyl groups of a glycoside or other similar carbohydrate can be replaced by the alkyl group of a hydrogel polymer to form another. Also, the hydroxyl groups of the glycosides can be reactivated to esterify the carboxyl groups of a polymeric hydrogel to form polymeric esters in situ. Another procedure is to employ the condensation of acetobromoglucose with colest-5-en-3-beta-ol on a maleic acid copolymer. The N-substituted polyacrylamides can be synthesized by the reaction of activated polymers with omega-aminoalkyl glycosides: (1) (carbohydrate-separator) (n) -polyacrylamide, pseudopolysaccharides; (2) (carbohydrate-separator) (n) -phosphatidylethanolamine (m) -polyacrylamide, neoglycolipids, phosphatidylethanolamine derivatives; and (3) (carbohydrate-separator) (n) -biotin (m) -polyacrylamide. These biotinylated derivatives can bind to the lectins on the mucosal surface to facilitate absorption of the biologically active agent (s), e.g., a glucose-regulating peptide encapsulated in polymer. Within more detailed aspects of the invention, one or more glucose regulating peptides, analogs and mimetics, and / or other biologically active agents described herein, optionally including secondary active agents such as protease inhibitor (s), cytosine (s) ), additional modulator (s) of the intercellular binding physiology, etc., are modified and bound to a vehicle or polymeric matrix. For example, this can be achieved by chemically joining a peptide or protein active agent and other optional agent (s) within a network of cross-linked polymer. It is also possible to chemically modify the polymer separately with an interactive agent such as a glycoside-containing molecule. In certain aspects, the biologically active agent (s), and the optional secondary active agent (s), can be functionalized, ie, where the appropriate reactive group is identified or chemically added to the active agent (s). More often a polymerizable ethylenic group is added and the functionalized active agent is then copolymerized with monomers and a crosslinking agent using a standard polymerization method such as solution polymerization (commonly in water), emulsion, suspension or dispersion polymerization. Frequently, the functionalizing agent is provided with a sufficiently high concentration of functional or polymerizable groups to ensure that several sites on the active agent (s) are functionalized. For example, in a polypeptide comprising 16 amine sites, it is generally desired to functionalize at least 2, 4, 5, 7, and up to 8 or more of the sites. After functionalization, the functionalized active agent (s) is mixed with monomers and a crosslinking agent comprising the reagents from which the polymer of interest is formed. The polymerization is then induced in this medium to create a polymer containing the bound active agent (s). The polymer is then cleaned with water or other suitable solvents and otherwise purified to remove unreacted trace impurities and, if necessary, crushed or fractured by physical means such as by agitation, forcing them through a mesh, ultrasonication or other suitable means up to a desired particle size. The solvent, commonly water, is then removed in a manner that does not denature or otherwise degrade the active agent (s). A desired method is lyophilization (freeze drying) but other methods are available and can be used (e.g., by vacuum drying, air drying, spray drying, etc.). In order to introduce the polymerizable groups into peptides, proteins and other active agents within the invention, it is possible to reactivate the amino, hydroxyl, thiol groups and other available reactive groups, with electrophiles containing unsaturated groups. For example, unsaturated monomers containing N-hydroxy succinimidyl groups, active carbonates such as p-nitrophenyl carbonate, trichlorophenyl carbonates, tresylate, oxycarbonylimidazoles, epoxide, isocyanates and aldehyde, and unsaturated carboxymethyl azides and unsaturated ortho-pyridyl disulfide, they belong to the category of the reactants. Illustrative examples of unsaturated reagents are glycidyl ether of allyl, allyl chloride, allylbromide, allyl iodide, acryloyl chloride, allyl isocyanate, allylsulfonyl chloride, maleic anhydride, copolymers of maleic anhydride, and allyl ether, and the like. All active derivatives of lysine, except aldehyde, can generally react with other amino acids such as the imidazole groups of histidine and the hydroxyl groups of tyrosine and thiol groups of cystine, if the local environment improves the nucleophilicity of these groups. Functionalization reagents containing aldehyde are specific for lysine. These types of reactions with available groups of Usins, cysteines and tyrosine have been extensively documented in the literature and are known to those skilled in the art. In the case of biologically active agents containing amine groups, it is convenient to reactivate such groups with an acyloyl chloride, such as acryloyl chloride, and to introduce the polymerizable acrylic group onto the reactivated agent. Then, during the preparation of the polymer, such as during the crosslinking of the acrylamide copolymer and the acrylic acid, the functionalized active agent, through the acrylic groups, binds to the polymer and binds thereto. In additional aspects of the invention, biologically active agents, including peptides, proteins, nucleosides and other molecules that are bioactive in vivo, are stabilized by conjugation by covalently linking one or more active agents to a polymer that is incorporated as an integral part. thereto both a hydrophilic residue eg, a linear polyethylene glycol, and a lipophilic residue (see, eg, U.S. Patent No. 5,681,811). In one aspect, a biologically active agent is covalently coupled to a polymer comprising (i) a linear polyethylene glycol residue, and (ii) a lipophilic residue, wherein the active agent, the linear polyethylene glycol residue, and the residue Lipophilic are arranged in a conformational manner in relation to one another such that the active therapeutic agent has improved resistance in vivo to enzymatic degradation (ie, in relation to its stability under similar conditions in a non-conjugated form devoid of the polymer coupled to it). In another aspect, the formulation stabilized by conjugation has a three-dimensional conformation comprising the biologically active agent covalently coupled to a polysorbate complex comprising (i) a linear polyethylene glycol residue, and (ii) a lipophilic residue, wherein the active agent, the linear polyethylene glycol residue, and the lipophilic residue are arranged in a conformational manner in relation to each other in such a way that (a) the lipophilic residue is available externally in the three-dimensional conformation, and (b) the active agent in the composition has improved resistance in vivo to enzymatic degradation. In a further related aspect, a conjugate complex is provided to the multiligand comprising a biologically active agent coupled to a residue of triglyceride structure through a polyalkylene glycol separation group attached at a carbon atom of the triglyceride structure residue, and minus a fatty acid residue covalently bound either directly to a carbon atom of the triglyceride structure residue or covalently bound through a glycol polyalkylene separation residue (see, eg, U.S. Patent No. 5,681,811). In such multiligand-conjugated therapeutic agent complex, the alpha and beta carbon atoms of the bioactive triglyceride residue may have fatty acid residues attached by covalent attachment either directly thereto or indirectly covalently bound thereto by residues of Glyco separation? polyalkylene. Alternatively, a fatty acid residue can be covalently linked directly or through a polyalkylene glycol separation residue to the alpha and alpha 'carbons of the triglyceride structure residue, the bioactive therapeutic agent being covalently coupled to the gamma-carbon of the triglyceride structure residue, either being covalently bound directly thereto or indirectly bound thereto by a polyalkylene separation residue. It will be recognized that a wide variety of structural, compositional and conformational forms are possible for the multiligand conjugate therapeutic agent complex comprising the triglyceride structure residue, within the scope of the invention. It is further noted that in such a multiligand-conjugated therapeutic agent complex, the biologically active agent (s) can advantageously be covalently coupled to the modified triglyceride structure residue through alkyl separation groups, or alternatively other acceptable separation groups within the scope of the invention. As used in such a context, the acceptability of the separation group refers to the spherical characteristics, composition and specific acceptability for the end-use application. In still further aspects of the invention, there is provided a complex conjugated stabilized comprising a polysorbate complex comprising a polysorbate residue including a triglyceride structure having covalently coupled to its alpha, alpha 'and beta carbon atoms and functionalization groups that include (i) a fatty acid group; and (ii) a polyethylene glycol group having a biologically active agent or residue covalently attached thereto, e.g., attached to an appropriate functionality of the polyethylene glycol group. Such a covalent linkage can be either direct, eg, to a hydroxy terminal functionality of the polyethylene glycol group, or alternatively, the covalent linkage can be indirect, eg, reactive capping of the hydroxy terminal of the polyethylene glycol group with a separation group of carboxy terminal functionality, such that the resulting polyethylene glycol capped group has a carboxy terminal functionality to which the biologically active agent or residue can be covalently bound. Still in further aspects of the invention, there is provided a stable complex, soluble in water, stabilized by conjugation comprising one or more peptides, proteins, analogs and glucose regulating mimetics and / or other biologically active agent (s) ( s) described herein, covalently coupled to a glycolipid residue modified with physiologically compatible polyethylene glycol (PEG). In such a complex, the biologically active agent (s) can be covalently coupled to the modified glycolipid residue with physiologically compatible PEG by labile covalent attachment to a free amino acid group of the active agent, wherein the labile covalent linkage it can be cut in vivo by hydrolysis and / or biochemical proteolysis. The glycolipid residue modified with physiologically compatible PEG may advantageously comprise a polysorbate polymer, eg, a polysorbate polymer comprising fatty acid ester groups selected from the group consisting of monopalmitate, dipalmitate, monolaurate, dilaurate, trilaurate, monoleate, dioleate , trioleate, monostearate, distearate and tristearate. In such a complex, the glycolipid residue modified with physiologically compatible PEG may suitably comprise a polymer selected from the group consisting of polyethylene glycol ethers of fatty acids and polyethylene glycol fatty acid esters, wherein the fatty acids comprise, for example, an acid fatty selected from the group consisting of lauric, palmitic, oleic, and stearic acids. Storage and Processing of the Material In certain aspects of the invention, combinatorial formulations and / or methods of co-ordinated administration herein, incorporate an effective amount of peptides and proteins that can adhere to the loaded glass thereby reducing the effective concentration in the container. Silanized containers, for example, silanized glass containers, are used to store the finished product to reduce the absorption of the polypeptide or protein into a glass container. Still in further aspects of the invention, a kit for the treatment of a mammalian subject comprises a stable pharmaceutical composition of one or more glucose regulating peptide compounds formulated for mucosal delivery to the mammalian subject, wherein the composition is effective in alleviating a or more symptoms of obesity, cancer or malnutrition or isting related to cancer in said subject without unacceptable adverse side effects. The kit further comprises a vial of pharmaceutical reagent for containing the one or more glucose regulating peptide compounds. The pharmaceutical reagent vial is composed of polymer, glass or other suitable pharmaceutical grade material. The pharmaceutical reagent vial, for example, is a silanized glass vial. The equipment further comprises an opening for delivery of the composition to a nasal mucosal surface of the subject. The supply opening is composed of a polymer, glass or other suitable pharmaceutical grade material. The supply opening, for example, is a silanized glass. A silanization technique combines a special cleaning technique to silanize the surfaces with a silanization process at low pressure. The silane is in the gas phase and at an elevated temperature of the surface to be silanized. The method provides reproducible surfaces with stable silane layers, homogeneous and functional that have characteristics of a monolayer. The silanized surfaces prevent the binding to the glass of the polypeptides or mucosal supply enhancing agents of the present invention. The method is useful for preparing silanized pharmaceutical reagent vials to contain the glucose regulatory peptide compositions of the present invention. Glass trays are cleaned by rinsing with double distilled water (ddH20) before use. The silane tray is then rinsed with 95% EtOH and the acetone tray is rinsed with acetone. The pharmaceutical reagent vials are sonicated in acetone for 10 minutes. After sonication with acetone, the reagent vials are washed in the ddH20 tray at least twice. The reagent vials are sonicated in 0.1 M NaOH for 10 minutes. While the reagent vials are sonicated in NaOH, the silane solution is prepared under a hook. (Silane solution: 800 ml of 95% ethanol; 96 1 glacial acetic acid; 25 ml of glycidoxypropylmethoxy silane). After sonication with NaOH, the reagent vials are washed in the ddH20 tray at least twice. The reagent vials are sonicated in the silane solution for 3 to 5 minutes. The reagent vials are washed in the 100% EtOH tray. The reagent vials are dried with pre-purified N2 gas and stored in an oven at 100 ° C for at least 2 hours before use. The nasal spray product preparation process can include the preparation of a diluent for the nasal spray, which includes 85% water plus the components of the nasal spray formulation without the glucose regulating peptide. The pH of the diluent is then measured and adjusted to pH 4.0 + 0.3 with sodium hydroxide or hydrochloric acid, if necessary. The nasal spray is prepared by non-aseptic transfer of -85% of the final target volume of the diluent into a screw cap bottle. An appropriate amount of the glucose regulatory peptide is added and mixed until completely dissolved. The pH is measured and adjusted to a pH of 7.0 + 0.3 with sodium hydroxide or hydrochloric acid, if necessary. A sufficient amount of diluent is added to achieve the final target volume. The screw cap bottles are filled and the caps are fixed. The above description of the manufacturing process represents a method used to prepare initial clinical batches of the drug product. This method can be modified during the development process to optimize the manufacturing process. The currently marketed glucose regulator peptide requires sterile processing conditions to comply with FDA regulations. Parenteral administration, including insulin for injection or infusion, requires a sterile (aseptic) manufacturing process. Good processing practices (GMP) - - Current for the preparation of sterile drug include standards for design and construction characteristics (21 CFR 211.42 (April 1, 2005)); standards for testing and approval or rejection of components, drug product containers, and caps (211.84); standards for the control of microbiological contamination (211.113); and other special test requirements (211.167). non-parenteral (non-aseptic) products, such as the intranasal product of the invention, do not require these specialized sterile processing conditions. As can be readily appreciated, the requirements for a sterile processing and processing are substantially higher and correspondingly more expensive than those required for a non-sterile product manufacturing process. These costs include much higher capitalization costs for facilities, as well as a more expensive processing cost; extra facilities for sterile processing include additional rooms and ventilation; The extra costs associated with sterile processing include increased manpower, extensive quality control and quality assurance, and administrative support. As a result, the costs of making a glucose regulating peptide product, such as that of the invention, are much lower than those of a parenterally administered glucose regulating peptide product. The present invention satisfies the need for a non-sterile manufacturing process for a glucose regulating peptide. Sterile solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of the ingredients listed above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the other required ingredients from those enumerated above. In the case of sterile powders, the methods of preparation include vacuum drying and freeze drying which produce a powder of the active ingredient plus any additional desired ingredients of a previously sterile-filtered solution thereof. The prevention of the action of microorganisms can be achieved by various antibacterial and anti-fungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, trimerosal and the like. The mucosal administration according to the invention allows the effective self-administration of the treatment by the patients, provided sufficient safeguards are in place to control and monitor the dosage and side effects. The mucosal administration also solves certain disadvantages of other forms of administration, such as injection, which are painful and expose the patient to possible infections and may present bioavailability problems of the drug. Bioadhesive Delivery Methods and Vehicles In certain aspects of the invention, the combinatorial formulations and / or the methods of co-ordinated administration herein incorporate an effective amount of a non-toxic bioadhesive as a compound or adjunct vehicle to improve the mucosal delivery of one. or more biologically active agents. The bioadhesive agents in this context exhibit a general or specific adhesion to one or more components or surfaces of the target mucosa. The bioadhesive maintains a desired concentration slope of the biologically active agent within or through the mucosa to ensure the penetration of even larger molecules (e.g., peptides and proteins) into or through the mucosal epithelium. Typically, the use of a bioadhesive within the methods and compositions of the invention produces an increase of two to five times, often five to ten times in permeability for peptides and proteins within or through the mucosal epithelium. This improvement of the epithelial permeation frequently allows the effective transmucosal supply of large macromolecules, for example to the basal portion of the nasal epithelium or within adjacent extracellular compartments or to a blood plasma or tissue or fluid of the CNS. This improved delivery provides greatly improved effectiveness of delivery of bioactive peptides, proteins and other macromolecular therapeutic species. These results will depend in part on the hydrophilicity of the compound, whereby a greater penetration with hydrophilic species will be achieved in comparison with the insoluble compounds in water. In addition to these effects, the use of bioadhesives to improve the persistence of the drug on the mucosal surface can emit a deposition mechanism for a prolonged drug supply, whereby the compounds not only penetrate through the mucous tissue, but also they retro-diffuse towards the mucous surface once the material on the surface is exhausted. A variety of suitable bioadhesives are described in the art for oral administration, US Patents. 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; and the U.S. Patent Reissue. No. 33,093, which find their use within the new methods and compositions of the invention. The potential of several bioadhesive polymers as a mucosal delivery platform, eg, nasal, within the methods and compositions of the invention can be easily achieved by determining their ability to retain and release the glucose regulating peptide, as well as by their ability to interact with glucose. in addition, well-known methods for determining the biocompatibility of the selected polymers with the tissue at the site of mucosal administration will be applied in addition to the mucosal surfaces after incorporation of the active agent therein. When the target mucosa is covered by mucus (i.e., in the absence of mucolytic treatment or mucus clearance), it can serve as a connecting link with the underlying mucosal epithelium. Accordingly, the term "bioadhesive" as used herein, also covers mucoadhesive compounds useful for improving the mucosal delivery of biologically active agents within the invention. However, adhesive contact to mucosal tissue mediated through adhesion to a mucosal gel layer can be limited by incomplete or transient binding between the mucus layer and the underlying tissue, particularly on nasal surfaces where rapid cleansing occurs. of the mucus. In this regard, the mucin glycoproteins are secreted continuously and, immediately after their release from the cells or glands, form a viscoelastic gel. The luminal surface of the adherent gel layer, however, is continuously eroded by enzymatic and / or ciliary mechanical action. When such additives are more prominent or when longer adhesion times are desired, the methods of co-ordinated administration and combinatorial formulation methods of the invention may further incorporate mucolytic and / or ciliostatic methods or agents as described hereinabove. Typically, the mucoadhesive polymers for use in the invention are natural or synthetic macromolecules that adhere to moist mucosal tissue surfaces by complex, but non-specific, mechanisms. In addition to these mucoadhesive polymers, the invention also provides methods and compositions incorporating bioadhesives that adhere directly to a cell surface, rather than to mucus, by means of specific interactions including mediated by the receptor. An example of bioadhesives that work in this specific manner is the group of compounds known as lectins. These are the glycoproteins with an ability to specifically recognize and bind sugar molecules, e.g., glycoproteins or glycolipids, which are part of the intranasal epithelial cell membranes and can be considered as "lectin receptors". In certain aspects of the invention, bioadhesive materials for improving the intranasal delivery of biologically active agents comprise a matrix of a hydrophilic polymer, e.g., soluble or swellable in water, or a mixture of polymers that can adhere to a moist mucosal surface. These adhesives can be formulated as ointments, hydrogels (see above) thin films, and other forms of application. Frequently, these adhesives have a biologically active agent mixed therewith to effect the slow release or local delivery of the active agent. Some are formulated with additional ingredients to facilitate the penetration of the active agent through the nasal mucosa, e.g., within the circulatory system of the individual. Several polymers, both natural and synthetic, show significant binding to mucous and / or mucous epithelial surfaces under physiological conditions. The strength of this interaction can easily be measured by mechanical peel or cut tests. When applied to a moist mucous surface, many dry materials will adhere spontaneously, at least lightly. After such initial contact, some hydrophilic materials begin to attract water by absorption, swelling or capillary forces, and if this water is absorbed from the underlying substrate or the polymer-tissue interface, adhesion may be sufficient to achieve the objective of improving mucosal absorption of biologically active agents. Such "adhesion by hydration" can be quite strong, but formulations adapted to employ this mechanism must have the swelling that continues as the dose is transformed into a hydrated mucilage. This is projected for many useful hydrocolloids within the invention, especially some cellulose derivatives, which are generally non-adhesive when applied in the pre-hydrated state. However, bioadhesive drug delivery systems for mucosal administration are effective within the invention when such materials are applied in the form of a dry polymer powder, microsphere, or a film-like delivery form. Other polymers adhere to mucosal surfaces not only when applied dry, but also in a fully hydrated state, and in the presence of excessive amounts of water. The selection of a mucoadhesive is required, therefore, due to the consideration of physiological as well as physical-chemical conditions, under which the contact with the tissue will be formed and maintained. In particular, it is known that the amount of water or moisture commonly present at the intended site of adhesion, and the prevailing pH, greatly affect the strength of the mucoadhesive bond of different polymers. Various bioadhesive polymeric drug delivery systems have been manufactured and studied in the past 20 years, not always successfully. However, a variety of such vehicles are currently used in clinical applications involving dental, orthopedic, ophthalmological, and surgical applications. For example, acrylic-based hydrogels have been widely used for bioadhesive devices. Acrylic based hydrogels are very suitable for bioadhesion due to their flexibility and non-abrasive characteristics in the partially swollen state, which reduces the attrition that causes damage to the tissues in contact. In addition, its high permeability in the swollen state allows the non-reactivated monomer, the non-crosslinked polymer chains and the initiator to be washed to the outside of the matrix after polymerization, which is an important feature for the selection of materials bioadhesives for use within the invention. The acrylic-based polymer devices exhibit a very high adhesive bond strength. For the controlled mucosal delivery of peptide and protein drugs, the methods and compositions of the invention optionally include the use of carriers, eg, polymeric delivery vehicles that function in part to protect the biologically active agent from proteolytic cleavage, while At the same time they provide improved penetration of the peptide or protein into or through the nasal mucosa. In this context, bioadhesive polymers have shown considerable potential to improve the oral supply of drugs. As an example, the bioavailability of 9-desglicinamide, 8-arginine vasopressin (DGAVP) administered intraduodenally to rats, together with a 1% (w / v) saline dispersion of polycarbofyl mucoadhesive derived from poly (acrylic acid), it is increased 3-5 times compared to an aqueous solution of the peptide drug without this polymer. Mucoadhesive polymers of the poly (acrylic acid) type are potent inhibitors of some intestinal proteases. The mechanism of inhibition of enzymes is explained by the strong affinity of this class of polymers for bivalent cations, such as calcium or zinc, which are essential cofactors of metallo-proteinases, such as trypsin and chymotrypsin. By depriving the proteases of their cofactors by poly (acrylic acid) it is reported that irreversible structural changes of the enzyme proteins are induced which is accompanied by a loss of enzymatic activity. At the same time, other mucoadhesive polymers (e.g., some cellulose derivatives and cytosan) may not inhibit proteolytic enzymes under certain conditions. In contrast to other enzyme inhibitors contemplated for use within the invention (eg, aprotinin, bestatin), which are relatively small molecules, it is likely that the trans-nasal absorption of the inhibitory polymers is minimal in view of the size of these molecules , and consequently eliminates the possible adverse side effects. Therefore, mucoadhesive polymers, particularly of the poly (acrylic acid) type, can serve as an absorption promoter adhesive or as an enzyme protective agent to improve the controlled delivery of peptide and protein drugs, especially when they are considered the security problems. In addition to protection against enzymatic degradation, bioadhesives and other polymeric or non-polymeric absorption promoter agents for use within the invention can directly increase mucosal permeability to biologically active agents. To facilitate the transport of large and hydrophilic molecules, such as peptides and proteins, through the nasal epithelial barrier, mucoadhesive polymers and other agents have been postulated to produce improved permeation effects beyond what is counted for a time. of prolonged pre-mucosal residence of the delivery system. The time course of plasma concentrations of the drug reportedly suggested that bioadhesive microspheres cause an acute but transient increase in insulin permeability through the nasal mucosa. Other mucoadhesive polymers for use within the invention, for example, cytosan, reportedly improve the permeability of certain mucosal epithelia even when applied as a solution or an aqueous gel. Another mucoadhesive polymer that reported directly affecting epithelial permeability is hyaluronic acid and ester derivatives thereof. A particularly useful bioadhesive agent within the coordinated administration and / or the methods and compositions of the combinatorial formulation of the invention is cytosan, as well as its analogues and derivatives. The cytosan is a non-toxic, biocompatible and biodegradable polymer that is widely used for pharmaceutical and medical applications due to its favorable properties of low toxicity and good biocompatibility. It is a polyaminessaccharide prepared from citin by N-deacetylation with alkali. As used within the methods and compositions of the invention, cytosan increases the retention of glucose regulatory peptides, proteins, analogs and mimetics and other biologically active agents described herein, at the mucosal site of application. This mode of administration can also improve patient compliance and acceptance. As further provided herein, the methods and compositions of the invention will optionally include a novel cytosan derivative or a chemically modified form of cytosan. A new derivative such for use within the invention is denoted as a polymer of beta- [1-4] -2-guanidino-2-deoxy-D-glucose (poly-GuD). The cytosan is the N-deacetylated product of citin, a polymer of natural origin that has been used extensively to prepare microspheres for oral and intra nasal formulations. The cytosan polymer has also been proposed as a soluble vehicle for parenteral drug delivery. Within one aspect of the invention, o-methylisourea is used to convert a cytosan amine to its guanidinium residue. The guanidinium compound is prepared, for example, by the reaction between equi-normal solutions of cytosan and o-methylisourea at a pH above 8.0. Additional compounds classified as bioadhesive agents for use within the present invention act by mediating specific interactions, typically classified as "receptor-ligand interactions" between the complementary structures of the bioadhesive compound and a mucosal epithelial surface component. Many natural examples illustrate this form of specific binding bioadhesion, as exemplified by lectin-sugar interactions. The lectins are (glyco) proteins of non-immune origin that bind to polysaccharides or glycoconjugates. Several plant lectins have been investigated as possible pharmaceutical agents promoting absorption. A plant lectin, Phaseolus vulgaris hemagglutinin (PHA), exhibits high oral bioavailability of more than 10% after feeding to rats. The tomato lectin (Lycopersicon esculeutum) (TL) seems safe for several forms of administration. In summary, the above bioadhesive agents are useful in the combinatorial formulations and methods of coordinated administration of the present invention, which optionally incorporate an effective amount and a bioadhesive agent form that prolongs persistence or otherwise increases mucosal absorption. one or more peptides, proteins, analogs and mimetic glucose regulators and other biologically active agents. The bioadhesive agents can be administered co-ordinately as adjuncts or as additives within the combinatorial formulations of the invention. In certain embodiments, the bioadhesive agent acts as a "pharmaceutical glue", while in other embodiments the adjunct supply of the combinatorial formulation of the bioadhesive agent serves to intensify the contact of the biologically active agent with the nasal mucosa, in some cases promoting the specific interactions of receptor-ligand with epithelial cell "receptors", and in others increasing epithelial permeability to significantly increase the drug concentration slope measured at a target delivery site (eg, liver, blood plasma, or tissue or CNS). Still further bioadhesive agents for use within the invention, act as inhibitors of enzymes (e.g., protease) to improve the stability of mucosally administered biotherapeutic agents supplied in a coordinated manner or in a combinatorial formulation with the bioadhesive agent. Liposomes and Micellar Supply Vehicles The methods of coordinated administration and combinatorial formulations of the present invention optionally incorporate lipid or fatty acid-based vehicles, processing agents or delivery vehicles, to provide improved formulations for the mucosal delivery of peptides, proteins , glucose regulating analogs and mimetics, and other biologically active agents. For example, a variety of formulations and methods for mucosal delivery are provided comprising one or more of these active agents, such as a peptide or protein, mixed or encapsulated by, or co-ordinated with, a liposome, a mixed micellar carrier, or emulsion, to improve chemical and physical stability and increase the half-life of biologically active agents (eg, reducing the susceptibility to proteolysis, modification and / or chemical denaturation) to their mucous supply.

Within certain aspects of the invention, specialized delivery systems for biologically active agents comprise small lipid vesicles known as liposomes. These are typically produced from natural, biodegradable, non-toxic and non-immunogenic lipid molecules and can trap or efficiently bind to drug molecules, including peptides and proteins, in or on their membranes. The attractiveness of liposomes as a peptide and protein delivery system within the invention is enhanced by the fact that the encapsulated proteins can remain in their preferred aqueous environments within the vesicles, while the liposomal membrane protects them against proteolysis and other factors of destabilization. Although not all known liposome preparation methods are viable in the encapsulation of peptides and proteins due to their unique physical and chemical properties, several methods allow the encapsulation of these macromolecules without substantial deactivation. A variety of methods are available to prepare liposomes for use within the invention, US Patents. Nos. 4,235,871; 4,501,728; and 4,837,028. For use with the liposome delivery, the biologically active agent is typically trapped within the liposome, or lipid vesicle, or attached to the outside of the - - vesicle Like liposomes, the long chain unsaturated fatty acids, which also have an enhanced activity for mucosal absorption, can form closed vesicles with bilayer-like structures (called "ufasomes"). These can be formed, for example, by using oleic acid to trap biologically active peptides and proteins for the mucosal, e.g., intranasal delivery within the invention. Other delivery systems for use within the invention, combine the use of polymers and liposomes to align the advantageous properties of both vehicles such as encapsulation within the natural fibrin of the polymer. Additionally, the release of biotherapeutic compounds from this delivery system is controllable through the use of covalent crosslinking and the addition of antifibrinolytic agents to the fibrin polymer. More simplified delivery systems for use within the invention include the use of cationic lipids as delivery vehicles or carriers, which can be effectively employed to provide an electrostatic interaction between the lipid carrier and charged biologically active agents such as proteins and nucleic acids polyanionic This allows efficient packaging of the drugs in a form suitable for mucosal administration and / or subsequent delivery to the systemic compartments. Additional supply vehicles for use within the invention include long and medium chain fatty acids, as well as mixed micelles of surfactant with fatty acids. Most lipids of natural origin in the form of esters have important implications with respect to their own transport through mucosal surfaces. The free fatty acids and their monoglycerides, which have bound polar groups, have been shown to act on the intestinal barrier, in the form of mixed micelles, as permeation enhancers. This discovery of the barrier modifying function of free fatty acids (carboxylic acids with a chain length varying from 12 to 20 carbon atoms) and their polar derivatives, has stimulated extensive research in the application of these agents as enhancers of absorption. For use within the methods of the invention, long chain fatty acids, especially fusogenic lipids (unsaturated fatty acids and monoglycerides such as oleic acid, linoleic acid, monoolein, etc.), provide useful vehicles for improving mucosal delivery of Glucose regulating peptides, analogs and mimetics and other biologically active agents described herein. The medium chain fatty acids (C6 to C12) and the monoglycerides have also been shown to have an enhancing activity in the intestinal absorption of the drug and can be adapted for use within the mucosal delivery formulations and methods of the invention. Additionally, the sodium salts of medium and long chain fatty acids are effective delivery vehicles and absorption enhancing agents for the mucosal delivery of biologically active agents within the invention. Therefore, fatty acids can be used in soluble forms of sodium salts or by the addition of non-toxic surfactants, e.g., polyoxyethylated hydrogenated castor oil, sodium taurocholate, etc. Other mixed fatty acid and micelle preparations which are useful within the invention include, but are not limited to, Na-caprylate (C8), Na-caprate (CIO), Na-laurate (C12) or Na-oleate (C18), optionally combined with bile salts such as glycocholate and taurocholate. Pegylation The additional methods and compositions provided within the invention involve the chemical modification of biologically active peptides and proteins by the covalent attachment of polymeric materials, for example dextrans, polyvinylpyrrolidones, glycopeptides, polyethylene glycol and polyamino acids. The resulting peptides and conjugated proteins retain their biological activities and solubility for mucosal administration. In alternate embodiments, peptides, proteins, analogs and glucose regulating mimetics and other biologically active peptides and proteins are conjugated to polyalkylene oxide polymers, particularly polyethylene glycols (PEG). Patent of E.U. No. 4,179,337. PEG polymers reactive to amine for use within the invention include SC-PEG with molecular masses of 2000, 5000, 10000, 12000, and 20 000; U-PEG-10000; NHS-PEG-3400-biotin; T-PEG-5000; T-PEG-12000; and TPC-PEG-5000. Pegylation of biologically active peptides and proteins can be achieved by modification of carboxyl sites (e.g., aspartic acid or glutamic acid groups in addition to the carboxyl terminus). The utility of PEG-hydrazide in the selective modification of carboxyl groups of carbodiimide-activated proteins under acidic conditions has been described. Alternatively, the bifunctional PEG modification of biologically active peptides and proteins can be employed. In some methods, charged amino acid residues, including lysine, aspartic acid, and glutamic acid, have a marked tendency to be accessible solvents on protein surfaces. Other Stabilization Modifications of Active Agents In addition to pegylation, biologically active agents such as peptides and proteins for use within the invention can be modified to improve the half-life in circulation by protecting the active agent by conjugation to other protective or protective compounds. known stabilizers, for example by the creation of fusion proteins with an active peptide, protein, analog or mimetic, linked to one or more carrier proteins, such as one or more immunoglobulin chains. Formulation and Administration The mucosal delivery formulations of the present invention comprise peptides, analogs and glucose-regulating mimetics, typically combined in combination with one or more pharmaceutically acceptable carriers and, optionally, other therapeutic ingredients. The vehicle (s) must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the formulation and of not emitting an unacceptable harmful effect on the subject. Such vehicles are described hereinbefore or otherwise are well known to those skilled in the art of pharmacology. Desirably, the formulation should not include substances such as enzymes or oxidizing agents with which the biologically active agent to be administered is known to be compatible. The formulations can be prepared by any of the methods well known in the pharmacy art. Within the compositions and methods of the invention, the peptides, proteins, analogs and mimetics and other biologically active agents described herein can be administered to subjects by a variety of modes of mucosal administration, including by oral, rectal, vaginal, intranasal delivery , intrapulmonary, or transdermal, or by topical delivery to the eyes, ears, skin or other mucosal surfaces. Optionally, the glucose regulating peptides, proteins, analogs and mimetics and other biologically active agents described herein, may be administered in coordination or in conjunction by non-mucosal pathways, including by intramuscular, subcutaneous, intravenous, intra-atrial, intra-articular routes. , intraperitoneal or parenteral. In other alternative embodiments, the biologically active agent (s) can be administered ex vivo by direct exposure to cells, tissues or organs originating from a mammalian subject, for example as a component of a formulation of ex vivo treatment of a tissue or organ containing the biologically active agent in a suitable liquid or solid carrier. The compositions according to the present invention may be administered in an aqueous solution such as a nasal or pulmonary spray and may be dispensed in the form of a spray by a variety of methods known to those skilled in the art. Preferred systems for dispensing liquids such as a nasal spray are described in the U.S. Patent. No. 4,511,069. The formulations can be presented in multi-dose containers, for example, in the sealed delivery system described in the U.S. Patent. No. 4,511,069. Additional aerosol delivery forms may include, eg, compressed air, jet, ultrasonic and piezoelectric nebulizers that supply the biologically active agent dissolved or suspended in a pharmaceutical solvent, eg, water, ethanol or a mixture thereof, an aerosol formulation of this invention may have drops which have diameters from 1 to 700 microns in size. The compositions and formulations of this invention can have an osmolarity of from 50 to 350 mOsm / 1 or 50 to 300 mOsm / 1. A toner can be used to adjust the osmolarity or tonicity of a formulation. The nasal and pulmonary spray solutions of the present invention typically comprise the drug or drug to be delivered, optionally formulated with a surfactant, such as a nonionic surfactant (e.g., polysorbate 80) and one or more buffers. In some embodiments of the present invention, the nasal spray solution further comprises a propellant. The pH of the nasal spray solution is optionally between about 3.0 and 9, preferably 7.0 + 0.5. Shock absorbers suitable for use within these compositions are as described above or otherwise as known in the art. Other components can be added to improve or maintain chemical stability, including preservatives, surfactants, dispersants or gases. Suitable preservatives include, but are not limited to, phenol, methyl paraben, paraben, m-cresol, thiomersal, chlorobutanol, benzylalkonium chloride, sodium benzoate and the like. Suitable surfactants include, but are not limited to, oleic acid, sorbitan trioleate, polysorbates, lecithin, phosphatidyl cholines, and various long chain diglycerides and phospholipids. Suitable dispersants include, but are not limited to, ethylenediaminetetraacetic acid, and the like. Suitable gases include, but are not limited to, nitrogen, helium, chlorofluorocarbons (CFCs), hydrofluorocarbons (HFCs), carbon dioxide, air and the like. Within alternate embodiments, the mucosal formulations are administered as dry powder formulations comprising the biologically active agent in a commonly lyophilized dry form of an appropriate particle size, or within an appropriate range of particle size, for intranasal delivery. The minimum particle size suitable for its deposition within the nasal or pulmonary passages is frequently approximately 0.5 μm mass average equivalent aerodynamic diameters (MMEAD), commonly of approximately MMEAD of 1 u, and more typically of approximately 2 μM MDAAD . The maximum appropriate particle size for its deposition within the nasal passages is frequently approximately MMEAD of 10 u, commonly of approximately MMEAD of 8 u, and more typically of approximately MMEAD of 4 u. Breathable powders can be produced intranasally within these size ranges by a variety of conventional techniques, such as jet grinding, spray drying, solvent precipitation, supercritical fluid condensation, and the like. These dry powders of an appropriate MMEAD can be administered to a patient through a conventional dry powder inhaler (DPI) which is based on the patient's breathing, lung or nasal inhalation, to disperse the powder within an aerosolized amount. Alternatively, the dry powder can be administered by air-assisted devices that use an external energy source to dispense the powder into an aerosolized amount, e.g., a piston pump. Dry powder devices typically require a powder pass in the range of from about 1 mg to 20 mg to produce a single aerosolized dose ("puff"). If the required or desired dose of the biologically active agent is less than this amount, the powdered active agent will typically be combined with a dry volume pharmaceutical powder to provide the total mass of powder required. Preferred dry bulk powders include sucrose, lactose, dextrose, mannitol, glycine, trehalose. Human serum albumin (HSA) and starch. Other suitable dry volume powders include cellobiose, dextrans, maltotriose, pectin, sodium citrate, sodium ascorbate and the like. To formulate the compositions for mucosal delivery within the present invention, the biologically active agent can be combined with various pharmaceutically acceptable additives as well as a base or carrier for the dispersion of the active agent (s). The desired additives include, but are not limited to, pH control agents, such as arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid, acetic acid, etc. Additionally, local anesthetics (eg, benzyl alcohol), isotonizing agents (eg, sodium chloride, mannitol, sorbitol), absorption inhibitors (eg, Tween 80), solubility enhancing agents (eg, cyclodextrins and their derivatives) may be included, stabilizers (eg, serum albumin) and reducing agents (eg, glutathione). When the composition for mucosal delivery is a liquid, the tonicity of the formulation, measured with reference to the tonicity of 0.9% (weight / volume) of physiological saline taken as a unit, is typically adjusted to a value at which none will be induced. Substantial irreversible damage to the tissue in the nasal mucosa at the site of administration. Generally, the tonicity of the solution is adjusted to a value of about 1/3 to 3, more typically of ^ to 2 and more frequently of H to 1.7. The biologically active agent can be dispersed in a base or carrier, which can comprise a hydrophilic compound having the ability to disperse the active agent and any desired additive. The base may be selected from a wide range of suitable vehicles, including but not limited to copolymers of polycarboxylic acids or their salts, carboxylic anhydrides (eg, maleic anhydride) with other monomers (eg, (meth) acrylate, acrylic acid, etc. ), hydrophilic vinyl polymers such as polyvinyl acetate, polyvinyl alcohol, polyvinyl pyrrolidone, cellulose derivatives such as hydroxymethyl cellulose, hydroxypropyl cellulose, etc., and natural polymers such as cytosine, collagen, sodium alginate, gelatin, hyaluronic acid, and metal salts non-toxic ones. Frequently, a biodegradable polymer is selected as a base or carrier, for example, polylactic acid, poly (lactic acid-glycolic acid) copolymer, polyhydroxybutyric acid, poly (hydroxybutyric acid-glycolic acid) copolymer, and mixtures thereof. Alternatively or additionally, synthetic fatty acid esters such as polyglycerin fatty acid esters, sucrose fatty acid esters, etc. may be employed as vehicles. Hydrophilic polymers and other vehicles can be used alone or in combination, and improved structural integrity can be imparted to the vehicle by partial crystallization, isotonic bonding, crosslinking and the like. The vehicle can be provided in a variety of ways, including fluid or viscous solutions, gels, pastes, powders, microspheres and films for direct application to the nasal mucosa. The use of a vehicle selected in this context may result in the promotion of the absorption of the biologically active agent. The biologically active agent can be combined with the base. or vehicle according to a variety of methods, and the release of the active agent can be by diffusion, vehicle disintegration, or associated formulation of water channels. In some circumstances, the active agent is dispersed in microcapsules (microspheres) or nanocapsules (nanospheres) prepared from a suitable polymer, eg, isobutyl-2-cyanoacrylate and dispersed in a biocompatible dispersion medium applied to the nasal mucosa, which produces sustained supply and biological activity for a long time. To further improve the mucosal delivery of pharmaceutical agents within the invention, the formulations comprising the active agent may also contain a low molecular weight hydrophilic compound as a base or excipient. Such low molecular weight hydrophilic compounds provide a means of passage through which a water soluble active agent, such as a physiologically active peptide or protein, can diffuse through the base to the body surface where the active agent is absorbed. . The low molecular weight hydrophilic compound optionally absorbs moisture from the mucosa or atmosphere of administration and dissolves the water-soluble active peptide. The molecular weight of the low molecular weight hydrophilic compound is generally not more than 10,000 and preferably no more than 3000. Exemplary low molecular weight hydrophilic compounds include polyol compounds, such as oligo, di and monosaccharides such as sucrose, mannitol , sorbitol, lactose, L-arabinose, D-erythrose, D-ribose, D-xylose, D-mannose, trehalose, D-galactose, lactulose, cellobiose, gentibiose, glycerin and polyethylene glycol. Other examples of low molecular weight hydrophilic compounds useful as carriers within the invention include N-methylpyrrolidone and alcohols (e.g., oligovinyl alcohol, ethanol, ethylene glycol, propylene glycol, etc.). These hydrophilic low molecular weight compounds may be used alone or in combination with each other or with other active or inactive components of the intranasal formulation. The compositions of the invention may alternatively contain as pharmaceutically acceptable carriers, the substances required to approximate physiological conditions such as pH adjusting agents and buffers, tonicity adjusting agents, wetting agents and the like, eg, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. For solid compositions, conventional non-toxic pharmaceutically acceptable carriers can be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate and the like. The therapeutic compositions for administering the biologically active agent can also be formulated as a solution, microemulsion, or other ordered structure suitable for the high concentration of the active ingredients. The vehicle can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol and the like), and suitable mixtures thereof. Proper fluidity for the solutions can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of a desired particle size in the case of dispersible formulations, and by the use of surfactants. In many cases, it will be desirable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the biologically active agent can be achieved by including in the composition an agent that retards absorption, for example, salts of monostearate and gelatin. In certain embodiments of the invention, the biologically active agent is administered in a time release formulation, for example in a composition that includes a slow release polymer. The active agent can be prepared with vehicles that will protect against rapid release, for example, a controlled release vehicle such as a polymer, a microencapsulated delivery system or a bioadhesive gel. The prolonged supply of the active agent, in various compositions of the invention, can be achieved by including in the composition agents that retard absorption, for example, aluminum monostearate hydrogels and gelatin. When controlled release formulations of the biologically active agent are desired, the controlled release linkers suitable for use in accordance with the invention include any biocompatible controlled release material that is inert to the active agent and that is capable of incorporating the biologically active agent. Numerous such materials are known in the art. Useful controlled release linkers are materials that are metabolized slowly under physiological conditions after intranasal delivery (e.g., on the mucosal surface, or in the presence of body fluids after transmucosal delivery). Suitable linkers include, but are not limited to, biocompatible polymers and copolymers previously used in the art in sustained release formulations. Such biocompatible compounds are non-toxic and inert to the surrounding tissues, and do not trigger significant side effects such as nasal irritation, immune response, inflammation or the like. They are metabolized into metabolic products that are also compatible and easily eliminated from the body. Exemplary polymeric materials for use in this context, include, but are not limited to, polymer matrices derived from copolymeric and homopolymeric polyesters having hydrolysable ester linkages. A number of these are known in the art to be biodegradable and to lead to the degradation of products having low or no toxicity. Exemplary polymers include polyglycolic acids (PGA) and polylactic acids (PLA), poly (DL-lactic acid-co-glycolic acid) (DL PLGA), poly (D-lactic acid-coglycolic acid) (D PLGA) and poly (L-lactic acid-co-glycolic acid) (L PLGA). Other useful biodegradable or bioerodible polymers include, but are not limited to polymers such as poly (epsilon-caprolactone), poly (epsilon-caprolactone-CO-lactic acid), poly (epsilon-aprolactone-CO-glycolic acid), poly (beta) hydroxybutyric acid), poly (alkyl-2-cyanoacrylate), hydrogels such as poly (hydroxyethyl methacrylate), polyamides, poly (amino acids) (ie, L-leucine, glutamic acid, L-aspartic acid and the like), poly ( urea ester), poly (2-hydroxyethyl DL-aspartamide), polyacetal polymers, polyorthoesters, polycarbonate, polymaleamides, polysaccharides and copolymers thereof. Many methods for preparing such formulations are generally known to those skilled in the art. Other useful formulations include controlled release compositions, e.g., microcapsules, Patents of E.U. Nos. 4,652,441 and 4,917,893, copolymers of lactic acid-glycolic acid useful in the preparation of microcapsules and other formulations, US Patents. Nos. 4,677,191 and 4,728,721 and sustained release compositions for water soluble peptides, US Pat. No. 4,675,189. For nasal and pulmonary delivery, systems for the controlled aerosol delivery of therapeutic liquids as a spray are well known. In one embodiment, the measured doses of the active agent are delivered by means of a specially constructed mechanical pump valve, US Pat. No. 4,511,069. Dosage For prophylactic and treatment purposes, the biologically active agent (s) described herein, can be administered to the subject in a single rapid supply, by continuous delivery (eg, transdermal delivery). , mucosal or continuous intravenous), over an extended period of time, or in a repeated administration protocol (eg, by a repeated administration protocol per hour, daily or weekly). In this context, a therapeutically effective dose of the glucose regulatory peptide may include repeated doses within a prolonged regimen of prophylaxis or treatment that will produce clinically meaningful results to alleviate one or more symptoms or detectable conditions associated with a disease or objective condition as established. previously. The determination of effective doses in this context is typically based on studies in animal models followed by clinical trials in humans and is guided by the determination of effective doses and administration protocols that significantly reduce the occurrence or severity of symptoms or conditions of the target disease in the subject. Suitable models in this regard include, for example, murine, rat, porcine, feline, non-human primate and other accepted animal model subjects known in the art. Alternatively, effective doses can be determined using in vitro models (e.g., immunological and histopathological analyzes). In using such models, only ordinary calculations and adjustments are typically required to determine an appropriate concentration and dose to administer a therapeutically effective amount of the biologically active agent (s) (eg, amounts that are intranasally effective, transdermally effective, intravenously effective or intramuscularly effective to emit a desired response). In an alternative embodiment, the invention provides compositions and methods for the intranasal delivery of the glucose regulatory peptide, wherein the The glucose regulator peptide compound (s) is repeatedly administered by an effective intranasal dose regimen involving multiple administrations of the glucose regulatory peptide to the subject during a daily or weekly schedule to maintain a high therapeutically effective level and Decreased pulsatile glucose regulator peptide over an extended dosing period. The compositions and methods provide glucose regulator peptide compound (s) that self-administer (s) by the subject in a nasal formulation between one and six times daily to maintain a high therapeutically effective and decreased pulsatile level of the glucose regulatory peptide. during an extended dosing period of 8 hours to 24 hours. Equipment The present invention also includes equipment, packages, and multi-package units containing the pharmaceutical compositions, active ingredients, and / or means described above for administering the same for use in the prevention or treatment of diseases and other conditions in mammalian subjects. Briefly, these kits include a package or formulation containing one or more glucose regulating peptides, proteins, analogs or mimetics and / or other biologically active agents in combination with the mucosal delivery enhancing agents described herein, formulated in a pharmaceutical preparation. for the mucosal supply. The intranasal formulations of the present invention can be administered using any spray bottle or syringe or by instillation. An example of a nasal spray bottle is the "Nasal Spray Pump w / Safety Clip", (Nasal spray pump with safety lock), Pfeiffer SAP # 60548, which supplies a dose of 0.1 ml per square and has a length of 36.05 mm tube. It can be purchased from Pfeiffer of America of Princeton, NJ. Aerosol Nasal Administration of a Glucose Regulating Peptide It has been discovered that GRPs can be administered intranasally using a nasal spray or spray. This is surprising because many proteins and peptides have been shown to be cut or denatured due to the mechanical forces generated by the triggering in the spray or spray. In this area the following definitions are useful. 1. Aerosol - A product that is packaged under pressure and that contains therapeutically active ingredients that are released upon activation of an appropriate valve system. 2. Measured aerosol - A form of pressurized dose comprising metered dose valves, which allow the supply of a uniform amount of spray at each activation. 3. Powdered aerosol - A product that is packaged under pressure and contains therapeutically active ingredients in the form of a powder, which are released upon activation of an appropriate valve system. 4. Spray aerosol - An aerosol product that uses a compressed gas as an impeller to provide the force needed to expel the product as a wet scrub; it is generally applicable to solutions of medicinal agents in aqueous solvents. 5. Roclo - A liquid that is minutely divided by a jet of air or current. Nasal spray drug products contain therapeutically active ingredients dissolved or suspended in solutions or mixtures of excipients in non-pressurized dispensers. 6. Measured spray - A non-pressurized dosage form consisting of valves that allow a specified amount of spray to be dispensed at each activation. 7. Dewdrop - A liquid preparation containing solid particles dispersed in a liquid vehicle and in the form of drops or as finely divided solids. The dynamic characterization of aerosol spray fluid is emitted by nasal spray pumps measured as a drug delivery device ("DDD"). The characterization of the roclo is an integral part of the regulatory submissions necessary for the approval of the Food and Drug Administration ("FDA") of research and development, of safety of the quality and of the procedures of test of stability for pumps of nasal spray new and existing. Through the characterization of the roclo geometry, it has been discovered that it is the best indicator of the general performance of nasal spray pumps. In particular, it has been found that measurements of the divergence angle of the spray (boom geometry) as it exists in the device; the cross-section ellipticity of the roclo, the uniformity and the particle / drop distribution (dew pattern); and the evolution in time of the developed spray, are the most representative performance quantities in the characterization of a nasal spray pump. During quality assurance and stability testing, boom geometry and brush pattern measurements are key identifiers to verify consistency and compliance with approved data criteria for nasal spray pumps. Definitions Boom height - the measurement from the tip of the boom to the point at which the boom angle becomes non-linear due to the fracture of the linear flow. Based on a visual examination of digital images, and to establish a measuring point for the width that is consistent with the measurement point furthest away from the spray pattern, a height of 30 mm is defined for this study. Axis Largest - the largest rope that can be traced within the tight loop pattern that crosses the COMW in base units (mm).

Minor Axis - the smallest rope that can be traced within the adjusted dew pattern that crosses the COMw in base units (mm). Ellipticity Ratio - the ratio of the major axis to the minor axis, preferably between 1.0 and 1.5, and more preferably between 1.0 and 1.3. It gave the drop diameter for which 10% of the total volume of liquid in the sample consists of droplets of smaller diameters (um). D50 - the droplet diameter for which 50% of the total volume of liquid in the sample consists of drops of smaller diameters (um), also known as the average mass diameter. D9o - the droplet diameter for which 90% of the total volume of liquid in the sample consists of droplets of smaller diameters (um). Scope - measuring the width of the distribution, the smallest value, the narrowest distribution. The scope is calculated as: (? 90 ~ ^ 10). D 50 RDS% - relative percentage standard deviation, the standard deviation divided by the series mean and multiplied by 100, also known as CV%. Volume - the volume of liquid or powder discharged from the delivery device with each actuation, preferably between 0.02 ml and approximately 2.5 ml, and most preferably between 0.02 ml and 0.25 ml. All publications, references, patents, patent publications and patent applications cited herein are hereby incorporated by reference in their entirety. Although this invention has been described in connection with certain embodiments, and many details have been established for purposes of illustration, it will be apparent to those skilled in the art that this invention includes additional embodiments and that some of the details described herein may vary considerably. without departing from this invention. This invention includes such modalities, modifications and additional equivalents. In particular, this invention includes any combination of characteristics, terms, or elements of the various components and illustrative examples. The use in the present of the terms "a", "an", "the" and similar terms to describe the invention, and the claims, should be construed to include both the singular and the plural. The terms "comprising", "having", "including" and "containing" should be interpreted as open ended terms, which means, for example, "including, but not limited to". the citation of a range of values herein refers individually to each value separately that falls within the range as it will be cited individually herein, whether or not some of the values within the range are expressly cited. The specific values employed herein will be construed as exemplary and not to limit the scope of the invention. The examples provided herein, and the exemplary language used herein, are for purposes of illustration only, and are not intended to limit the scope of the invention. EXAMPLES EXAMPLE 1 Insulin aspart formulations Table 1 describes the twelve aspart insulin formulations tested using the EpiAirway in vitro model system for transepithelial resistance analysis (TER), cell viability analysis (MTT), cell death analysis lactate dehydrogenase (LDH), and tissue permeation analysis. The results were used to determine which formulation achieved the highest degree of tissue permeation and reduction of TER, while resulting in no cell toxicity. Insulin aspart is an insulin analogue that is homologous to regular human insulin except for a single substitution of aspartic acid for proline at position B28. NovoLog (NovoLog ™, Novo Nordisk Pharmaceuticals) is a sterile, aqueous, clear and colorless solution containing insulin aspart (regular human insulin analog B28 asp) 100 Units / ml, glycerin 16 mg / ml, phenol 1.50 mg / ml, metacresol 1.72 mg / ml, zinc 19.6 ug / ml, disodium hydrogen phosphate dihydrate 1.25 mg / ml, and sodium chloride 0.58 mg / ml. NovoLog has a pH of 7.2-7.6. New formulations of insulin aspart were generated. A total volume of 0.5 ml was made for each formulation. The formulations contained varying concentrations of insulin aspart, NovoLog diluent, and the excipients methyl-beta-cyclodextrin (M-beta-CD), didecanoil of L-alpha-phosphatidylcholine (DDPC), and disodium edetate (EDTA), alone or in combination. Controls without excipients were also included in the study. small amounts of 2N HCl or NaOH were added, when necessary, to the formulations until the desired pH was achieved. The reagents used to prepare the formulations are shown in Table 2.

- - TABLE 1: Aspart Insulin Formulations for In Vitro Studies TABLE 2: Insulin Formulations Reagents aspart EXAMPLE 2 Nasal Mucosal Supply - Permeation Kinetics and Cytotoxicity The following methods are generally useful for evaluating the parameters, kinetics and side effects of the nasal mucosal delivery, for insulin within the formulations and method of the invention, as well as for determining the efficacy and the characteristics of the various mucosal delivery enhancing agents described herein for the combinatorial formulation or coordinated administration with insulin aspart. In an exemplary protocol, permeation kinetics and lack of unacceptable cytotoxicity are demonstrated for an intranasal delivery enhancing agent as described above in combination with a biologically active therapeutic agent, exemplified by insulin aspart. Cell Cultures The EpiAirway system was developed by MatTek Corp.

(Ashland MA) as a model of the pseudostratified epithelium that lines the respiratory tract. The epithelial cells are cultured in porous membrane bottom cell culture inserts at an air-liquid interface, which results in the differentiation of the cells into a highly polarized morphology. The apical surface is ciliated with a microvellose structure and the epithelium produces mucus (the presence of mucin has been confirmed by immunoblotting). The inserts have diameters of 0.875 cm, providing a surface area of 0.6 cm2. The cells are plated on the inserts at the factory approximately three weeks before shipment. The EpiAirway ™ culture membranes were received the day before the start of the experiments. They embarked in half Dulbecco's Modified Eagle's Medium (DMEM) free of red phenol and hydrocortisone-free. Each tissue insert was placed into a well of a 6-well plate containing 0.9 ml of serum-free DMEM. The membranes were then cultured for 24 hours at 37 ° C / 5% C02 to allow tissue to equilibrate. The inserts are fed for each day of recovery. The DMEM-based medium is free of serum but is supplemented with epidermal growth factor and other factors. The medium was tested by the endogenous levels of some cytosine or growth factor considered for intranasal delivery, and was found free of all cytosine and factors studied to date except insulin. The volume was insufficient to provide contact with the bottoms of the units in their bases, but the epic surface was allowed to remain in direct contact with the air. Sterile clamps were used in this stage and in all subsequent stages involving the transfer of units to wells containing liquid to ensure that air was not trapped between the bottoms of the units and the environment. An EpiAirway ™ model system was used to evaluate the effect of each formulation containing NovoLog on TER, cell viability (MTT), cytotoxicity (LDH) and permeation. These analyzes are described below in detail. In all experiments, the nasal mucosal supply formulation studied was applied to the apical surface of each unit in a volume of 100 ul, which was sufficient to cover the total apical surface. An appropriate volume of the test formulation in the concentration applied to the apic surface (no more than 100 ul is necessary) was set aside for the subsequent determination of the concentration of the active material by ELISA or other designated analysis. Transepithelial Electrical Resistance (TER) The TER measurements were read using a Tissue Resistance Measurement Chamber connected to an Epithelial Voltometer with electrode signals, both from World Precision Instruments. First, the background TER was read for each insert on the day of the start of the experiment. After reading the TER, 1 ml of fresh medium was placed in the bottom of each well in a 6-well plate. The inserts were drained on paper towels and placed into fresh wells with fresh medium, while the numbered inserts were maintained to correlate them with background TER measurements. 100 ul of the experimental formulation was added to each insert. The inserts were placed in a shaking incubator at 100 rpm and at 37 ° C for 1 hour. The electrodes and the blank insert of the tissue culture were equilibrated for at least 20 minutes in fresh medium with the power disconnected prior to checking the calibration. The background resistance was measured with 1.5 ml of medium in the Endohm tissue chamber and 300 ul of the medium in a blank Millicell-CM insert. The upper electrode was adjusted so that it was immersed in the medium but without contacting the upper surface of the insert membrane. The background resistance of the blank insert was 5-20 ohms. For the determination of TER, 300 ul of the medium was added to the insert followed by a 20 minute incubation at room temperature before placement in the Endohm chamber to read the TER. The resistance was expressed as (measured-white resistance) x 0.6 cm2. All TER values were reported as a function of the surface area of the tissue. TER was calculated as: TER = (R? -Rb) x A Where R? is the resistance of the insert with a membrane, R is the resistance of the insert in white, and A is the area of the membrane (0.6 cm2). A decrease in the value of TER relative to the control value (control = approximately 1000 ohms-cm2; normalized to 100) indicates a decrease in cell membrane resistance and an increase in mucosal epithelial cell permeability. After completing the 1 hour incubation, the tissue inserts were removed from the incubator. 200 ul of the fresh medium was placed in each well of a 24-well plate and the tissue inserts were transferred to the 24-well plate. 200 ul of the fresh medium was gently added to each tissue insert. The TER was measured again for each insert. After transferring the tissue culture inserts from the 6 well plate to the 24 well plate, the basal medium was subdivided into three parts and stored in eppendorfs. All three subdivisions were placed at -80 ° C until use. Lactate Dehydrogenase (LDH) Analysis The amount of cell death was analyzed by measuring the release of LDH from the cells using CytoTox 96 cytotoxicity analysis equipment from Promega Corp. Samples were run in triplicate for face insert tissue in the study. 50 ul of the harvested medium (stored at 4 ° C) were loaded in triplicate into a 96-well plate. Fresh cell-free medium was used as target. 50 ul of the substrate solution (12 ml of assay buffer added to a new bottle of the substrate mixture, prepared according to the kit), were added to each well and the plates were incubated for 30 minutes at room temperature in Darkness. After incubation, 50 ul of the stop solution was added to each well and the plates were read in a uQuant optical density plate reader at 490 nm using KCJr software. MTT analysis The cell viability of each tissue insert was tested by MTT analysis (MTT-100, MatTek equipment) that tests for mitochondrial reductase activity. This equipment measures the absorption and transformation of the tetrazolium salt to the formazan dye. The MTT concentrate was frozen and diluted with the medium at a ratio of 2 ml of MTT: 8 ml of the medium. The diluted MTT concentrate was pipetted (300 ul) into a 24-well plate. The tissue inserts were gently dried, placed in the wells of the plate and incubated for three hours in the dark at 37 ° C. After incubation, each insert was removed from the plate, colored gently and placed in a 24-well extraction plate. The cell culture inserts were then immersed in 2.0 ml of the extractant solution per well (to completely cover the sample). The extraction plate was covered and sealed to reduce the evaporation of the extractant. After an overnight incubation at room temperature in the dark, the liquid within each insert was decanted back to the well from which it was taken, and the inserts were discarded. The extractant solution (50 ul) from each well was pipetted in triplicate into a 96-well microtiter plate, together with the extract blanks and diluted with the addition of 150 ul of fresh extractant solution. The optical density of the samples was measured at 550 nm in an uQuant optical density plate reader using KCJr software. TER results The TER measurements (ohms x cm2) before and after the 1 hour incubation at 37 ° C for the experimental formulations were compared with the controls. The results show that formulations containing breeders, except for # 7 and # 8, have a significant reduction of TER after one hour of incubation. MTT results Almost all formulations showed a good to good viability compared to the controls. The MTT% for most formulations was greater than 80% (except # 1, # 6 and # 12). LDH results Most of the formulations tested showed very little LDH, indicating a very low cytotoxicity. Summary The results of the TER, MTT and LDH analyzes indicate that formulations # 2, # 6, 39, # 10, and # 11 all show a significant reduction in TER without increased toxicity. EXAMPLE 3 Permeability of Insulin aspart ELISA was used to quantify the amount of insulin or insulin analog that permeates through the apex to the basolateral side of the insert. The insulin is present in the MaTek medium so that the raw data was corrected by subtracting the average concentration present in the medium sample from all the other samples. The Iso-Insulin ELISA kits are purchased from Alpco Diagnostics, (Windham, NH, Cat # 08-10-1128-01). The samples were diluted with assay buffer that was provided with the equipment. Dilution was mixed in clear silanized HPLC vials with Teflon coated covers by gentle inversion. The optical density of the samples was measured at 450 nm (as indicated in the protocol) in a uQuant optical density plate reader using KCJr software. The loading volume was 100 ul per insert and the permeation sample time was 60 minutes. Each formulation, as well as controls, were tested using n = 3 inserts. Controls for this study included baseline MatTek and Triton X-100 at 9%. Each tissue insert was placed in an individual well containing 0.95 ml of - MatTek basal medium. At the apex surface of the inserts, 100 ul of the test formulation was applied according to the study design, and the samples were placed on a shaker (-100 rpm) for 1 hour at 37 ° C. At the end of the incubation period, 50 ul of -20,000 Kl Units of aprotinin were added to each sub-sample of the culture medium and stored at 2-8 ° C for the ELISA analysis. Table 3 shows the permeability results of the basolateral samples analyzed by ELISA. The averages were corrected from the experimental samples. TABLE 3: Permeation% Results Testing Aspartin Insulin Formulations These permeation results show that permeation enhancers can be employed to deliver insulin aspart by intranasal formulations. All formulations containing improver resulted in at least 9% (-9-21%) permeation compared to formulations with enhancers that yielded at most -1-2%. The samples with the most improvement in permeability included # 1, # 2, # 9, # 10, # 11 and # 12. When taken together with the results of TER, MTT and LDH, samples # 2 (5 U / ml insulin aspart, 45 mg / ml of Me-beta-CD, 1 mg / ml of DDPC, 1 mg / ml of EDTA , 5% NovoLog diluent, pH 4); # 9 (20 U (insulin aspart, 45 mg / ml Me-beta-CD, 1 mg / ml DDPC, 1 mg / ml EDTA, 20% NovoLog diluent, pH 4), # 10 (20 U / ml aspartic isulin, 45 mg / ml Me-beta-CD, 1 mg / ml DDPC, 1 mg / ml EDTA, 20% NovoLog diluent, pH 3), and # 11 (5 U / ml of insulin aspart, 0 mg / ml of Me-beta-CD, 0 mg / ml of DDPC, 10 mg / ml of EDTA, 5% of NovoLog diluent, pH 4) have the greatest improvement in permeability with the lowest cellular toxicity. EXAMPLE 4 Effects of NPG and PN159 on Insulin Permeability In vitro experiments evaluated the effect of both small molecule and peptide permeation enhancers on insulin permeability Seven different treatments were applied to EpiAirway 96-well plates during 1 hour at 37 ° C with 0.1 U of insulin applied to the apical side.The standard curve and the high, low controls were as expected, and the insulin peak in the basolateral medium showed a recovery of almost 100%. they included: PBS + insulin 0.1 U; 25 uM PN159 + insulin 0.1 U; 50 uM PN159 + insulin 0.1 U; PDF + insulin 0.1 U; PBS + insulin 0.1 U + 150 mM NPG; 25 uM PN159 + insulin 0.1 U + 150 mM NPG; and PDF + insulin 0.1 U 150 mM NPG. The insulin used in this study is the natural sequence of recombinant human insulin (derived from yeast) Sigma. This recombinant human insulin is derived from pro-insulin and is chemically, physically and biologically identical to pancreatic human insulin. PBS is saline buffered with phosphate. PDF is a mixture consisting of 45 mg / ml of methyl-beta-cyclodextrin, 1 mg / ml of ethylenediamine tetraacetate, 1 mg / ml of didecanoylphosphatidyl choline and 10 mM of acetate, pH 5.5. The monomeric stabilizer (NPG) is N-pivaloyl glucosamine. PN159 is a peptide described in the U.S. Patent Application. Copending No. 11 / 233,239. The results are shown in Table 4 as follows: TABLE 4: Permeability Results With PDF, PN159 and NPG Formulations PN159 increased insulin permeability: 25 uM of PN159 resulted in 0.8% permeability, while 50 uM of PN159 increased permeability to 2.5%. combined with NPG, 25 uM of PN159 resulted in a 1.8% permeability. The results showed that PDF alone provided 3% permeability, approximately 12 times greater than PBS alone. When the PDF was combined with NPG the permeability was increased to 7.6% a 30-fold increase over PBS. An additional study was conducted to test the TER, LDH, MTT and the permeability of the formulations shown in Table 5, including the formulations containing PN159. All formulations were tested with inserts n = 3 in the EpiAirway model. The regular insulin was approximately 28 U / mg (i.e., 200 U / ml = -7.14 mg / ml) TABLE 5: Formulations Abbreviations: Arg = Arginine, Me-ß-CD = methyl-beta-cyclodextrin, EDTA = sodium edetate, NaCl = sodium chlorido, MP = methylparaben sodium, PP = propylparaben sodium, PG = propylene glycol Abbreviations: Arg = Arginine, Me-beta-CD = methyl-beta-cyclodextrin, EDTA = disodium edetate, NaCl = sodium chloride, MP = sodium methylparaben, PP- = sodium propylparaben, PG = propylene glycol The results of TER measurements (ohms x cm2 ) before and after the 1 hour incubation at 37 ° C for the experimental formulations were compared with the controls. The results show that the formulations containing improvers, had a significant reduction of TER after one hour of incubation. Formulations # 5, # 6 and # 8 showed cell viability and all formulations except # 7 had minimal cell toxicity, similar to the PBS control. The permeation results are shown in Table 6. TABLE 6: Permeation Results Formulations # 1 /4.47%) and # 7 / 4.66%) had the highest percentage permeability. These data show that the addition of PN159 did not significantly improve insulin permeation over the PDF formulation (Me-beta-CD, DDPC, and EDTA) in vitro.

EXAMPLE 5 Effect of Alternative Shock Absorbers on Insulin Permeability A permeability study was carried out to compare the alternative shocks in combination with the PDF formulation (Me-beta-CD, DDPC and EDTA). These permeability data were generated by ELISA (from LINCO Research Inc., Catalog # EZHI-14K) after a 60-minute incubation and using a loading volume of 50 ul. All the formulations listed in Table 7 had good cell viability and low toxicity as measured by MTT and LDH analysis.

TABLE 7: Permeability Results With Alternate Shock Absorbers The arginine buffer was a buffer of modest performance with percent permeation of insulin at 3% to 6%. The acetate buffer formulations achieved a% permeability of 7% to 12%. % Permeability for phosphate buffer formulations ranged from 5% to 28%. The highest percent permeabilization, 28%, was achieved with 10 mM phosphate buffer, 45 mg / ml Me-beta-CD, 1 mg / ml DDPC, 1 mg / ml EDTA, 280 U / ml insulin a pH of 7 (# 4). EXAMPLE 6 In Vitro Visualization Studies for the Optimal Intranasal Insulin Formulation A preliminary in vitro visualization was carried out for insulin formulations of 280 U / ml and 840 U / ml at varying component concentrations and pH ranges. The base formulation of IX PDF included 45 mg / ml Me-beta-CD, 1 mg / ml DDPC, 1 mg / ml EDTA, 10 mM acetate, 10 mM phosphatase, and 220 mOdm / kg NaCl. The formulation components of study 1 in vitro are shown in Table 8.

TABLE 8: Study 1 In Vitro of Formulation Components The time course tested for the first in vitro experiment included incubations of 30, 60, and 120 minutes. Incubations were carried out in PBS with Ca + X and Mg ++ (note that insulin is present in the standard MatTek medium). The conditions of the analysis included turns of 100 rpm at 37 ° C and the application of 50 ul of the tested formulation. A significant reduction of TER was observed with all the formulations. High toxicity was observed with the MTT analysis, and low cell viability was observed with the LDH analyzes. The permeation data for the insulin concentration of 280 U / ml were taken at 30 and 60 minutes and IX PDF was compared; pH 3; 2X PDF, pH 3; IX PDF, pH 4; and 0.5 X PDF, pH 3. Approximately 10% permeation was achieved with IX PDF, pH 3; IX PDF, pH 4; and 0.5 X PDF, pH 3 at 60 minutes. 2X PDF, pH 3 resulted in a 2% permeation. Control without improver showed less than 1% permeation. The permeation data for the concentrations of 280 U / ml of insulin and 840 U / ml of insulin were compared at 30 and 60 minutes in the formulation of 2X PDF, pH 3. No significant differences in permeation were observed due to the high variability , possibly caused by precipitation at high insulin concentration. The permeation data for the concentration of 280 U / ml of insulin taken at 30 and 60 minutes in IX PDF, pH 3 were compared with IX PDF, pH 7. The results showed that the formulation at pH 7 performed better than pH 3. The percentage permeation for IX PDF, pH 3 was 10% while the permeation for IX PDF, pH 7 was 35%. Summary of Study 1 in vitro Permeation studies with incubations of 30, 60 and 120 minutes in PBS (with Ca ++ and Mg ++) resulted in high toxicity and low cell viability. IX and 0.5 X PDF resulted in a better permeation than 2X PDF. PDF was able to solubilize 10 mg / ml (i.e., 280 U / ml), but not 30 mg / ml (i.e., 840 U / ml). The percentage permeation results were higher for pH 7 than for pH 3. A second study was conducted using IX PDF (45 mg / ml Me-beta-CD, 1 mg / ml DDPC, 1 mg / ml EDTA, 10 mM acetate, 10 mM phosphatase, and 220 mOsm / kg NaCl) as the base formulation. The components of the formulation of Study 2 in vitro are shown in Table 9.

TABLE 9: In Vitro Study 2 of Formulation Components The time course for the second in vitro experiment included an incubation 60 minutes. Incubation was carried out in PBS with Ca ++ and Mg ++. The conditions of the analysis included turns of 100 rpm at 37 ° C and the application of 50 ul of the tested formulation. A significant reduction of TER was observed with all the formulations. High toxicity was observed with the MTT analysis, and low cell viability was observed with the LDH analyzes, even without solubilizers. The permeation data for the insulin concentration of 280 U / ml were taken at 60 minutes and IX PDF; pH 3.5; IX PDF, pH 7; IX PDF + 0.5% CEL, pH 3.5; IX PDF + 1% CEL, pH 3.5; IX PDF + 0.1% Tween 80, pH 3.5; and IX PDF + 1% Tween 80, pH 3.5. At least 10% permeation was achieved with all formulations at a pH of 3.5. The permeation was increased in the formulations at a pH of 7 compared to a pH of 3.5. The permeation data for the concentrations of 280 U / ml and 840 U / ml of insulin were compared at 60 minutes in the formulations of IX PDF, pH 7; IX PDF + 0.5% CEL, pH 3.5; IX PDF + 0.5% CEL, pH 7; IX PDF + 1% CEL, pH 3.5; IX PDF + 0.1% Tween 80, pH 3.5; and IX PDF + 1% Tween 80, pH 3.5 .. Formulations of 840 U / ml of insulin were visually soluble on the surface of the insert when the solubilizers were present. All formulations had similar permeability, with the exception of formulations at pH 7 that have a higher permeability than formulations at a pH of 3.5. Summary of In Vitro Study 2 High concentration insulin formulations (840 U / ml) were successfully stabilized (and solubilized) in the presence of an additional surfactant (i.e., Tween or Cremophor). Even in a 60-minute incubation, increased cytotoxicity and decreased cell viability were observed for all in vitro formulations. A third study was conducted to determine the in vitro effects on the permeation of three different surfactants (Tween 80, Tween 20 and Pluronic F68) using insulin formulations of IX PDF of 280 U / ml and 840 U / ml at a pH of 7 The components of the formulation of Study 3 in vitro are shown in Table 10. TABLE 10: Study 3 In Vitro of Formulation Components The time course for the third in vitro experiment included an incubation 60 minutes. Incubation was carried out in PBS with Ca ++ and Mg ++. The conditions of the analysis included turns of 100 rpm at 37 ° C and the application of 50 ul of the tested formulation. A significant reduction of TER was observed with all the formulations. High toxicity was observed with the MTT analysis, and low cell viability was observed with the LDH analyzes. The permeation data for the insulin concentration of 280 U / ml were taken at 60 minutes and formulations of IX PDF + 1% Tween 80, pH 7; IX PDF + 0.01% Pluronic F68, pH 7; and IX PDF + 0.1% Pluronic F68, pH 7. The results showed that Pluronic F68 does not solubilize insulin sufficiently to improve permeation. The effects of Tween 80 and Tween 20 on the permeation of insulin were tested in the formulation of IX PDF (insulin concentration of 280 U / ml and 840 U / ml). The percentage permeation data were compared at 60 minutes in the formulations of IX PDF + 1% Tween 80, pH 7; IX PDF + 0.1% Tween 80, pH 7; IX PDF (without DDPC) + 1% Tween 80, pH 7; IX PDF (without DDPC) + 0.1% Tween 80, pH 7; IX PDF + 1% Tween 20, pH 7; IX PDF + 0.1% Tween 20, pH 7; and IX PDF + 1% Tween 80, pH 7 (hypotonic). The results showed that 1% Tween (both Tween 80 and Tween 20) provides higher permeation than 0.1% Tween. The permeation results for Tween 20 were the same as for Tween 80. The removal of the DDPC had no effect on the% permeation in these formulations. Increased amounts of Tween 80 (0.01%, 0.1%, 0.5% and 1%) were tested for the effect on permeation for 280 U / ml of insulin in IX PDF formulations at pH 7. The amount of Tween 80 in the formulation effected% permeation. The permeation was increased with an increased concentration of Tween 80. Additional analyzes of the effect of the Tween concentration on the permeation were made in formulations from IX PDF to 1%, 2% and 5% of Tween 80. Additionally, permeation was tested with the 2X PDF formulations with 1% and 2% Tween 80. The results showed that once the Tween concentration was above 1% whether in IX or 2X PDF, there was no further improvement of in vitro permeation. The effect of removing the Me-beta-CD in the permeation was tested with the formulations of 0.1% Tween and 1% Tween (containing 1 mg / ml and 10 mg / ml EDTA). Removal of Me-beta-CD from the formulation resulted in a dramatic decrease in permeation. The results were observed with the formulations of both 280 U / ml and 840 U / ml. Summary of Study 3 in vitro Tween 80 and Tween 20 both resulted in a good in vitro permeation when used in combination with IX PDF formulations. Pluronic F68 did not improve the permeation. Tween 80 or 20 alone is not sufficient to achieve an increase in insulin permeation in vitro. The removal of Me-beta-CD from the formulation resulted in a significant decrease in insulin permeation. The increase in Tween up to 1% resulted in an increase in permeation, but above 1% no additional benefit was observed. A formulation of 1% Tween is lower than some commercialized nasal products containing Tween. EXAMPLE 7 Stability Data of the Insulin Formulation A stability study in use was conducted for up to 28 days at 5 ° C, 25 ° C, 40 ° C and 50 ° C, for the insulin spray formulations of IX PDF ( 45 mg / ml Me-beta-CD, 1 mg / ml DDPC, 1 mg / ml EDTA, 10 mM acetate, 10 mM phosphatase and 220 mOsm / kg NaCl). HPLC was used to analyze the% recovery of peptide. The parameters of the formulation evaluated in the study are shown in Table 11. TABLE 11: Parameters of the Formulation of the Preliminary Study of Stability of Insulin The insulin-based formulations of IX PDF remained more stable compared to insulin stored only with salt and buffer. The presence of Tween did not affect the stability of IX PDF formulations. The formulations at a pH of 7.0 maintained much better stability than the formulations at pH 3.5. The stability of the insulin stored in IX PDF was very good during 28 days at 5 ° C, 25 ° C, 40 ° C and 50 ° C, a recovery of approximately 100% was observed for insulin concentrations of 280 U / ml as of 840 U / ml. Additional analyzes in use (unit dose and 8 days) were conducted to evaluate the stability of the IX PDF PDF spray formulations. The "unit dose" was used to evaluate the stability of the insulin coat after priming and a drive. The conditions for the study in use of 8 days included drive for 8 days, three times a day (TID) at 5 ° C and 30 ° C in storage. The studies were analyzed by peptide content. The results of the peptide content studies in use showed that PDF insulin spray formulations demonstrate good stability for both the unit dose and 8 days in use. The stability at a storage temperature of 30 ° C appears to be as stable as at a storage temperature of 5 ° C in this study.

EXAMPLE 8 Results of Insulin Pharmacokinetics Intranasally Administered in Rabbits The pharmacokinetic values (PK, i.e., insulin measurements) were measured for white rabbits from New Zealand treated with insulin at specified time points of up to 240 minutes. Four (4) intranasal groups (IN), one (1) subcutaneous group (SC), and one (1) intravenous group (IV) that generated the bioavailability data were included in the study. Each group included male rabbits All data calculations are of standardized dose and PK data are corrected baseline. The treatment and dose details for PK Study 1 are shown in Table 12. TABLE 12: The results of PK Study 1 are shown in Table 13 and Figure 1. IN administration of insulin resulted in a T max faster than that of normal SC insulin. IN / 1X PDF with 1% Tween (dose of 6 IU / kg), # 3 showed the highest peak of the intranasal formulations. The percentage bioavailability (BA) of insulin was -3-5% for formulations of IN / 1X PDF with 1% Tween both # 2 and # 3, (in relation to SC). The absolute% of BA for SC was 30% while IN was 1%. The% CV for IN was 50% while SC was 20% based on the AUC. TABLE 13: Results of Study 1 PK in Rabbits A second PK study was conducted to compare the intranasal formulations of PDF plus Tween with the fast-acting NovoLog formulation (the NovoLog diluent consists of: 16 mg / ml glycerin, 1.5 mg / ml phenol, 1.72 mg / ml m-cresol, 19.6 μg / ml zinc, 1.25 mg / ml disodium hydrogen phosphate dihydrate, and 0.58 mg / ml NaCl, pH 7.2-7.6). The parameters of PK Study 2 are shown in Table 14. TABLE 14: Formulation Parameters for PK Study 2 (and PD) Table 15 shows the Tmax,% Cmax, AUCuitima, UCinf and% of BA in relation to the results of SC-Novolog for Study 2 with the base line of subtracted PK; The results of Study 1 are also included for the formulations of IN / 1X PDF 1% of Tween (# 3) and SC-Normal (# 5). The results of PK Study 2 are shown in Figure 2. The PK curve for Study 2 is similar to the curve observed in Study 1, showing that IN / 1X PDF results in a fast-acting PK profile. A second insulin peak was observed in some animals treated IN. TABLE 15: Results of PK Study 2 in Rabbits The results of the PK 2 Study show that the IN / 1X PDF 2% Tween has the highest% BA, Cmax and AUCuitima of the intranasal formulations tested. The BA%, Cmax and AUCúitima decreased when the DDCP was removed. The results of IN / 1X PDF 1% of Tween for Study 2 were consistent with the results of Study 1. Insulins SC-Normal, SC-Novolog and SC-PDF resulted in a similar bioavailability. For% BA, intranasal formulations resulted in a bioavailability of approximately 2-5%. IN / 1X PDF 2% Tween showed the highest bioavailability at 5%. EXAMPLE 9 Pharmacodynamic Data of Insulin Administered Intranasally in Rabbits Pharmacokinetic values (PK); i.e., glucose measurements) were measured for New Zealand white rabbits treated with insulin at specified time points of up to 240 minutes. Glucose was measured at each time point in duplicate with a glucometer (One-Touch Ultra). The results of Study 1 of PD (the test groups are shown in Example 8, Table 12 above) are shown in Table 16 and Figure 3. All calculations were of normalized dose,% BA were based on the values of the nominal analysis of the test article.

TABLE 16: Results of PD Study 1 In Study 1, the% of Cm? N for the insulin formulations of IN / PDF-Tween (# 2 and # 3), SC (# 5) and IV (# 6) was approximately 40%. The Tm? N was faster for IN / PDF-Tween (30-45 minutes) than for SC (120 minutes). The% BA of glucose was -8% for IN PDF with 1% Tween (relative to SC). A second PD study was conducted to compare intranasal PDF formulations with NovoLog's rapid-acting formulation (NovoLog's diluent consists of: 16 mg / ml glycerin, 1.5 mg / ml phenol, 1.72 mg / ml m -cresol, 19.6 μg / ml zinc, 1.25 mg / ml disodium hydrogen phosphate dihydrate, and 0.58 mg / ml NaCl, pH 7.2 -7.6). The parameters of Study 2 of PK are shown above in Example 8, Table 14. The results of Cm? N and Tm? N of Study 2 of PD are shown in Table 17. TABLE 17: Results Cm? N and Tm? N for Study 2 of PD * Results of PD Study 1 In Figures 4, the results of PD for Study 2 are compared with Study 1. The Tm? N for IN / 1X PDF 5% Tween, IN / 2X PDF 1% Tween, SC NovoLog It was about 30 minutes. The Tm? N for SC-Normal * was approximately 40 minutes. The ^ n for another formulation was approximately 45 minutes. The results of Study 2 of PD showed that 2X PDF 1% Tween had the greatest effect on PD of all intranasal formulations. The presence of DDCP in the formulation did not affect PD results.

Intranasal irritation was absent or silent in rabbits for IN administration. The PD data described are supports for the intranasal formulations that deliver insulin for a rapid action profile. The best performance formulations contained a solubilizing agent and a surfactant. A description of insulin IN formulations for subsequent in vivo administration is shown in Table 18. TABLE 18: Formulations for In Vitro Studies EXAMPLE 10 Preclinical Study 3: PK and PD Results after the Intravenous, Subcutaneous and Intranasal Administration of Insulin in Rabbits Table 19 shows the dose groups in Study 3. The following abbreviations were used: PDF = 45 mg / ml of Me-beta-CD, 1 mg / ml of DDPC, 1 mg / ml EDTA, 10 mM arginine pH 7.0 with added NaCl to achieve approximately 220 mPsm / kg; 2X PDF = 90 mg / ml of Me-beta-CD, 2 mg / ml of DDPC, 2 mg / ml of EDTA (other components remained the same as in PDF); the conservative (Pre), in this case was a combination of 10 mg / ml of propylene glycol, 0.33 mg / ml of methyl paraben and 0.17 mg / ml of propyl paraben. Polysorbate 80 (Tween) was added to various formulations at 1% or 2% (10 or 20 mg / ml) as indicated. Two SC groups were dosed, one with regular insulin in the absence of enhancers and one with regular insulin in the presence of PDF. TABLE 19: Description of the Dosed Groups in the Preclinical Study 3 The PD data for the groups dosed in the Preclinical Study 3 are shown in Table 20 and Figure 5. TABLE 20: PD Data for the Dosed Groups in the Preclinical Study 3 All the intranasal groups showed approximately the same effect of PD (Tm? N and% Cm? N). Regular insulin delivered subcutaneously in the absence and in the presence of PDF had a similar PD effect (and a slower Tm? n and higher% Cm? n as expected). The data showed that the onset (as indicated by the Tm? N) is faster for regular insulin in the PDF formulations (45 minutes for SC, 30 minutes for intranasal) compared to the control formulation (60 minutes for SC). The data show that regular insulin in intranasal PDF formulations is consistent with a rapid-acting insulin profile. The PK data for the groups dosed in the Preclinical Study 3 are shown in Figure 6 and Table 21, Table 22 and Table 23. TABLE 21: PK Parameters for the Dosed Groups in the Preclinical Study 3 TABLE 22: PK (biodisonibility) data for the Dosed Groups in the Preclinical Study 3 TABLE 23: CVC% for PK Parameters for Dosed Groups in Preclinical Study 3 The% CV for the various PK parameters was similar for the various groups. The% F (bioavailability relative to the SC control) for PDF with Tween formulations compared to the regular SC insulin control was approximately 2-6% and the Tmax was found in the range of 12-36 minutes. The IN formulation with the highest bioavailability% was IX PDF / 2% Tween without DDCP (5.8%). These PD data suggest that DDPC is not necessary in PDF formulation to achieve improved bioavailability. EXAMPLE 11 Preclinical Study 4: PK and PD Results after Oral and Intranasal Administration of Insulin in Rabbits Table 24 describes the dose groups in Study 4. The following abbreviations were used: PDF = 45 mg / ml Melanin beta-CD, 1 mg / ml DDPC, 1 mg / ml EDTA, 10 mM arginine pH 7.0 with added NaCl to achieve approximately 220 mOsm / kg; 2X PDF = 90 mg / ml of Me-beta-CD, 2 mg / ml of DDPC, 2 mg / ml of EDTA (other components remained the same as in PDF); TDM = 2.5 mg / ml tetradecylmaltoside. Polysorbate 80 (Tween) was added to several formulations at 1% (10 mg / ml) as indicated. Propylene glycol (PG) was added to various formulations at 1% or 2.5% (10 to 25 mg / ml). The 0.2% gelatin effect was tested in the IN formulations. Three oral groups were dosed, one with regular insulin in the absence of improvers (# 8), one with regular insulin in the presence of PDF (# 9) and one with regular insulin in the presence of PDF without DDCP (# 7). TABLE 24: Description of the Dosed Groups in the Preclinical Study 4 The PD data for the groups dosed in the Preclinical Study 4 are shown in Figure 7. The PD data were similar among all nasal formulations, but the SC dosage had an extended PD effect against the nasal. No PD effect was observed for the oral dose groups. Regular insulin delivered subcutaneously in the absence and presence of PDF had a similar PD effect (and a slower Tmin and higher% Cmin as expected). The data showed that the onset (as indicated by the Tm? N) is faster for regular insulin in the PDF formulations. The PK data for the groups dosed in Study 4 Preclinic are presented in Figure 8 and Table 25, Table 26 and Table 27.

TABLE 25: PK Parameters for Dosed Groups in Preclinical Study 4 TABLE 26: PK (bioavailability) data for Dosed Groups in Preclinical Study 4 TABLE 27: PK Parameters for Dosed Groups in Preclinical Study 4 For intranasal groups containing PDF with or without PG, as well as for groups containing TDM, PK data were similar, with% F (bioavailability compared to regular insulin SC) at approximately 5.4-10.6% and a Tma ? in the range of 18-59 minutes. In the case of IX PDF with 1% Tween in the presence of 0.2% gelatin, there was an increase in bioavailability, approximately 14.9%. For intranasal groups containing PDF with or without PG, as well as for groups containing TDM, the% CV for Cma? and AUC were between 50,200%. in contrast, for IX PDF with 1% Tween in the presence of 0.2% gelatin, there was a decrease in Cmax and AUC to 21.3% and 35.3%, respectively. It was noted that the% CV for Craax and AUC of the formulation of IX PDF with 1% of Tween in the presence of 0.2% of gelatin were lower than those observed for the SC injection. These data show that initiation (as indicated by Tmin) is faster for regular insulin in PDF formulations than in SC formulations, as a result insulin has the rapid-acting insulin profile. The addition of gelatin improves the effect of PD and PK (14.9% bioavailability relative to SC control) for PDF formulations. EXAMPLE 12 PK and PD Results for Formulations Containing Viscosity Enhancing Agents PK and PD were evaluated for rabbits dosed with intranasal insulin formulations containing different viscosity improving agents. The viscosity improvement agents included gelatin, HPMC, MC and Carbomer. Carbomer is a generic name for a polymer family known as Carbopol®. The time points were taken at 5, 10, 15, 30, 45, 60, 120 and 240 minutes. The glucose was measured at each time point with a glucometer (One-Touch Ultra). Small amounts of 2N HCl or NaOH were added to the formulation when necessary to achieve the desired pH. The insulin used in the study was found at a concentration of approximately 28 U / mg. Table 28 shows the formulations used in this study.

TABLE 28: Insulin Formulations Containing a Viscosity Improvement Agent Abbreviations: Me-ß-CD = methyl-beta-cyclodextrin, EDTA = disodium edetate, HPMC = hydroxypropyl methylcellulose (100 cps), MC = methylcellulose (15 cps), CMC = sodium carboxymethylcellulose (low viscosity), MP = methylparaben sodium, PP = propylparaben sodium, PG = propylene glycol, NaCl = sodium chloride. 15 ml of each formulation were manufactured and stored in clear 3 silanized glass vials. All tested insulin formulations were stored at 2-8 ° C. All formulations were dosed at 6.0 IU / kg. Table 29 describes the dosage groups used in this study.

TABLE 29: Agent Dosing Groups that Improve Viscosity The PD results for% glucose from the start are shown in Figure 9. Figure 9 shows the average change in% glucose over time for the 8 groups tested. Group 6 (IX PDF / 1% Tween / (0.25% Carbopol)) showed the greatest reduction in% glucose from the start compared to all other groups. The glucose depressions for the 8 groups were presented within 90 minutes as shown in Figure 9. Group 8 (which contained a tonicity agent) had the greatest reduction in% glucose from the start compared to the other gelatine formulations The formulations containing Carbomer (0.25% Carbopol) and CMC had the greatest reduction in% glucose from the start compared to the other non-gelatin formulations. The PK results for the average data per time point are shown in Figure 10. In Figure 10, the average insulin concentration (ulU / ml) over time is shown for the 8 groups tested. Figure 10 shows that Cmax was higher for Group 6, IX PDF / 1% Tween / (0.25% Carbopol) compared to the other formulations. Peak serum insulin levels for the 8 groups were presented within 60 minutes as shown in Figure 10. The PK parameters are summarized in Table 30. TABLE 30: Parameters in PK Rabbits of the Viscosity Enhancing Agent The results of% CV are shown in Table 31 TABLE 31: Results in Rabbits% CV of Agent that Improves Viscosity The results of% F (bioavailability) are shown in Table 32. TABLE 32: Results in Rabbits% F of the Viscosity Enhancing Agent Summary The PK and PD results show that the intranasal insulin formulations tested had rapid-acting insulin profiles, with peak serum insulin levels within 60 minutes and glucose depressions within 90 minutes. Bioavailability was increased when viscosity improvers were added to PDF intranasal insulin formulations. The increased tonicity increased the bioavailability in formulations containing gelatin. The formulation containing gelatin showed an improved performance with isotonic conditions (Group # 8, 0.2% gelatin including NaCl) compared to hypotonic conditions (Group # 2, 0.2% gelatin without NaCl). Formulations containing Carbomer and CMC showed the highest increase in PK and PD results for intranasal insulin formulations. Bioavailability as shown by% F was 19.7% and 17.8% for Carbomer and CMC, respectively. The effect of PD as shown by the% glucose from the start was improved with the addition of viscosity improving agents, such as Carbomer and CMC, to the intranasal insulin formulations. The PK and PD data in the rabbit confirm the in vitro results of an increase in insulin permeation through the nasal epithelium in the presence of formulation improvers. In vitro drug permeation data and rabbit PK data in vivo showed a substance correlation for intranasal insulin formulations. Using representative intranasal formulations, an XY trace analysis with AUC last (min * uU / ml) on the X axis and% permeation on the Y axis showed an R2 = 0.8994, y = 0.0007 x + 0.4191. EXAMPLE 13 AET Studies 1-8: Antimicrobial Effectiveness Test (AET) ETA Study 1 ETA Study 1 was conducted to determine the antimicrobial effectiveness (ETA) of a nasal insulin dextrose placebo with sodium methylparaben, sodium propylparaben, and propylene glycol. Additionally, Study 1 of AET examined the increased EDTA AET alone. The formulations evaluated in Study 1 of AET are shown in Table 33. Approximately 120 ml of each formulation were manufactured and tested in duplicate (n = 2 analyzes per sample). TABLE 33: Formulations Evaluated in the AET Study Abbreviations: Me-ß-CD = methyl-beta-cyclodextrin, DDPC = L to didecanoyl phosphatidylcholine, EDTA = sodium edetate, MP = methylparaben sodium, PP = propylparaben sodium, PG = propylene glycol, NaCl = sodium chloride. The TEA methods used were in compliance with the requirements for the TEA of U.S. Pharmacopeial (USP) and the European Pharmacopeial (EP) and are described in Tables 34 and 35, respectively. The formulations were also tested for pH (by SOP 403), appearance (visual) and osmolarity (by SOP 4000). TABLE 34: USP AET Requirements (USP < 51 >) TABLE 35: EP AET Requirements (EP < 5.1.3 >) The combination of 0.33 mg / ml sodium methylparaben, 0.17 mg / ml sodium propylparaben, and at least 25 mg / ml propylene glycol was an effective preservative combination and meets USP standards. These formulations passed all the requirements of the USP, but failed for EP (par S. aureus and A. niger). The increased EDTA alone did not appear to be effective for the requirements of the USP and the EP. AET Study 2 Study 2 of AET was conducted to determine if a humectant (propylene glycol) improves antimicrobial effectiveness when sodium methylparaben and sodium propylparaben were used as preservatives. Other preservatives were also evaluated, such as benzalkonium chloride (BAK), benzyl alcohol and sodium benzoate. Two groups of insulin were tested at the levels of 500 U / ml or 1000 U / ml. The formulations evaluated in Study 2 of AET are listed in Table 36.

TABLE 36 Formulations Evaluated in Study 2 of AET Abbreviations: Me-ß-CD = methyl ß cyclodextrin DDPC = L to didecanoyl phosphatidylcholine EDTA = Disodium of edetate MP = sodium of methylparaben PP = sodium of propilparabneo BAK = chloride of benzalconio NaCl = chloride of sodium The methods for Study 2 of AET were conducted as described for Study 1 of AET. Additionally, a positive control (PBS with 5 mg / ml sodium benzoate) and a negative control (PBS alone) were included. The results of Study 2 of AET showed that sodium methylparaben and sodium propylparaben were not effective preservatives without humectant (such as propylene glycol) included in the formulation. Benzalkonium chloride was an excellent preservative with respect to the antimicrobial effectiveness test of USP and PD, but it was found to be incompatible with insulin (i.e., its presence within the formulation caused precipitation of insulin). Benzyl alcohol and sodium benzoate were also not effective preservatives at a neutral pH and consequently they were not suitable for use within the nasal spray formulations of insulin. Study 3 of AET The purpose of Study 3 of AET was to evaluate other preservatives, such as benzaconium chloride (BAK), benzyl alcohol, and sodium benzoate. Two groups were tested with insulin at the 500 U / ml or 1000 U / ml levels. The formulations for Study 3 of AET are listed in Table 37. TABLE 37: Formulations Evaluated in Study 3 of AET Abbreviations: Me-ß-CD = methyl ß cyclodextrin DDPC = L to didecanoyl phosphatidylcholine EDTA = Disodium of edetate MP = sodium of methylparaben PP = sodium of propilparabneo BAK = chloride of benzalconio NaCl = sodium chloride The analysis in Study 3 of AET was conducted as described for Study 1 of AET. The results of Study 3 of AET showed that banzaconium chloride was incompatible with insulin (caused precipitation), but maintained the best antimicrobial performance. Benzyl alcohol and benzyl alcohol / sodium methylparaben / sodium propylparaben were effective as antimicrobial reagents in this study. AET Study 4 The purpose of ETA Study 4 was to determine if satisfactory results of USP ETA and EP could be achieved with lower levels of humectant (propylene glycol) when used with sodium methylparaben and sodium propylparaben. Additionally, the alternative preservatives m-cresol and benzyl alcohol were evaluated. The formulations of Study 4 of AET are listed in Table 38.

TABLE 38: Formulations Evaluated in Study 5 of AET Abbreviations: Me-ß-CD = methyl ß cyclodextrin EDTA = Disodium of edetate MP = sodium of methylparaben PP = sodium of propilparabneo PG = propilenglicol The methods used for Study 4 of AET were conducted as described for Study 1 of AET. Formulations containing sodium methylparaben and sodium propylparaben with a low concentration of humectant (i.e., propylene glycol) do not achieve antimicrobial effectiveness. The optimal level for sodium methylparaben and sodium propylparaben with a propylene glycol was found between 1 and 25 mg / ml of propylene glycol. Additionally, benzyl alcohol and m-cresol were not effective preservatives for insulin nasal spray formulations. AET Study 5 The purpose of Study 5 of AET was to conduct the antimicrobial effectiveness test (ETA) of insulin spray formulations (placebo and active) to determine if both sodium methylparaben and sodium propylparaben are required for optimal preservative effectiveness and if one is more effective than the other. Additionally, the increased levels of sodium methylparaben and sodium propylparaben were evaluated to determine if the increase of their content within the formulation increased the antimicrobial effectiveness. The formulations used in Study 5 of AET are listed in Table 39. TABLE 39: Formulations Evaluated in Study 5 of AET Abbreviations: Me-ß-CD = methyl ß cyclodextrin DDPC = L to didecanoyl phosphatidylcholine EDTA = Disodium of edetate MP = sodium of methylparaben PP = sodium of propilparabneo BAK = chloride of benzalconio NaCl = sodium chloride The methods used in Study 5 of AET were conducted as described for Study 1 of AET. The results of Study 5 of AET showed that the increase in sodium methylparaben and sodium propylparaben levels to at least ten times of 0.33 mg / ml sodium methylparaben and 0.17 mg / ml sodium propylparaben increased the antimicrobial effectiveness. Additionally, it was evident that 0.33 mg / ml sodium methylparaben alone had the same antimicrobial effectiveness as 0.17 mg / ml sodium propylparaben alone, which also had the same antimicrobial effectiveness of the combination. AET Study 6 The purpose of Study 6 of AET was to conduct the antimicrobial effectiveness test (ETA) of insulin spray formulations (placebo) to determine the optimal level of propylene glycol needed for its use with sodium methylparaben and sodium propylparaben. In addition, the increased levels of sodium methylparaben and sodium propylparaben with a static propylene glycol level were evaluated to determine if the increase of their content within the formulation increased the antimicrobial effectiveness. Finally, ethanol was also evaluated as a potential conservative. The formulations evaluated in Study 6 of AET are listed in Table 40.

TABLE 40: Formulations Evaluated in a Study 6 of AET Abbreviations: Me-ß-CD = methyl ß cyclodextrin DDPC = L to didecanoyl phosphatidylcholine EDTA = Disodium of edetate MP = sodium of methylparaben PP = sodium of propilparabneo BAK = chloride of benzalconio NaCl = sodium chloride The methods used in Study 6 of AET were conducted as described for Study 1 of AET. The results of Study 6 of AET show that the optimum level of propylene glycol was 10 mg / ml. The AET results of the nasal spray formulations of insulin with 10, 15, 20 and 25 mg / ml of propylene glycol were very similar; but the TEA results were less successful when the propylene glycol level was less than 10 mg / ml. All formulations passed the requirements of the USP AET except for the requirement of P. aeruginosa. With respect to this category, the formulations were bacteriostatic (i.e., there was no indication of microbial growth). All the formulations failed to the requirements of the EP for the most previous time points for each required body. Ethanol alone (at 1% or 2%) appeared to have an antimicrobial activity similar to that of sodium methylparaben / sodium propylparaben / propylene glycol. AET Study 7 The purpose of ETA Study 7 was to conduct the antimicrobial effectiveness test (ETA) of the insulin spray formulations (placebo) containing 20 mg / ml of Tween 80. An in vivo pharmacokinetic study showed that the Increased content of Tween 80 at 20 mg / ml can help increase bioavailability, but it is also known that Tween 80 micelles interact with conservatives (specifically with parabens). Additionally, Study 7 of AET was conducted to determine the optimal level of propylene glycol needed for its use with sodium methylparaben and sodium propylparaben. Increased levels of sodium methylparaben and sodium propylparaben were evaluated with a static propylene glycol level to determine whether increasing their content within the formulation increases antimicrobial effectiveness. Finally, ethanol was also evaluated as a potential conservative. The formulations evaluated in Study 7 of AET are listed in Table 41. TABLE 41: Formulations Evaluated in Study 7 of AET Abbreviations: Me-ß-CD = methyl ß cyclodextrin DDPC = L to didecanoyl phosphatidylcholine EDTA = Disodium of edetate MP = sodium of methylparaben PP = sodium of propilparabneo BAK = chloride of benzalconio NaCl = sodium chloride The methods for Study 7 of AET were conducted as described for Study 1 of AET. The results of Study 7 of AET showed that the increase in Tween 80 content from 10 mg / ml to 20 mg / ml reduced the antimicrobial activity, even when the highest level of propylene glycol (i.e., 25 mg / ml) was added. Ethanol was not an effective conservative (at 1%) when used in combination with 20 mg / ml of Tween 80. Study 8 of AET Study 8 of AET was conducted to test the antimicrobial effectiveness test (AET) of spray formulations. of insulin (active) with formulations containing propylene glycol levels at 1, 5, 10 and 25 mg / ml. Additionally, the three concentrations of nasal spray of insulin were tested: 250, 500 and 1000 U / ml. The formulations evaluated in Study 8 of AET are listed in Table 42. TABLE 42 Formulations Evaluated in Study 8 of AET Abbreviations: Me-ß-CD = methyl ß cyclodextrin DDPC = L to didecanoyl phosphatidylcholine EDTA = Disodium of edetate MP = sodium of methylparaben PP = sodium of propilparabneo BAK = chloride of benzalconio NaCl = chloride He sodium The methods used for the analysis are described for Study 1 of AET. The results of Study 8 of AET showed that the addition of insulin to the formulations improved the performance of AET. Additionally, when the propylene glycol level was high (i.e., 25 mg / ml or 10 mg / ml), the formulations containing insulin with sodium methylparaben and sodium propylparaben passed the requirements of the USP AET. However, the EP requirements were not met. Summary of Studies 1 to 8 of AET Data from Studies 1-8 showed that with respect to TEA the combination of sodium methylparaben, sodium propylparaben and propylene glycol humectant resulted in an increased preservative effectiveness compared to sodium methylparaben and the sodium propylparaben alone. Additionally, several other preservatives were evaluated over the course of these studies, such as benzalkonium chloride, sodium benzoate, benzyl alcohol, ethanol, increased EDTA, benzethonium chloride and meta-cresol; however, the best results were achieved with sodium methylparaben / sodium propylparaben / propylene glycol. Each of the other preservatives was either incompatible with insulin (as in the case of benzalkonium chloride, which caused the precipitation of insulin) or they probably became ineffective due to interactions with methyl-beta-cyclodextrin and / or polysorbate 80. Although sodium methylparaben and sodium propylparaben also interacted with methyl-beta-cyclodextrin and / or polysorbate 80, the addition of the humectant, propylene glycol, interferes with their interaction, thus making parabens more available for antimicrobial activity. The results showed that 10 mg / ml of humectant added to the formulations of 0.17 mg / ml of sodium propylparaben, 0.33 mg / ml of sodium methylparaben, produced good antimicrobial activity. One aspect of the invention herein consists of a combination of preservative for use within the nasal spray formulations of insulin which provides a bacteriostatic effect when treated for the antimicrobial effectiveness test (ETA) of the U.S. Pharmacopeial and European Pharmacopeial. The formulation with the best performance of AET contained water, solubilizer (s), surfactant agent (s), buffer, chelator, toner, and preservative. The preferred solubilizer was Me-beta-CD. The preferred surfactants were a combination of DDPC and a polysorbate (such as Tween 80) or polysorbate alone. The preferred chelator was EDTA. The preferred tonifier was sodium chloride. The preferred preservatives were sodium methylparaben and sodium propylparaben. The formulation also contained a humectant such as propylene glycol, which provided an excellent AET performance. EXAMPLE 14 Stability of the Insulin Formulation Stability was tested for intranasal insulin formulations at 5 ° C / ambient humidity (routine storage), 25 ° C / 60% RH (accelerated storage), accelerated storage with agitation, and routine storage or accelerated in combination with aerosolization three times daily (TID) (to mimic the use of the patient). After three months (84 days) of storage, the HPLC results showed no significant change in the insulin content at 5 ° C / ambient humidity (99.2% insulin recovery) and a minor loss of insulin content was observed at 25 ° C / 60% RH (96.3% insulin recovery). There was no significant insulin loss to the TID aerosolization for short incubation times (11 days) with the formulations containing 250 U / ml, 500 U / ml or 1000 U / ml. No significant decrease in stability was observed after agitation at 100 rpm for 24 hours at accelerated temperature, in contrast, a commercial product of insulin showed a reduction of at least 20% of the insulin content under the same conditions. EXAMPLE 15 Human PD Clinical Study A human study was completed to measure the pharmacodynamic (PD) data after nasal administration of the insulin formulations with enhancers compared to the administration of currently commercially available glucose regulator pharmacists, NovoLog and Exubera . A glucometer was used to measure glucose levels. A summary of the percentage reduction in glucose for each treatment group is shown in Table 43. The incidence of 30%, 20% and 10% in the reduction in the percentage of glucose for each treatment group is shown in Table 44 TABLE 43 Reduction of Glucose Percentage by Treatment Group TABLE 44 sa The results of this initial study of PD show that intranasal administration of insulin is effective in reducing the percentage of glucose in a patient. Nasal administration of 50 IU and 100 IU resulted in a glucose reduction similar to Exubera, a glucose marketer currently marketed. Although the above invention has been described in detail by way of example for purposes of clarity and understanding, it will be apparent to the skilled person that certain changes and modifications to the description are understood and may be practiced without undue experimentation within the scope of the appended claims, which are presented by way of illustration and not of limitation.

Claims (52)

1. CLAIMS 1. A pharmaceutical formulation for the intranasal delivery of insulin to a patient, comprising an aqueous mixture of a monomeric insulin, a solubilizing agent, and a surfactant.
2. 2. The formulation of claim 1, wherein the insulin is a human insulin.
3. 3. The formulation of claim 1, wherein the insulin is a human insulin of rapid action.
4. The formulation of claim 1, wherein the insulin is selected from the group consisting of natural human insulin, human insulin (LysB3, GluB29), human insulin (LysB3, IleB28), human insulin (GlyA21, HisB31, HisB32), human insulin (AspB28), human insulin (AspBlO), human insulin (LysB28, ProB29) and mixtures thereof.
5. The formulation of claim 4, wherein the insulin is a human insulin (AspB28).
6. The formulation of claim 1, wherein the solubilizing agent is selected from the group consisting of a cyclodextrin, hydroxypropyl-beta-cyclodextrin, sulfobutylether-beta-cyclodextrin, methyl-beta-cyclodextrin and mixtures thereof.
7. The formulation of claim 6, wherein the solubilizing agent is methyl-beta-cyclodextrin.
8. 8. The formulation of claim 1, wherein the surfactant is selected from the group consisting of nonionic polyoxyethylene ether, fusidic acid and its derivatives, sodium taurodihydrofusidate, L-alpha-phosphatidylcholine didecanoyl, polysorbate 80, polysorbate 20, polyethylene glycol, cetyl alcohol, polyvinyl pyrrolidone, polyvinyl alcohol, lanolin alcohol, sorbitan monooleate, and mixtures thereof.
9. The formulation of claim 8, wherein the surfactant is L-alpha-phosphatidylcholine didecanoyl.
10. The formulation of claim 8, wherein the surfactant is polysorbate 80.
11. 11. The formulation of claim 1, further comprising a chelating agent selected from the group consisting of ethylenediamine tetraacetic acid, ethylene glycol tetraacetic acid, and mixtures thereof. thereof.
12. The formulation of claim 1, further comprising one or more polyols.
13. The formulation of claim 12, wherein the polyol is selected from the group consisting of sucrose, mannitol, sorbitol, lactose, L-arabinose, D-erythrose, D-ribose, D-xylose, D-mannose, trehalose, D-galactose, lactulose, cellobiose, gentibiose, glycerin, polyethylene glycol and mixtures thereof.
14. 14. The formulation of claim 12, wherein the polyols are lactose and sorbitol.
15. 15. The formulation of claim 1, further comprising a preservative.
16. 16. The formulation of claim 15, wherein the preservative is selected from the group consisting of chlorobutanol, methyl paraben, propyl paraben, butyl paraben, benzalkonium chloride, benzethonium chloride, sodium benzoate, sorbic acid, phenol, ortho- cresol, metacresol, para-cresol and mixtures thereof.
17. The formulation of claim 15, wherein the preservative is methyl paraben and propyl paraben.
18. 18. The formulation of claim 1, further comprising an aerosol of droplets having diameters of l to 700 microns in size.
19. The formulation of claim 17, further comprising a humectant.
20. The formulation of claim 19, wherein the humectant is selected from the group consisting of propylene glycol, glycerin, glyceryl triacetate, a polyol, a polymeric polyol, lactic acid, urea and mixtures thereof.
21. The formulation of claim 20, wherein the humectant is propylene glycol.
22. 22. The formulation of claim 1, further comprising a buffer.
23. The formulation of claim 22, wherein the buffer is selected from the group consisting of glutamate, acetate, glycine, histidine, arginine, lysine, methionine, lactate, formate, glycolate, and mixtures thereof.
24. The formulation of claim 23, wherein the buffer is arginine.
25. 25. The formulation of claim 22, wherein the buffer has a pKa of 5 to 9.
26. The formulation of claim 22, wherein the buffer has a pKa of 6 to 8.
27. 27. The formulation of claim 1, further comprising an enhancer agent. viscosity.
28. The formulation of claim 27, wherein the viscosity improving agent is selected from the group consisting of gelatin, hydroxypropyl methylcellulose, methylcellulose, carbomer, carboxymethylcellulose and mixtures thereof.
29. 29. The formulation of claim 28, wherein the viscosity improving agent is carbomer.
30. 30. The formulation of claim 28, wherein the viscosity enhancing agent is carboxymethylcellulose.
31. The formulation of claim 28, wherein the viscosity improving agent is gelatin.
32. 32. The formulation of claim 1, which has a pH of 7.0 + 0.5.
33. 33. The formulation of claim 1, further comprising a toner.
34. 34. The formulation of claim 1, having an osmolarity of from 50 to 350 mOsm / 1.
35. 35. The formulation of claim 1, characterized by a bioavailability greater than about 15%.
36. 36. A pharmaceutical formulation comprising an aqueous solution of a human insulin, methyl-beta-cyclodextrin, L-alpha-phosphatidylcholine didecanoyl, disodium edetate, polysorbate 80, arginine buffer and carbomer.
37. 37. The formulation of claim 36, wherein the human insulin is a human insulin of rapid action.
38. 38. The formulation of claim 36, wherein the human insulin is human insulin (AspB28).
39. 39. The use of the formulation of any of the preceding claims in the preparation of a medicament for treating the signs and symptoms of a disease or condition in a human, including diabetes mellitus, hyperglycemia, dyslipidemia, induction of satiety in an individual, promotion of weight loss in an individual, obesity, cancer, colon cancer and prostate cancer.
40. 40. The use of a pharmaceutical formulation comprising an aqueous mixture of a monomeric insulin, a solubilizing agent, and a surfactant in the manufacture of a medicament for treating the signs and symptoms of a disease or condition in a human, including diabetes mellitus. , hyperglycemia, dyslipidemia, induction of satiety in an individual, promotion of weight loss in an individual, obesity, cancer, colon cancer and prostate cancer.
41. 41. The use of claim 40, wherein the insulin is selected from the group consisting of natural human insulin, human insulin (LysB3, GluB29), human insulin (LysB3, IleB28), human insulin (GlyA21, HisB31, HisB32), human insulin (AspB28), human insulin (AspBlO), human insulin (LysB28, ProB29) and mixtures thereof.
42. 42. The use of claim 40, wherein the insulin is a human insulin (AspB28).
43. 43. The use of claim 40, wherein the disease is diabetes mellitus.
44. 44. The use of claim 40, wherein the disease is diabetes mellitus and the medicament is administered as an aerosol of drops having diameters of from 1 to 700 microns in size.
45. 45. The use of claim 40, wherein the solubilizing agent is methyl-beta-cyclodextrin and the surfactant is L-alpha-phosphatidylcholine didecanoyl.
46. 46. The use of claim 40, wherein the pharmaceutical formulation further includes a viscosity improving agent, a preservative, a buffer and a toner.
47. 47. The use of claim 40, wherein the medicament elevates the level of insulin in blood in a human for at least about 6 hours after administration.
48. 48. The use of claim 40, wherein the medicament reduces the percentage of glucose in a human by more than about 10%.
49. 49. The use of claim 40, wherein the medicament is administered as an aerosol of droplets having diameters of from 1 to 700 microns in size.
50. 50. The use of a pharmaceutical composition comprising an aqueous solution of a human insulin, methyl-beta-cyclodextrin, L-alpha-phosphatidylcholine didecanoyl, disodium edetate and polysorbate 80 in the preparation of a medicament for treating diabetes mellitus or hyperglycemia in a human.
51. 51. The use of claim 50, wherein human insulin is a human insulin of rapid action.
52. 52. The use of claim 50, wherein the human insulin is human insulin (AspB28).