JP2009518315A - Pharmaceutical formulations for increasing epithelial permeability of glucose-regulating peptides - Google Patents

Abstract

Disclosed is a pharmaceutical formulation comprising a pharmaceutically effective amount of a glucose-regulating peptide (GRP) and a mixture of promoters for use in the treatment of metabolic syndrome.
[Selection] Figure 1

Description

  Glucose-regulating peptides ("GRP") have therapeutic potential in the treatment of insulin-dependent diabetes (IDDM), gestational diabetes, or non-insulin-dependent diabetes (NIDDM), the treatment of obesity and the treatment of dyslipidemia The type of peptide. See U.S. Patent No. 6,506,724, U.S. Patent Application Publication No. 20030036504A1, European Patent No. 1083924B1, WO 98 / 30231A1, and WO 00 / 73331A2. These GRPs include, for example, glucagon-like peptides (GLP) such as GLP-I, exendins also known as exenatide, especially exendin-4, and amylin analogs such as amylin peptides and pramlintide. To date, however, these peptides have been administered to humans only by injection.

  The main disadvantage of drug administration by injection is that in many cases, trained personnel are required to administer the drug. In addition, trained personnel are at risk when administering drugs by injection. For self-administered drugs, many patients do not want or can not inject themselves regularly. Injection is also associated with an increased risk of infection. Other disadvantages of drug injection include variability in delivery results among individuals, as well as unpredictable strength and duration of drug efficacy.

  Oral administration can be used as an alternative. However, certain therapeutic agents, when administered by this route, exhibit very low bioavailability and significant time delay during action due to hepatic first pass metabolism and degradation in the gastrointestinal tract. Thus, it is necessary to develop GRP administration methods other than injection and / or oral administration.

  Mucosal administration of therapeutic compounds provides certain advantages over injection and other methods of administration, for example, in terms of convenience and delivery rate, as well as reducing or eliminating compliance issues and side effects associated with delivery. However, mucosal delivery of bioactive substances is limited by mucosal barrier function and other factors. Epithelial cells constitute the mucosal barrier and provide an important interface between the external environment and the mucosal and submucosa and extracellular compartments. One of the most important functions of mucosal epithelial cells is to determine and control mucosal permeability. In this situation, epithelial cells create a selective permeability barrier between different physiological compartments. Selective permeability is the result of controlling the transport of molecules through the cytoplasm (transcellular pathway) and the permeability of the intercellular space (paracellular pathway).

  It is known that cell-cell junctions between epithelial cells are involved in both the maintenance and control of epithelial barrier function and cell-cell adhesion. Tight junctions (TJ) of epithelial and endothelial cells are particularly important for cell-cell junctions that control the permeability of paracellular pathways and also divide the cell surface into apical and basolateral compartments. Tight junctions form a continuous circular cell-cell contact between epithelial cells and form a regulatory barrier to paracellular movement of water, solutes and immune cells. They also provide a second type of barrier that contributes to cell polarity by limiting membrane lipid exchange between the apical and basolateral domains of the membrane.

  In the context of drug delivery, the ability of a drug to penetrate the epithelial cell layer of the mucosal surface without the aid of a delivery enhancer appears to be related to many factors including molecular size, lipid solubility and ionization. In general, small molecules of less than about 300 to 1000 daltons can often penetrate the mucosal barrier, but permeability decreases rapidly as the molecules become larger. Transdermal drug delivery allows large molecules to penetrate through the epithelial cell layer of the skin. Transdermal administration, such as skin patches, is another alternative delivery route for larger polymeric drugs. However, transdermal delivery may still be more limited in size than injection. For these reasons, mucosal and epithelial drug administration usually requires a larger amount of drug than administration by injection. Other therapeutic compounds, including large molecule drugs, are often resistant to mucosal delivery. In addition to the inherent low permeability, large macromolecular drugs are susceptible to limited diffusion and enzymatic degradation by lumens and cells, and rapid clearance at the mucosa. Thus, in order to deliver therapeutically effective amounts of these large molecules, cell permeation enhancers are required, passing across these mucosa and skin surfaces, and systemic circulation that can act quickly on the target tissue Assist in passing to.

Reduction of blood glucose concentration (corrected for endogenous glucose) after administration of GLP-I in rats compared to exenatide (SQ) and saline control (IN) Insulin response after nasal administration of GLP-I in rats Gastric emptying after nasal administration of GLP-I in rats Exendin-4 enhanced drug administered 2 × accelerator in IN, 2 × accelerator + gelatin in IN, and 1 × accelerator + gelatin in IN compared to IN control and IV in rabbits Dynamics

DETAILED DESCRIPTION OF THE INVENTION One aspect of the present invention includes GRP, GRP analogs, fragments of GRP, and functional GRP across the epithelial surface for the treatment of human diseases, including obesity and diabetes. It includes the therapeutic utility of pharmaceutical formulations for delivering derivatives.

  The present invention promotes an individual's satiety center to treat insulin-dependent diabetes (IDDM), gestational diabetes or non-insulin-dependent diabetes (NIDDM), dyslipidemia, hyperglycemia, obesity, GLP and GLP analogs that promote weight loss, amylin or amylin analogs, and novel and effective methods for transepithelial, particularly transmucosal, delivery of GRP such as exendin or exendin analogs, By using and providing the composition, the above-mentioned demand is satisfied, and further additional objects and benefits are satisfied.

  In an exemplary embodiment, the enhanced delivery methods and compositions of the present invention are therapeutically effective to cross a layer of biological cells with a GRP agonist for the prevention or treatment of obesity and eating disorders in a subject mammal. Provide a convenient delivery method. In one aspect of the present invention, there is provided a pharmaceutical formulation suitable for transepithelial administration, comprising a therapeutically effective amount of GRP and one or more transepithelial delivery accelerators described herein, the formulation Is useful in the epithelial delivery method of the present invention for preventing the onset or progression of obesity or eating disorders in a subject mammal. Transepithelial delivery of a therapeutically effective amount of a GRP agonist and one or more epithelial delivery enhancers produces high therapeutic concentrations of the GRP agonist within the subject.

  The enhanced delivery methods and compositions of the present invention provide therapeutically effective delivery of GRP for the prevention or treatment of various diseases and conditions in a subject mammal. GRP can be administered via various epithelial routes, for example, GRP is intranasal mucosal epithelium, bronchial or pulmonary mucosal epithelium, buccal surfaces in the oral cavity, mucosal surfaces of the oral and small intestines, or epidermis It can be administered by contacting the surface. In exemplary embodiments, the methods and compositions are intended for or formulated for intranasal delivery (eg, nasal mucosal delivery or intranasal mucosal delivery).

  The aforementioned GRP formulations, preparation methods and delivery methods of the present invention improve epithelial delivery of GRP to the subject mammal. These compositions and methods may include co-administration of a combination formulation or one or more GRPs with one or more epithelial delivery enhancers. Epithelial delivery enhancers to be selected to achieve the above formulations and methods include (A) lytic agents; (B) charge modifiers; (C) pH regulators; (D) degrading enzyme inhibitors; E) mucolytic or mucosal removing agent; (F) cilia static agent; (G) membrane permeation enhancer (eg (i) surfactant, (ii) bile salt, (iii) phospholipid) Or additives that are fatty acids, mixed micelles, liposomes or carriers, (iv) alcohol, (v) enamines, (iv) NO donor compounds, (vii) long-chain amphiphilic molecules, (viii) small molecule hydrophobic permeation enhancement (Ix) sodium or salicylic acid derivative; (x) glycerol ester of acetoacetic acid, (xi) cyclodextrin or β-cyclodextrin derivative, (xii) medium chain fatty acid, (Xiii) a chelating agent, (xiv) an amino acid or a salt thereof, (xv) an N-acetylamino acid or a salt thereof, (xvi) an enzymatic degradation agent for a selected membrane component, (xvii) an inhibitor of fatty acid synthesis, (xviii) Inhibitors of cholesterol synthesis; or (xiv) (i)-(xviii) any combination of membrane permeation enhancers); (H) nitric oxide (NO) stimulators, epithelia such as chitosan and chitosan derivatives (I) a vasodilator; (J) a selective transport enhancer; and (K) a stabilized delivery vehicle, carrier, support for stabilizing active agents for accelerated epithelial delivery Or complex-forming species in which GRP or GRP is effectively formulated, conjugated, entrapped, encapsulated or bound )

  In various embodiments of the present invention, GRP is combined with 1, 2, 3, 4 or 5 or more of the epithelial delivery promoters listed in (A)-(K) above. These epithelial delivery-enhancing agents are mixed with GRP alone or together with others, or are combined with them in a pharmaceutically acceptable formulation or delivery vehicle. Formulating GRP with one or more epithelial delivery enhancers according to the teachings herein (optionally of two or more epithelial delivery enhancers selected from (A)-(K) above) Any combination may be provided to provide increased bioavailability of the glucose-regulating binding peptide after delivery of these formulations to the epithelial surface of the subject mammal. In addition to adding an epithelial delivery enhancer to the formulation, modification of GRP, such as by addition of hydrophobic groups, can also be used to affect peptide bioavailability.

  Accordingly, the present invention relates to a method for treating obesity and / or diabetes, comprising transepithelially administering a preparation containing GRP, suppressing appetite, promoting weight loss, reducing food intake in a mammal It is.

  The invention further treats hyperglycemia, diabetes, metabolic syndrome, dyslipidemia, suppresses appetite, promotes weight loss, reduces food intake, or treats obesity in mammals There is provided the use of GRP in the manufacture of a medicament for transepithelial administration of GRP.

  For the pharmaceutical formulations of the present invention, effective doses of GRP in the mucosa are, for example, from about 0.001 pmol to about 100 pmol per kg body weight, from about 0.01 pmol to about 10 pmol per kg body weight, or 1 body weight. about 0.1 pmol to about 5 pmol per kg. In a further exemplary embodiment, the dosage of GRP is from about 0.5 pmol to about 1.0 pmol per kg body weight. In preferred embodiments, the dose for intranasal administration is in the range of 0.1-100 μg / kg, or about 7-7000 μg, more preferably 0.5-30 μg / kg, or 35-2100 μg. Will change. More specific doses of GRP for intranasal administration include 20 μg, 50 μg, 100 μg, 150 μg, 200 μg to 400 μg, 500 μg, 800 to 1000 μg and 1200 to 1800 μg. The pharmaceutical formulations of the present invention can be administered one or more times a day, or three times a week or once a week for one week to at least 96 weeks, or for the life of an individual patient or subject. . In certain embodiments, the pharmaceutical formulations of the invention are administered one or more times daily, twice daily, four times daily, six times daily, or eight times daily.

  Epithelial delivery enhancers that promote GRP delivery into or between cell layers including the nasal mucosal surface are used. For passively absorbed drugs, the relative contribution of paracellular and transcellular pathways to drug transport is the pKa, partition coefficient, molecular radius and charge of the drug, the pH of the luminal environment to which the drug is delivered, As well as the absorption area. The epithelial delivery accelerator of the present invention may be a pH adjuster. The pH of the pharmaceutical formulation of the present invention is a factor that affects GRP absorption through paracellular and transcellular pathways to drug transport. In one embodiment, the pharmaceutical formulation of the invention is adjusted to a pH of about pH 2-8. In a further aspect, the pharmaceutical formulation of the present invention is adjusted to a pH of about pH 3.0 to 6.0. In a further aspect, the pharmaceutical formulation of the present invention is adjusted to a pH of about pH 3.5 to 5.5. In general, the pH is 4.5 ± 0.5.

  As mentioned above, the present invention provides improved methods and compositions for epithelial delivery of GRP to a subject mammal for the treatment or prevention of various diseases and conditions. Examples of suitable subject mammals for performing treatment and prevention according to the methods of the present invention include, but are not limited to, livestock species such as humans and non-human primates, horses, cows, sheep, and goats, and Included are research and pet species including dogs, cats, mice, rats, guinea pigs, and rabbits.

  In order to better understand and understand the present invention, the following definitions are presented:

Glucagon-like peptide (GLP)
The present invention includes analogs, fragments, mimetics, and functional derivatives of GLP proteins and peptides. Incretin is an intestinal hormone that stimulates insulin secretion in response to nutrient intake (in a glucose-dependent manner). Two types of natural incretins include glucose-dependent insulinotropic peptide (GP) and glucagon-like peptide-1 (GLP-1). GLP-1 is released from cells in the intestine in response to food. GLP-1 binds to the GLP-1 receptor on pancreatic β cells and stimulates the release of insulin. GLP-1 [7-36] NH ₂ , also known as proglucagon [78-107], most commonly known as “GLP-1”, exhibits insulin secretion promoting action and promotes insulin secretion; , GLP-1 inhibits glucagon secretion [Orskov, et al, Diabetes 42: 658-61, 1993; D'Alessio, et al., J. Clin. Invest. P7: 133-38, 1996]. GLP-1 is also found in gastric emptying [Williams B., et al., J. Clin. Endocrinol Metab. 81 :(?): 327-32, 1996; Wettergren A., et al., Dig. ScL. 3S: (4): 665-73, 1993] and have been reported to inhibit gastric acid secretion. [Schjordager BT, et al., Dig. Dis. ScL 34 (5): 703-8, 1989; O'Halloran D.J., et al., J. Endocrinol 126 (1): 169-73. , 1990; Wettergren A., et al., Dig. Dis. ScL 3S: (4): 665-73, 1993]. GLP-1 [7-37], which has an additional glycine residue at its carboxy terminus, also promotes insulin secretion in humans [Orskov, et al., Diabetes 42: 658-61, 1993]. It has been reported that a transmembrane G protein adenylate cyclase-binding protein, which is considered to be responsive to the insulin secretion promoting action of GLP-1, has been cloned from the cell line of β cells [Thorens, Proc. Natl. Acad. ScL USA 59: 8641-45, 1992].

  A major limitation to the therapeutic use of GLP-1 is its rapid degradation by the widely distributed enzyme dipeptidyl peptidase-IV (DPP-IV). The use of a DPP-IV inhibitor (LAF237; MK-0431) improves the activity time of endogenous GLP-1. The US Food and Drug Administration (FDA) has approved JANUVIA ™ (sitagliptin phosphate) from Merck & Co., an oral DPP-IV inhibitor, and type 2 diabetes in the United States. It has become available for treatment. US Patent Application No. 20060234940 includes a compound capable of binding to a secondary binding site of DPP-IV and a DPP-IV-like enzyme and at least one anti-diabetic agent in a mammal. Methods for treating metabolic diseases are described.

  Incretin mimetics are a class of drugs that mimic the anti-diabetic or glucose-lowering effects of natural human incretin hormones such as GLP-1. Incretin mimetics include stimulation of the body's ability to produce insulin in response to increased blood sugar levels, inhibition of glucagon hormone release, delayed nutrient absorption into the bloodstream, and decreased gastric emptying rate This includes promoting satiety centers and reducing food intake. Incretin mimetics were developed for use in the treatment of type 2 diabetes and include the following: GLP-1 derivatives (Liraglutide and CJC-1131) and exenatide. CJC-1131 (ConjuChem, Montreal, Canada) has a reactive linker that allows for covalent attachment to serum albumin, which increases resistance to DPP-IV degradation. Liraglutide (Novo Nordisk, Copenhagen, Denmark) is a GLP-1 derivative and is designed to overcome the degradation effect of DPP-IV by acylation with a fatty acid chain. The structure of liraglutide is shown in WHO Drug information, Vol. 17, No. 2 (2003).

The present invention also includes modification of GRP by adding hydrophobic groups such as fatty acids to the peptide. Further examples of modified derivatives of GRP having desirable pharmacokinetic properties are described in Knudsen et al., J. Med. Chem. 43: 1664-1669, 2000, which is hereby incorporated by reference. Incorporated. These GLP-1 compounds were derivatized with fatty acids to prolong their action by promoting binding to serum albumin. The following parent peptide and acyl-substituted, are ^{described: K 8 R 26,34 -GLP-1} (7-37) (K 8: γ-Glu-C16); K 18 R 26 '34 -GLP-1 ^{(7-37) (K 18: γ} -Glu-C16); K 23 R 26,34 -GLP-1 (7-37) (K 23: γ-Glu-C16); R 34 -GLP-1 (7 ^{-37) (K 26: γ-} Glu-C16); K 27 R 26,34 -GLP-1 (7-37) (K 27: γ-Glu-C16); R 26 -GLP-1 (7-37 ^{) (K 34: γ-Glu} -C16); K 36 R 26,34 -GLP-1 (7-37) (K 36: γ-Glu-C16); R 26,34 -GLP-1 (7-38 ^{) (K 38: γ-Glu} -C16); GLP-1 (7-37) (K 26,34: bis -C16- diacid); GLP-1 (7-37) (K 26,34: bis - γ- lu-C16); GLP-1 (7-37) (K 26,34: bis -γ-Glu-C14; GLP- 1 (7-37) (K 26,34: bis -C12- diacid); R ^{34 -GLP-1 (7-37) (} K 26: C16- ^{diacid); R 34 GLP-1 (} 7-37) (K 26: C14- ^{diacid); R 34 -GLP-1 (} 7-37 ^{) (K 26: γ-Glu} -C18); R 34 -GLP-1 (7-37) (K 26: γ-Glu-C14); R 34 -GLP-1 (7-37) (K 26: γ -Glu-C12); Desamino-H ⁷ R ³⁴ -GLP-1 (7-37) (K ²⁶ : γ-Glu-C16); R ³⁴ -GLP-1 (7-37) (K ²⁶ : GABA-C16) ^{); R 34 -GLP-1 (} 7-37) (K 26: β-Ala-C16); R 34 -GLP-1 (7-37) (K 26: iso -Nip-C16); desamino -H ^{R 26 -GLP-1 (7-37)} (K 34: γ-Glu-C16); desamino ^{-H 7 R 26 -GLP-1 (} 7-37) (K 34: C8); desamino ^-H 7 ^{R 26 -GLP-1 (7-37) (K} 34: γ-Glu-C8); K 36,34 -GLP-1 (7-36) (K 36: C20- ^{diacid); K 36,34} -GLP- ^{1 (7-36) (K 36:} C16- ^{diacid); K 36,34 -GLP-1 (} 7-36) (K 36: γ-Glu-C18); R 26,34 -GLP-1 (7 ^-38) (K 38: C16- ^{diacid); R 26,34 -GLP-1 (} 7-38) (K 38: C12- ^{diacid); R 26,34 -GLP-1 (} 7-38) ( ^{K 38: γ-Glu-C18} ); R 26,34 -GLP-1 (7-38) (K 38: y-Glu-C14); C 8 R 26,34 -GLP-1 (7-38) ( K ³⁸ : γ-Gl ^{u-C16); E 37 R} 26,34 -GLP-1 (7-38) (K 38: γ-Glu-C16); E 37 C8R 26,34 -GLP-1 (7-38) (K 38: γ-Glu-C16); and ^{E 37 C 8 R 26,34 -GLP-} 1 (7-38) (K 38: γ-Glu-C18).

  The amino acid sequence of GLP-1 is presented, inter alia, in Schmidt, et al., Diabetologia 28: 704-707, 1985. Human GLP-1 is a peptide consisting of 37 amino acid residues and is derived from preproglucagon synthesized in L cells in the terminal ileum, pancreas and brain. Processing of preproglucagon into GLP-1 (7-36) amide, GLP-1 (7-37) and GLP-2 occurs primarily in L cells. In recent years, the interesting pharmacological properties of GLP-1 (7-37) and its analogs have attracted much attention, but the structures of these molecules are largely unknown. The secondary structure of GLP-1 in micelles is described in Thorton, et al., Biochemistry 33: 3532-539, 1994), but in normal solutions GLP-1 is a very flexible molecule. It is considered to be.

  GLP-1 and GLP-1 analogs and fragments thereof are particularly useful for the treatment of type 1 and type 2 diabetes and obesity. Hoist, J., Expert Opin. Emerg. Drugs 9 (1): 155-161, 2004; Rolin, R., et al., Am. J. Physiol. Endocrinol. Metab. , C, Diabetes 53: 2181-2187, 2004; Perry, T., et al., Trends Pharmacol. ScL 24 (7): 377-383, 2003; HoIz, G., et al., Curr. Med. 10 (22): 2471-2481, 2003; Naslund, K, et al., Regul. Pept. 706: 89-95, 2002; International Patent Application WO 87/06941; WO 90/11296; WO 91/11457; Described in European Patents EP 0708179-A2 and EP 069686-A2 The listed GLP-1 analogs, GLP-1 fragments and functional derivatives of GLP-1 are hereby incorporated by reference in their entirety.

  WO 87/06941 discloses GLP-1 fragments including GLP-1 (7-37) and functional derivatives thereof, and their use as insulin secreting agents.

  WO 90/11296 contains GLP-1 (7-37) which has GLP-1 (1-36) or GLP-1 (7-37) which has an insulin secretion promoting action which exceeds that of GLP-1 (1-37) Fragments and functional derivatives thereof and their use as insulin secreting agents are disclosed.

  WO 91/11457 discloses analogs of active GLP-1 peptides 7-34, 7-35, 7-36, and 7-37 that may also be useful as GLP-1 moieties.

EP 0708179-A2 (Eli Lilly & Co.) contains an N-terminal imidazole group, optionally containing an unbranched C ₆ -C ₁₀ acyl group appended to the 34th position lysine residue. Further good GLP-1 analogs and derivatives are disclosed.

  EP 069686-A2 (Eli Lilly & Co.) discloses certain N-terminal truncated fragments of GLP-1 which are reported to have biological activity.

The amino acid sequence of GLP-1 (1-37) is as follows:
HDEFERHAEGFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 1).

The amino acid sequence of GLP-1 (7-37) is as follows:
HAEGFTSDVSSYLEGQAAKEFIAWLVGKGRG (SEQ ID NO: 2).

The amino acid sequence of GLP-1 (7-36) is as follows:
HAEGFTSDVSSYLEGQAAKEFIAWLVKGR (SEQ ID NO: 3).

The amino acid sequence of GLP-1 (7-34) is as follows:
HAEGFTSDVSSYLEGQAAKEFIALVVK (SEQ ID NO: 4).

The amino acid sequence of GLP-1 (9-36) is as follows:
EGTFTSDVSSYLEGQAAKEFIAWLVKGR (SEQ ID NO: 5).

  The GLP-1 analogs listed below have improved DPP-IV resistance.

The amino acid sequence of GLP-1 analog GG is as follows:
HGEGFTSDVSSYLEGQAAKEFIAWLVKGR (SEQ ID NO: 6).

The amino acid sequence of GLP-1 analog GG ₁ is as follows:
HGEGFTSDVSSYLEGQAAKEFIAWLVKGRPS (SEQ ID NO: 7).

The amino acid sequence of GLP-1 analogues GG ₂ are as follows:
HGEGFTSDVSSYLEGQAAKEFIAWLVKGRPSSGAP (SEQ ID NO: 8).

The amino acid sequence of GLP-1 analogues GG ₃ are as follows:
HGEGFTSDVSSYLEGQAAKEFIAWLVKGGRPSSGAPPPS (SEQ ID NO: 9).

The amino acid sequence of the GLP-1 analog GLP-1 ET is as follows:
HAEGFTSDVSSYLEGQAAKEFIAWLVGKGGPSSGAPPPS (SEQ ID NO: 10).

The amino acid sequence of GLP-1 synthetic analog NN2211 is as follows:
HAEGFTSDVSSYLEGQAAK ^* EFIAWLVRRGRG (SEQ ID NO: 11), K ^* at the 26th position of the amino acid chain in the sequence is a hexadecanoyl side chain (ie, K-N-ε- (γ-Glu (N-α-hexa It has been modified by acylation to produce decanoyl))).

The amino acid sequence of GLP-1 synthetic analog CJC-1131 is as follows:
HA ^* EGTSDSDVSSYLEGQAAKEFIAWLVKGRK ^* (SEQ ID NO: 12), A ^* in the 8th position of the amino acid chain in the sequence is D-alanine substituted with L-alanine, and is in the 37th position of the amino acid chain K ^* has a [2- [2- [2-maleimidopropionamido (ethoxy) ethoxy] acetamide linker in its ε-amino group.

The amino acid sequence of the GLP-1 synthetic analog LY315902 is as follows:
des-HAEGFTSDVSSYLEGQAAREFIAWLVK ^* GRG (SEQ ID NO: 13), in which the N-terminal (des-H) histidine residue does not contain an amino group, and K ^* at the 34th position of the amino acid chain is the octanoyl side chain (Ie, K- (octanoyl)) has been modified by acylation to produce.

  In accordance with the present invention, GLP-1 includes free bases, peptide acid addition salts or metal salts such as potassium or sodium salts, and amidation, glycosylation, acylation, sulfation, phosphorylation, acetylation, ring Also included are GLP-1 peptides modified by processes such as conjugation and other well-known covalent modification methods.

Exendins and exendin agonists The present invention includes analogs, fragments, and functional derivatives of exendin proteins and peptides. Exendin is a peptide that was first isolated from the salivary secretions of the American lizard, a lizard found in Arizona, and the Mexican lizard. Exendin-3 is present in the salivary secretion of Heroderma horridum and exendin-4 is present in the salivary secretion of Heroderma suspectum [Eng, J. , et al., J. Biol. Chem. 265: 20259-62, 1990; Eng., J., et al., J. Biol. Chem. 267: 7402-05, 1992]. Exendin shows some sequence similarity with multiple members of the glucagon-like peptide family, with the highest 53% homology to the incretin hormone GLP-1 [7-36] NH ₂ [Goke, et al, J. Biol. Chem. 268: 19650-55, 1993].

  The generic name for synthetic exendin-4 is exenatide [WHO Drug lnformation, Vol. 18, No. 1, 2004]. Exenatide is a synthetic exendin-4. Exenatide reflects the effect of GLP-1, but is more potent because of its resistance to DPP-IV degradation. BYETTA® is a commercial version of Exenatide (Amylin & Lilly). The US FDA has approved BYETTA (exenatide) injections as an adjunct therapy in patients with type 2 diabetes when oral treatment with metformin and / or sulfonylurea is not sufficient to achieve glycemic control. In addition to improving glycemic control, subjects in the trial with exenatide also experienced weight loss.

  The present invention is directed to exendins, exendin analogs, exendin agonists, modified exendins, modified exendin analogs, modified exendin agonists, or any combination thereof (see, for example, below). Aiming at a novel method for treating diabetes and lowering plasma glucose or delaying and / or slowing gastric emptying or inhibiting food intake, including nasal administration And

Exendin-3:
His Ser Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Alu Val Arg Leu Phe Ile Glu Trp Leu Lys As

Or exendin-4 (synthetic exendin-4 (exenatide)):
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys As (SEQ ID NO: 15),

Or an insulin secretagogue fragment of exendin-4:
Exendin-4 (1-31) His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Les Glu Trp Le
y.sup.31 Exendin-4 (1-31) His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val ArG Le L I Phe ,

Or an inhibitory fragment of exendin-4:
Exendin-4 (9-39) Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Tip Leu Lys Asn Gly Gly Pro Ser Ser

Or other preferred exendin agonists:
Exendin-4 (1-30) His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Tip Le Glu Tip Le 1-30) Amides His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu TipLu G 4 (1-28) amide His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Alu Val Arg Leu Phe Ile Glu Tip Leu Lys Asn-NH.sub.2 (SEQ ID NO: 21), .sup.14 Leu, .sup.25 Phe exendin-4 amide His Gly Glu Gly Thr Phe Thr Phr Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Pro Ser-NH.sub.e. P. Din-4 (1-28) amide His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile lu Phe Leu Lys Asn-NH.sub.2 (SEQ ID NO: 23) and .sup.14 Leu, .sup.22 Ala, .sup.25 Phe exendin-4 (1-28) amide His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Ala Ile Glu Phe Leu Lys Asn-NH.sub.2 (SEQ ID NO: 24).

Or US Pat. No. 5,424,286; US Pat. No. 6,506,724; US Pat. No. 6,528,486; US Pat. No. 6,593,295; US Pat. No. 6,872,700 US Pat. No. 6,902,744; US Pat. No. 6,924,264; and US Pat. No. 6,956,026, incorporated by reference;

  Or a receptor that exerts the action of exendin, useful in the treatment of diabetes and lowering plasma glucose or delaying and / or slowing gastric emptying or inhibiting food intake Other compounds that bind effectively to The use of exendin-3 and exendin-4 as insulin secretagogues in the treatment of diabetes and the prevention of hyperglycemia is disclosed in US Pat. No. 5,424,286. Exendins have also been demonstrated to be useful in regulating triglyceride levels and treating dyslipidemia.

  Accordingly, the present invention provides a peptide or peptide fragment that encompasses the biological activity of exendin or a fragment thereof, as described herein, made synthetically or purified from a natural source.

  According to the present invention, exendins include free bases, acid addition salts of peptides or metal salts such as potassium or sodium salts, and amidation, glycosylation, acylation, sulfation, phosphorylation, acetylation, cyclization. Also included are exendin peptides modified by processes such as other well-known covalent modification methods.

  Thus, according to the present invention, the peptides described above are incorporated into formulations suitable for transepithelial delivery, particularly intranasal and dermal delivery.

Biological membrane A “biological membrane” is defined as a membrane material that resides within an organism, preferably an animal, more preferably a human, and separates one region of the organism from another. In many cases, biological membranes separate a living organism from its surroundings or environment. Non-limiting examples of biological membranes include human mucosa and skin.

Epithelial delivery enhancer “Epithelial delivery enhancer” is the maximum blood concentration, maximum serum concentration, or maximum cerebrospinal fluid concentration when added to a formulation containing water, salt and / or normal buffer and GRP (control formulation). (C _max ) or area under the curve in a plot of concentration versus time Defined as chemicals and other excipients that produce a formulation that significantly increases the transport of GRP between biological membranes as measured by AUC. Epithelial biofilms may include the nose, oral cavity, intestine, cheeks, bronchopulmonary, vagina, rectum and skin surfaces. Transepithelial delivery enhancers are sometimes called carriers.

Mucosal delivery enhancer “mucosal delivery enhancer” is the maximum blood concentration, maximum serum concentration, or maximum cerebrospinal fluid concentration when added to a formulation containing water, salt and / or normal buffer and GRP (control formulation). (C _max ) or area under the curve in a plot of concentration versus time Defined as chemicals and other excipients that produce a formulation that significantly increases transport of GRP between mucosa, as measured by AUC. The mucosa includes the surfaces of the nose, oral cavity, intestine, cheeks, bronchopulmonary, vaginal and rectal mucosa, and in fact includes all mucous secreting membranes that cover the body cavities or passages leading to the outside world. Mucosal delivery enhancers are sometimes referred to as carriers.

Endotoxin-free formulation "Endotoxin-free formulation" means a formulation that contains GRP and one or more epithelial delivery enhancers and is substantially free of endotoxins and / or related pyrogens. Endotoxins include toxins that are trapped inside microorganisms and are released only when the microorganisms are destroyed or die. Pyrogens include substances (glycoproteins) that generate heat from the outer membranes of bacteria and other microorganisms and exhibit thermal stability. Both of these substances can cause fever, hypotension and shock when administered to humans. To produce endotoxin-free preparations, special equipment and specialists may be required, and are significantly more expensive than non-endotoxin-free preparations There is also. Intravenous administration of GLP or amylin at the same time as endotoxin injection in rodents prevents hypotension and even death associated with administration of endotoxin alone (US Pat. No. 4,839,343). Manufacturing a therapeutic endotoxin-free formulation is not expected for non-parenteral (non-injection) administration.

Non-injection administration “Non-infusion administration” means any delivery method that does not involve direct injection into an artery or vein, typically inside something (typically by a needle, syringe or other invasive means). In particular, it means a method of pushing or feeding a liquid), in particular a method of introducing it into a body part. Non-injection administration includes subcutaneous injection, intramuscular injection, intraperitoneal injection, or non-injectable delivery methods to biological membranes.

Delivery Methods and Compositions Improved methods and compositions for epithelial administration of GRP to a subject mammal optimize the GRP dosing schedule. The present invention provides that the release of GRP dose is well normalized and / or about 0.1 to 2.0 hours; 0.4 to 1.5 hours; 0.7 to 1.5 after epithelial administration. Provided is a GRP formulation comprising one or more epithelial delivery enhancers, wherein the release of said GRP dose is sustained over an effective delivery time of GRP release ranging from time; or in the range of 0.8 to 1.0 hour To do. The sustained release of GRP achieved is facilitated by repeated administration of exogenous GRP utilizing the methods and compositions of the present invention.

Sustained Release Compositions and Methods Improved methods and compositions for epithelial administration of GRP to a subject mammal optimize the GRP dosing schedule. The present invention provides improved epithelial (eg, nasal) delivery of a formulation comprising GRP in combination with one or more epithelial delivery enhancers and optionally a sustained release enhancer. The epithelial delivery-enhancing agent of the present invention effectively improves delivery such that, for example, increasing the maximum plasma concentration (C _max ) increases the therapeutic activity of epithelially administered GRP. The second factor that affects the therapeutic activity of GRP in plasma and CNS is residence time (RT). The sustained release enhancer increases the C _max of GRP and increases the residence time (RT) in combination with an intranasal delivery enhancer. Disclosed herein are polymer delivery vehicles and other agents and methods of the present invention, such as polyethylene glycol (PEG), that produce sustained release enhancing formulations. The present invention relates to obesity, diabetes, hyperglycemia, metabolic syndrome, coronary syndrome, colorectal cancer, exendin cancer, breast cancer, myocardial inflation in a subject mammal. Provided are improved GRP delivery methods and dosage forms for treating symptoms, promoting neurogenesis, suppressing appetite, promoting weight loss and reducing food intake.

  In the epithelial delivery formulations and methods of the present invention, GRP is often combined or administered simultaneously with a carrier or vehicle suitable for epithelial delivery. As used herein, the term “carrier” means a pharmaceutically acceptable solid or liquid filler, diluent or encapsulating material. Water-containing liquid carriers include acidifying agents, alkalizing agents, antimicrobial preservatives, antioxidants, buffers, chelating agents, complexing agents, solubilizers, wetting agents, solvents, suspending and / or thickening. Pharmaceutically acceptable additives such as agents, isotonic agents, wetting agents or other biocompatible materials may be included. A summary of the components listed according to the above classifications can be found in the US Pharmacopoeia National Pharmaceuticals 1857-1859, 1990. Some examples of substances that can function as pharmaceutically acceptable carriers include: sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose derivatives such as cellulose and sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate Tragacanth powder; malt; gelatin; talc; additives such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; Polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid Pyrogen-free water; isotonic saline; Ringer's solution, ethyl alcohol and phosphate buffer solutions, as well as other compatible material non-toxic to be used in pharmaceutical compositions. Wetting agents such as sodium lauryl sulfate and magnesium stearate, emulsifiers and lubricants, and colorants, release agents, coating agents, sweeteners, flavoring agents and fragrances, preservatives and antioxidants are also desired by the formulator According to the present invention. Examples of pharmaceutically acceptable antioxidants include water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite; ascorbyl palmitate, butylated hydroxyanisole (BHA) Contains oil-soluble antioxidants such as butylated hydroxytoluene (BHT), lecithin, propyl gallate, and α-tocopherol; and metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid It is. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the particular mode of administration.

  In the epithelial delivery compositions and methods of the present invention, various delivery enhancers that facilitate delivery of GRP into or between cell layers can be used. In this regard, delivery of GRP between epithelia may occur “transcellularly” or “paracellularly”. The extent to which these pathways contribute to the total flux and biological effectiveness of GRP depends on the biomembrane environment, the physicochemical properties of the active agent, and the properties of the epithelium. Transcellular transport is only related to passive diffusion, but paracellular transport can also occur by passive, accelerated or aggressive processes. In general, hydrophilic, passively transported, polar solutes diffuse through the paracellular pathway, and more lipophilic solutes use the transcellular pathway. A variety of passively and actively absorbed solute absorptions and biological effectiveness (such as reflected in permeability coefficients or physiological assays) can be selected within the scope of the present invention. GRPs can be evaluated immediately for both paracellular and transcellular delivery components. For passively absorbed drugs, the relative contribution of paracellular and transcellular pathways to drug transport is the pKa, partition coefficient, molecular radius and charge of the drug, the pH of the luminal environment to which the drug is delivered, As well as the absorption area. The paracellular pathway represents a relatively small fraction of the accessible area of the nasal mucosal epithelium. In general, it has been reported that the cell membrane occupies a surface area several thousand times larger than the area occupied by the paracellular space. Thus, its small accessible area and size and charge-based exclusion to polymer permeation generally suggest that transcellular delivery may be preferable to paracellular routes for drug delivery. Let's go. Surprisingly, the methods and compositions of the present invention significantly facilitate the transport of biological therapeutic agents into or between mucosal epithelia via the paracellular pathway. Accordingly, the methods and compositions of the present invention target both paracellular and transcellular pathways well instead of within a single method or composition or within a single method or composition.

  As used herein, epithelial delivery-enhancing agents include GRP or other bioactive compound release or solubility (from the pharmaceutical delivery vehicle), diffusion rate, permeability and timing, uptake, residence time, Stability, effective half-life, peak or sustained concentration level, clearance and other desired epithelial delivery properties (eg, measured at the delivery site or selected target active site such as bloodstream or central nervous system) Such as). Thus, improvements in epithelial delivery may include, for example, the availability or action of calcium and other ions that regulate GRP diffusion, transport, increased persistence or stability, increased membrane fluidity, intracellular or paracellular permeability. Regulation of membrane components (eg lipids), changes in sulfhydryl levels of non-proteins and proteins in mucosal tissues, increased water flow between cell layers, regulation of physiology of epithelial junctions, mucosa covering mucosal epithelium This can be caused by any of a variety of mechanisms such as a decrease in viscosity, a decrease in mucociliary clearance rate, and other mechanisms.

  As used herein, an “effective amount of GRP” is intended for effective delivery of GRP to a target site of drug activity within a subject that would require various delivery or migration routes. For example, any active agent may find its way through the gaps between the mucosal cells and reach the adjacent vessel wall, while another route causes the agent to passively or actively enter the mucosa. It may be taken up into the cell and act inside the cell, or may be discharged or transported outside the cell to reach a secondary target site such as systemic circulation. The methods and compositions of the present invention facilitate the translocation of active agents along one or more such alternative pathways or act directly on mucosal tissue or surrounding vascular tissue, Absorption or permeation of the active drug can be facilitated. The promotion of absorption or permeation in this context is not limited to the mechanism described above.

As used herein, “peak concentration of GRP in plasma (C _max )”, “concentration of GRP in plasma versus area under time curve (AUC)”, “time to maximum plasma concentration of GRP in plasma (t “ _max )” is a pharmacokinetic parameter known to those skilled in the art. Laursen, et al., Eur. J. Endocrinology 755: 309-315, 1996. A “concentration versus time curve” measures the concentration of GRP in a subject's serum versus time after administration of a dose of GRP to a subject by either intranasal, intramuscular, subcutaneous or other parenteral routes of administration. Is. “C _max ” is the maximum concentration of GRP in the subject's serum after a single dose of GRP to the subject. “T _max ” is the time to reach the maximum concentration of GRP in the subject's serum after a single dose of GRP to the subject.

The “area of GRP in plasma versus area under time curve (AUC)” used in the present invention is calculated by adding the residual area according to the linear trapezoidal formula. A 23% decrease or a 30% increase between the two doses will be detected with a 90% likelihood (type 2 error β = 10%). “Delivery rate” or “absorption rate” is assessed by comparing the time to reach maximum concentration (C _max ) (t _max ). Both C _max and t _max are analyzed using non-parametric methods. Comparison of pharmacokinetics of intramuscular, subcutaneous, intravenous and intranasal GRP administration was performed by analysis of variance (ANOVA). For pairwise comparison, significance is evaluated using the Bonferroni-Holmes sequential processing method. The dose-response relationship between 3 nasal doses is assessed by regression analysis. P <0.05 is considered significant. Results are expressed as mean +/- standard error.

  Although the mechanism of enhanced absorption may be altered by the different epithelial delivery enhancers of the present invention, reagents useful in this context do not substantially adversely affect the tissue and are not specific to GRP or other It will be the reagent selected depending on the physicochemical properties of the active agent or delivery enhancer. In this regard, delivery enhancers that increase penetration or penetration of mucosal tissue will alter the mucosal protective penetration barrier to some extent. In order for these delivery enhancers to take values within the scope of the present invention, it is generally that any significant change in the permeability of the biological membrane is reversible within a time frame appropriate for the desired drug delivery time. Is desirable. Furthermore, prolonged use should not cause substantial cumulative toxicity or permanent degradation to the barrier properties of the biological membrane.

  In a particular aspect of the present invention, the absorption enhancer for co-administration or combination preparation of the GRP of the present invention is a low molecular weight hydrophilic agent comprising dimethyl sulfoxide (DMSO), dimethylformamide, ethanol, propylene glycol, and 2-pyrrolidone. It is selected from, but not limited to, sex molecules. Instead of the above, for example, diacylmethyl sulfoxide, azone, sodium lauryl sulfate, oleic acid, and bile salt can be used to promote GRP permeation through the biomembrane. In a further aspect, a surfactant (eg, polysorbate) is used as an auxiliary compound, processing agent or formulation additive to facilitate transepithelial delivery of GRP. Drugs such as DMSO, polyethylene glycol, and ethanol, when present at sufficiently high concentrations in the delivery environment (eg, by pre-administration or incorporation into therapeutic formulations) enter the mucosal aqueous layer and It changes the dissolution properties, thereby facilitating the separation of GRP from the medium into the biological membrane.

  Additional epithelial delivery enhancers useful in the co-administration and treatment methods and combination formulations of the present invention include, but are not limited to, mixed micelles; enamines: nitric oxide donors (eg, S-nitroso-N- Acetyl-DL-penicillamine, NOR1, N0R4-; these are preferably co-administered with a NO scavenger such as carboxy-PITO or diclofenac sodium; sodium salicylate; glycerol esters of acetoacetate (eg, glycerin-1,3- Diacetoacetate or 1,2-isopropylideneglycerin-3-acetoacetate); and other physiologically compatible release diffusion agents or intraepithelial or transepithelial permeation enhancers for epithelial delivery . Other absorption enhancers are selected from various carriers, bases and excipients that promote epithelial delivery, stability, activity or transepithelial penetration of GRP. These include, among others, cyclodextrins and β-cyclodextrin derivatives (eg, 2-hydroxypropyl-β-cyclodextrin and heptakis (2,6-di-O-methyl-β-cyclodextrin). These compounds, which may be conjugated with one or more active ingredients, and optionally optionally formulated in an oily base, improve bioavailability in the epithelial formulation of the present invention. Additional absorption enhancers applied for epithelial delivery include mono- and diglycerides (eg, sodium caprate extract of coconut oil, capmul), and triglycerides (eg, amylodextrin, Estaram 299, Miglyol 810). Contains medium chain fatty acids.

  The epithelial treatment and prevention composition of the present invention may be supplemented with any suitable permeation enhancer that promotes the absorption, dispersion or permeation of GRP between biological membrane barriers. The permeation enhancer can be any pharmaceutically acceptable enhancer. Accordingly, in a more detailed aspect of the present invention, sodium salicylate and salicylic acid derivatives (acetylsalicylic acid, choline salicylate, salicylamide, etc.); amino acids and salts thereof (eg, glycine, alanine, phenylalanine, proline, hydroxyproline, etc.) Acids; hydroxy amino acids such as serine; acidic amino acids such as aspartic acid and glutamic acid; and basic amino acids such as lysine—including alkali metal salts or alkaline earth metal salts thereof; and N-acetylamino acid (N-acetylalanine) Acetylphenylalanine, N-acetylserine, N-acetylglycine, N-acetyllysine, N-acetylglutamic acid, N-acetylproline, N-acetylhydroxyproline, etc.) and salts thereof (alkali metal salts and alkaline earths) A composition incorporating one or more permeation enhancers selected from the group of metal salts) is provided within the scope of the methods and compositions of the present invention. , Substances commonly used as emulsifiers (eg, sodium oleyl phosphate, sodium lauryl phosphate, sodium lauryl sulfate, sodium myristyl sulfate, polyoxyethylene alkyl ether, polyoxyethylene alkyl ester, etc.), caproic acid, lactic acid, There are malic acid and citric acid and their alkali metal salts, pyrrolidone carboxylic acid, alkyl pyrrolidone carboxylic acid ester, N-alkyl pyrrolidone, proline acyl ester.

  In various aspects of the invention, for improved nasal mucosal delivery that enables delivery of the GRP and other therapeutic agents of the invention between the biomembrane barriers that exist between the administration site and the selected target site. Formulations and methods are provided. Certain formulations are specially adapted for selected target cells, tissues or organs, or for specific disease states. In other aspects, the formulations and methods provide efficient and selective endocytosis or transcytosis of GRP, particularly through pathways along specific intracellular or intercellular pathways. Typically, GRP is effectively infused in an effective amount in a single or other delivery vehicle, for example, in the nasal mucosa and / or to the target site of drug action off the intracellular compartment and membrane. Delivered (eg, in the bloodstream or certain tissues, organs, or extracellular compartments) and held in a stable form. GRP may be provided as a delivery vehicle or modified (eg, in the form of a prodrug), where release or activation of GRP is triggered by a physiological stimulus (eg, a change in pH, a lysosomal enzyme, etc.). Be triggered. In some cases, GRP is pharmacologically inactive until it reaches its active target site. In most cases, GRP and other formulation ingredients are non-toxic and non-immunogenic. In this regard, the carrier and other formulation components are generally selected based on their ability to rapidly degrade and be excreted under physiological conditions. Similarly, the formulation is chemically and physically stable within the dosage form for efficient storage.

Peptide and protein analogs and mimetics The definition of bioactive peptides and proteins for use within the scope of the present invention includes peptides having two or more covalently linked, natural or synthetic, therapeutic or prophylactic activities. Protein, peptide or protein fragments, peptide or protein analogs, and chemically modified derivatives or salts of active peptides or proteins. The present invention contemplates using a wide variety of useful GRP analogs and mimetics that can be produced according to known methods and tested for their biological activity. In some cases, a GRP peptide or protein or other biological activity peptide or protein for use within the scope of the present invention is natural or natural (eg, wild type, natural mutant, or allelic variant). Etc.) muteins that are readily available by partial substitution, addition or deletion of amino acids in the peptide sequence or protein sequence. In addition, biologically active fragments of natural peptides or proteins are included. Such mutant derivatives and fragments retain the desired biological activity of the natural peptide or protein. When the peptide or protein has a sugar chain, biologically active mutants characterized by mutations in these sugar species are also encompassed by the present invention.

  The term “conservative amino acid substitution” as used herein refers to the general interchangeability of amino acids with similar side chains. For example, a commonly displaceable group of amino acids having an aliphatic side chain is alanine, valine, leucine, and isoleucine; a group of amino acids having an aliphatic-hydroxyl side chain is serine and threonine; The group of amino acids having chains is asparagine and glutamine; the group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; the group of amino acids having basic side chains is lysine, arginine, and Histidine; and a group of amino acids having sulfur-containing side chains are cysteine and methionine. Examples of conservative substitutions include substitution of a nonpolar (hydrophobic) residue such as isoleucine, valine, leucine or methionine with another nonpolar (hydrophobic) residue. Similarly, the present invention contemplates substitution of polar (hydrophilic) residues such as between arginine and lysine, between glutamine and asparagine, and between threonine and serine. In addition, substitution of basic residues such as lysine, arginine, or histidine to other basic residues, or substitution of acidic residues such as aspartic acid or glutamic acid to other acidic residues is also contemplated. Representative conservative amino acid substitution groups include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. By aligning the peptide or protein analog, optionally with the corresponding native peptide or protein, and using an appropriate assay to determine the selected biological activity, such as, for example, a protein adhesion assay or a receptor binding assay Thus, functional peptides and protein analogs for use in the methods and compositions according to the present invention can be readily identified. Functional peptide and protein analogs typically have specific immunoreactivity for antibodies raised against the corresponding natural peptide or protein.

  A method of stabilizing a solid protein formulation according to the present invention is to increase the physical stability of a purified protein, such as, for example, a lyophilized protein. This would inhibit aggregation due to hydrophobic interactions, as well as aggregation via covalent pathways that would increase as the protein unfolds. Formulation stabilization in such circumstances includes, for example, polymer-based formulations such as biodegradable hydrogel formulations / delivery systems. As mentioned above, the critical role of water in protein structure, function and stability is well known. Typically, proteins are relatively stable in the solid state from which bulk water has been removed. However, solid therapeutic protein formulations may be hydrated upon storage at high humidity, or may be hydrated during delivery from a sustained release composition or device. Protein stability generally decreases with increasing hydration. Water, for example, increases protein flexibility to increase accessibility to reactive groups, provides a mobile phase for reactants, and reacts in multiple detrimental processes such as beta elimination and hydrolysis By acting as a substance, it may also play an important role in the aggregation of solid proteins.

  Protein formulations containing about 6% to 28% water are the most unstable. Below this level, the mobility of the bound water and the internal movement of the protein will be low. Above this level, water mobility and protein movement are close to being fully hydrated. Increased sensitivity to solid phase aggregation with increasing hydration to a certain degree has been observed in several systems. However, at higher water content, a relatively low degree of aggregation is observed due to the dilution effect.

  In accordance with the above principles, an effective way to stabilize peptides and proteins against solid-phase aggregation for mucosal delivery is to control the water content in the solid formulation to ensure proper water activity in the formulation. Is to maintain the level. This level depends on the nature of the protein, but in general, proteins maintained below the protein's “monolayer” water coverage show excellent solid state stability. become.

  Various additives, diluents, bases and delivery vehicles that effectively adjust moisture content to improve protein stability are provided within the scope of the present invention. These reagents and carrier materials useful in this sense as anti-aggregating agents include polymers having various functionalities such as, for example, polyethylene glycol, dextran, diethylaminoethyl dextran, and carboxymethyl cellulose. Significantly increases the stability of peptides and proteins mixed with or bound to the polymer and decreases solid phase aggregation. In some cases, protein activity and physical stability can also be improved by various additives to aqueous solutions of the peptide or protein drug. For example, additives such as polyols (including sugars), amino acids, proteins including collagen and gelatin, and various salts can be used.

  Certain additives, particularly sugars and other polyols, also impart significant physical stability to dried proteins, such as lyophilized proteins. Within the scope of the present invention, these additives can be used to protect proteins against aggregation not only during freeze-drying but also during storage in a dry state. For example, sucrose and Ficoll 70 (polymer with sucrose units) show significant protection against peptide or protein aggregation during solid phase incubation under various conditions. These additives can also increase the stability of the solid protein embedded in the polymer matrix.

  For example, additional additives such as sucrose stabilize the protein against solid state aggregation under high temperature and humid atmosphere that may occur in certain sustained release formulations of the present invention. Proteins such as gelatin and collagen also function as stabilizers or fillers to reduce unstable protein denaturation and aggregation in this context. These additives can be incorporated into polymer dissolution processes and compositions within the scope of the present invention. For example, polypeptide microparticles can be prepared by simply lyophilizing or spray drying a solution containing the stabilizing additives described above.

  A variety of formulations for epithelial delivery of peptides and proteins that are prone to aggregation, using solubilizers to produce formulations in which the peptides or proteins in the formulation are substantially pure and stable in non-aggregated form Additional preparative ingredients and methods of preparation, as well as special pharmaceutical additives, are provided herein. A wide range of ingredients and additives are intended to be used in these methods and formulations. A typical example of these stabilizers is cyclodextrin (CD) that selectively binds to the hydrophobic side chain of a polypeptide. These CDs have been found to bind to hydrophobic patches of proteins so as to significantly inhibit aggregation. This inhibition is selective for both CD and the proteins involved. Such selective inhibition of protein aggregation provides further benefits in the intranasal delivery methods and compositions according to the present invention. Additional agents used in this context include CD dimers, trimers, and tetramers with various linker controlled geometries that specifically block peptide and protein aggregation. Is included. Furthermore, solubilizers and methods incorporated within the scope of the present invention include selectively blocking protein-protein interactions with peptides and peptidomimetics. In one aspect, the specific binding to hydrophobic side chains reported for CD multimers is extended to proteins by the use of peptides and peptidomimetics that also block protein aggregation. A wide range of suitable methods and anti-aggregating agents are available for incorporation into the compositions and methods according to the present invention.

Charge modification and pH adjustment agents and methods To improve transport properties of bioactive agents (GRP, other active peptides and proteins, and high and low molecular weight drugs) for improved delivery between hydrophobic biomembrane barriers The present invention also provides techniques and reagents for modifying the charge of selected bioactive agents or delivery enhancers described herein. In this regard, the relative permeability of a polymer is generally correlated with its partition coefficient. degree of ionization of molecules depends on the pK _a, and the pH of the biological membrane surface also affects permeability of the molecules. Penetration and distribution of bioactive agents for epithelial delivery, including GRP and analogs of the present invention, is facilitated by charge change or charge diffusion of the active agent or penetrant, where charge change or charge diffusion is For example, it can be accomplished by changing the charged functional group, adjusting the pH of the delivery vehicle or solution that delivers the active agent, or co-administering the active agent and the charge or pH changing agent.

  In accordance with these general teachings, epithelial delivery of charged macromolecular species, including GRP and other bioactive peptides and proteins, that are within the scope of the methods and compositions of the present invention, the active agent is substantially It is fully enhanced when delivered to the epithelial surface in a non-ionized or neutral charge state.

  Certain GRP and other bioactive peptide and protein components of epithelial formulations used within the scope of the present invention will undergo charge modification to increase the positive charge density of the peptide or protein. These modifications extend to the cationization of peptide and protein conjugates, carriers and other delivery forms disclosed herein. Cationization provides a convenient means of changing the biodistribution and transport properties of proteins and macromolecules within the scope of the present invention. Cationization is performed to substantially maintain the biological activity of the active agent and limit potentially harmful side effects including tissue damage and toxicity.

  A “buffer” is generally used to maintain the pH of the solution approximately constant. The buffering agent maintains the pH of the solution by preventing or neutralizing large changes in hydrogen or hydroxide ion concentration, even when a small amount of strong acid or base is added to the solution. In general, the buffer consists of a weak acid and its appropriate salt (or a weak base and its appropriate salt). Suitable salts for weak acids include the same anions present in the weak acid (Lawski, McCillan Encyclopedia of Chemistry, Vol. 1, Simon & Schuster, New York, 1997-p. 1997, p. 3). reference). The description of the buffer includes the Henderson-Hasselbalch formula,

が用いられ、この式は、弱酸の解離に関する標準的な等式ＨＡ＝Ｈ^＋＋Ａ⁻に基づく。一般的に用いられる緩衝剤供給源の代表例には、以下のものが含まれる：グルタメート、アセテート、シトレート、グリシン、ヒスチジン、アルギニン、リジン、メチオニン、ラクテート、ホルメート、グリコレート、タルトレート及びこれらの混合物。

This equation is based on the standard equation for weak acid dissociation HA = H ⁺ + A ⁻ . Representative examples of commonly used buffer sources include: glutamate, acetate, citrate, glycine, histidine, arginine, lysine, methionine, lactate, formate, glycolate, tartrate and these blend.

  “Buffer volume” means the amount of acid or base that can be added to a buffer solution until a significant pH change occurs. If the pH is within the pK-1 and pK + 1 ranges of the weak acid, the buffer dose can be evaluated, but if it is outside this range, the buffer dose can be mostly evaluated. I can't. Thus, any system exhibits a useful buffering action within the range of 1 pH unit on either the positive or negative side of the pK of a weak acid (or weak base) (Dawson, Data for Biochemical Research, Third Edition, Oxford Science Publications, 1986). , p. 419). In general, an appropriate concentration is selected such that the pH of the solution is close to the pKa of the weak acid (or weak base) (Lide, CRC Handbook of Chemistry and Physics, 86th Edition, Taylor & Frances Group, 2005). -2006, p. 2-41). Furthermore, strong acid and strong base solutions are not normally classified as buffer solutions, and they do not exhibit a buffer capacity at a pH value of 2.4 to 11.6.

Degrading enzyme inhibitors and methods of inhibition Other excipients that may be included in transepithelial formulations include degrading enzyme inhibitors. Exemplary mucoadhesive polymer-enzyme inhibitor complexes useful in epithelial delivery formulations and methods according to the present invention include, but are not limited to: Carboxymethylcellulose-pepstatin (Having anti-pepsin activity); poly (acrylic acid) -Bowman-Birk inhibitor (anti-chymotrypsin); poly (acrylic acid) -chymostatin (anti-chymotrypsin); poly (acrylic acid) -elastinal ( Anti-elastase); carboxymethylcellulose-elalastatin (anti-elastase); polycarbophil-elalastatin (anti-elastase); chitosan-antipain (antitrypsin); poly (acrylic acid) -bacitracin (anti-aminopeptidase N); Chitosan-EDTA (anti-aminopeptidase N, anti-carboxypeptidase A); San -EDTA- antipain (anti-trypsin, anti-chymotrypsin, anti-elastase). As described in further detail below, certain embodiments of the present invention will optionally incorporate novel chitosan derivatives or chemically modified forms of chitosan. One such novel derivative for use within the scope of the present invention is represented as β- [1 → 4] -2-guanidino-2-deoxy-D-glucose polymer (polyGuD).

  Any inhibitor that inhibits the activity of the enzyme to protect the bioactive agent can be beneficially used in the compositions and methods of the invention. Useful enzyme inhibitors for protecting biologically active proteins and peptides include, for example, dipeptidyl aminopeptidase (DPP) IV inhibitor, soybean trypsin inhibitor, exendin trypsin inhibitor, chymotrypsin inhibitor and potato (Solanum Trypsin and chymotrypsin inhibitor isolated from tuber of Solanum tuberosum L. are included. Combinations or mixtures of inhibitors can also be used. Further inhibitors of proteolytic enzymes for use within the scope of the present invention include ovomucoid enzyme, gabaxate mesylate, α1-antitrypsin, aprotinin, amastatin, bestatin, puromycin, bacitracin Leupepsin, α2-macroglobulin, pepstatin and egg white or soy trypsin inhibitor. These inhibitors and other inhibitors can be used alone or in combination. Inhibitors may be incorporated or bound to a carrier, such as a hydrophilic polymer, in combination with a bioactive agent or in a separately administered (eg pre-administered) formulation. It may be coated on the surface of the dosage form in contact with the surface, or may be incorporated in the surface layer of the surface.

  The amount of an inhibitor that may optionally be incorporated into the composition of the present invention, such as a proteolytic enzyme inhibitor, is (a) specific inhibitor properties, (b) functional groups present in the molecule. The number of (functional groups capable of introducing unsaturation of the ethylene group necessary for copolymerization of the hydrogel-forming monomer by reaction) and (c) lectin groups such as glycosides present in the inhibitor molecule Will vary depending on the number of It will also depend on the specific therapeutic agent to be administered. Generally speaking, an effective amount of enzyme inhibitor is from about 0.1 mg to about 50 mg per ml of formulation (ie, separate protease inhibitor formulation or combination of inhibitor and bioactive agent). Often about 0.2 mg to about 25 mg, and more typically about 0.5 mg to about 5 mg.

  In the case of trypsin inhibition, suitable inhibitors are, for example, aprotinin, BBI, soybean trypsin inhibitor, chicken ovomucoid, chicken ovo inhibitor, human exendin trypsin inhibitor, camostat mesylate, flavonoid inhibitor, antipine, leupeptin, p-amino Benzamidine, AEBSF, TLCK (tosyllysine chloromethylketone), APMSF, DFP, PMSF, and poly (acrylate) derivatives can be selected. In the case of chymotrypsin inhibition, suitable inhibitors include, for example, aprotinin, BBI, soybean trypsin inhibitor, chymostatin, benzyloxycarbonyl-Pro-Phe-CHO, FK-448, chicken ovo inhibitor, sugar biphenylboronic acid complex, DFP, PMSF, phenyl β-propionate, and poly (acrylate) derivatives can be selected. In the case of elastase inhibition, suitable inhibitors are, for example, elastatinal, methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone (MPCMK), BBI, soybean trypsin inhibitor, chicken ovo inhibitor, DFP, and PMSF, You can choose from.

  Further enzyme inhibitors for use in the present invention are selected from a wide range of non-protein inhibitors that differ in their potency and degree of toxicity. Immediate implementation of immobilization of these adjuncts to a matrix or other delivery carrier, or development of a chemically modified analog, as described in more detail below, if they encounter an inhibitor Furthermore, it is possible to reduce or eliminate the toxic effects. Among the broad group of enzyme inhibitor candidates for use within the scope of the present invention, among others, diisopropylfluorophosphate (DFP), a potent and irreversible inhibitor of serine proteases (eg, trypsin and chymotrypsin), There are organophosphorus inhibitors such as phenylmethylsulfonyl fluoride (PMSF). Further inhibition of acetylcholinesterase by these compounds makes them very toxic in an uncontrolled delivery environment. Another inhibitor candidate, 4- (2-aminoethyl) -benzenesulfonyl fluoride (AEBSF), has similar inhibitory activity to DFP and PMSF. Compared with these, toxicity is remarkably low. (4-Aminophenyl) -methanesulfonyl fluoride hydrochloride (APMOSF) is another effective inhibitor of trypsin but is toxic in an uncontrolled environment. In contrast to these inhibitors, 4- (4-isopropylpiperazinocarbonyl) phenyl 1,2,3,4, -tetrahydro-1-naphthoate methanesulfonate (4- (4-isopropylpiperadinocarbonyl) phenyl 1, 2,3,4, -tetrahydro-1-naphthaneate sulfonate (FK-448) is a low toxic substance and represents an effective and specific inhibitor of chymotrypsin. Further representatives of this non-protein group of candidate inhibitors and those showing a low toxicity risk are camostat mesylate (N, N′-dimethylcarbamoylmethyl-p- (p′-guanidinobenzoyloxy) phenyl acetate Methanesulfonate (N, N′-dimethyl carbamoylmethyl-p- (p′-guanidino-benzoyloxy) phenylacetate methane-sulfonate)).

  Furthermore, other types of enzyme inhibitors for use in the methods and compositions of the present invention are amino acids or modified amino acids that interfere with the enzymatic degradation of certain therapeutic compounds. For use in this context, amino acids and modified amino acids are substantially non-toxic and can be produced at low cost. However, due to their low molecular size and good solubility, they are readily diluted and absorbed in the mucosal environment. Even so, under appropriate conditions, amino acids can act as reversible and competitive inhibitors of protease enzymes. Certain modified amino acids can exhibit more potent inhibitory activity. Preferred modified amino acids in this context are known as “transition state” inhibitors. The potent inhibitory activity of these compounds is based on structural similarity to the substrate in its transition state structure, but they have a higher affinity for the active site of the enzyme than the substrate itself. Selected. Transition state inhibitors are reversible competitive inhibitors. Examples of this type of inhibitor include α-aminoboronic acid derivatives such as boro-leucine, boro-valine and boro-alanine. The boron atom in these derivatives can form a tetrahedral boronate ion, which is thought to be similar to the transition state of the peptide during hydrolysis by aminopeptidases. These amino acid derivatives are potent reversible inhibitors of aminopeptidases, and boroleucine has over 100 times more effects than bestatin and over 1000 times more than puromycin in enzyme inhibition. It has been reported. Another modified amino acid that has been reported to have potent protease inhibitory activity is N-acetylcysteine, which inhibits the enzymatic activity of aminopeptidase N. This adjuvant also exhibits mucosal degradation properties that can be used in the methods and compositions of the present invention to reduce the effectiveness of the mucosal diffusion barrier.

  Still other useful enzyme inhibitors for use in the coordinated administration method and combination formulations of the present invention can be selected from peptides and modified peptide enzyme inhibitors. An important representative of this type of inhibitor is the cyclic dodecapeptide bacitracin obtained from Bacillus licheniformis. In addition to these types of peptides, certain dipeptides and tripeptides also exhibit weak, nonspecific inhibitory activity against certain proteases. Due to the similarity to amino acids, their inhibitory activity can be improved by chemical modification. For example, phosphinic acid dipeptide analogs are also “transition state” inhibitors with potent inhibitory activity against aminopeptidases. It has been reported that nasally administered leucine enkephalin has been stabilized by using these. Another example of a transition state analog is the modified pentapeptide pepstatin, which is a very potent pepsin inhibitor. Structural analysis of pepstatin by testing the inhibitory activity of several synthetic analogues demonstrated the main structural and functional properties of the molecule involved in inhibitory activity. Another special class of modified peptides includes inhibitors with aldehyde function located at the end of the structure. For example, the sequence benzyloxycarbonyl-Pro-Phe-CHO, which satisfies the known primary and secondary specificity requirements of chymotrypsin, is a potent reversible inhibitor of this target proteinase. It became clear that. The chemical structures of additional inhibitors with terminally located aldehyde functions such as antipine, leupeptin, chymostatin and elastatinal are also known from other known reversibles such as phosphoramidon, bestatin, puromycin and amastatin. Similar to the structure of sex modified peptide inhibitors, it is known in the art.

  Polypeptide protease inhibitors are more suitable for concentrated delivery in drug-carrier matrices than small compounds because of their relatively large molecular weight. Additional protease inhibitors within the scope of the formulations and methods according to the invention include the use of complexing agents. These agents mediate enzyme inhibition by depriving the intranasal environment (or pharmaceutical or therapeutic composition) of divalent cations that are cofactors for many proteases. For example, as an adjunct administered synchronously or as an adjunct formulated in combination at the appropriate concentration, the complexing agents EDTA and DTPA sufficiently inhibit the selected protease, thereby allowing the present invention to Facilitates intranasal delivery of such bioactive agents. Further representative examples of this type of inhibitor are EGTA, 1,10-phenanthroline and hydroxyquinoline. In addition, due to their tendency to chelate divalent cations, these and other complexing agents are useful as direct absorption enhancers in the present invention.

As described in more detail elsewhere herein, various polymers may be used as enzyme inhibitors in methods and compositions of synchronous administration, multi-processing and / or combination formulations according to the present invention. In particular, it is also contemplated to use mucoadhesive polymers. For example, poly (acrylate) derivatives such as poly (acrylic acid) and polycarbophil can affect the activity of various proteases including trypsin, chymotrypsin. The inhibitory action by these polymers may also be based on the complexation of divalent cations such as Ca ²⁺ and Zn ²⁺ . It is further contemplated that these polymers will function as conjugation partners or carriers for further inhibitors, as described above. For example, a chitosan-EDTA complex has been developed and is useful in the present invention in that it exhibits a strong inhibitory action on the enzyme activity of a zinc-dependent protease. The mucoadhesive properties of polymers in this context should not be substantially attenuated after other enzyme inhibitors are covalently added and are within the scope of the present invention for such polymers. The general utility as a delivery vehicle for drugs cannot be reduced. In contrast, the distance between the delivery vehicle and the mucosal surface shortened by the mechanism of mucoadhesion minimizes pre-systemic metabolism of the active agent, and covalently bound enzyme inhibitors allow drug delivery. Because it remains concentrated at the site, the unwanted dilution effect of the inhibitor and the resulting toxic effects and other side effects are also minimized. In this way, the effective amount of enzyme inhibitor administered synchronously can be reduced by eliminating the dilution effect.

  Exemplary mucoadhesive polymer-enzyme inhibitor complexes useful in the mucosal formulations and methods of the present invention include, but are not limited to: carboxymethylcellulose-pepstatin (anti Poly (acrylic acid) -Bowman-Birk inhibitor (anti-chymotrypsin); poly (acrylic acid) -chymostatin (anti-chymotrypsin); poly (acrylic acid) -elastinanal (anti-elastase); carboxymethylcellulose- Elastatinal (anti-elastase); polycarbophil-elastatinal (anti-elastase); chitosan-antipine (anti-trypsin); poly (acrylic acid) -bacitracin (anti-aminopeptidase N); chitosan-EDTA (anti-aminopeptidase) N, anti-carboxypeptidase A); chitosan-EDTA-antipie (Anti-trypsin, anti-chymotrypsin, anti-elastase).

Drugs and methods of mucolysis and mucosal removal Effective delivery of biotherapeutic agents by intranasal administration includes drug loss due to binding of the mucosal layer to glycoproteins, as well as low drug transport between the protective mucosa covering the nasal mucosa Efficiency must be considered. The normal mucous membrane is a viscoelastic gel-like substance containing water, electrolytes, mucins, macromolecules and shed epithelial cells. The mucosa functions primarily as a lubricious coating for cell protection of the underlying mucosal tissue. Mucus is located in the nasal epithelium and other mucosal epithelia and is secreted from randomly distributed secretory cells. The structural unit of mucus is mucin. This glycoprotein is mainly involved in the viscoelasticity of mucus, but other polymers may also contribute to the viscoelasticity. In airway mucus, these macromolecules also include locally produced secretory IgA, IgM, IgE, lysozyme and bronchotransferrin, which also play an important role in host defense mechanisms. Plays.

  The synchronized administration method of the present invention may optionally incorporate an effective mucosal degrading agent or mucosal removing agent that degrades or thins mucus on the intranasal mucosal surface. Or removing it serves to promote the absorption of intranasally administered biotherapeutic agents. In these methods, a mucosal degradant or mucosal remover will be administered synchronously as an adjunct compound to facilitate intranasal delivery of the bioactive agent. Alternatively, an effective amount of a mucosal degrading agent or mucosal removing agent is incorporated as an effective amount of the mucosal degrading agent or mucosal removing agent in the multiple treatment method of the present invention, or an additive in the combination preparation of the present invention. Is provided as an improved formulation that facilitates intranasal delivery of biotherapeutic compounds by reducing the barrier effect of the intranasal mucosa.

  Various mucosal degradation agents or mucosal removal agents are available for incorporation into the methods and compositions of the present invention. Mucosal degradation agents or mucosal removal agents can often be divided into the following groups based on their mechanism of action: proteases that cleave the protein core of mucin glycoprotein (eg, pronase, papain); Sulfhydryl compounds that break disulfide bonds; and surfactants that break non-covalent bonds in mucus (eg, Triton X-100, Tween 20). Additional compounds in this context include, but are not limited to, bile salts and surfactants such as, for example, sodium deoxycholate, sodium taurodeoxycholate, sodium glycocholate and lysophosphatidylcholine. Absent.

The efficiency of causing structural degradation of bile salt mucus is in the following order: deoxycholate>taurocholate> glycocholate. Other effective agents that reduce mucus viscosity or adhesion and facilitate intranasal delivery according to the methods of the present invention include, for example, short chain fatty acids and N-acyl collagen peptides, bile salts and saponins Etc. (the latter function in part by the chelation of Ca ²⁺ and / or Mg ²⁺ , which plays an important role in the maintenance of the mucosal layer structure).

  Additional mucosal degradation agents for use in the methods and compositions of the present invention are potent mucosal degradation agents that reduce both bronchopulmonary mucus viscosity and adhesion, and human growth hormone in anesthetized rats. N-acetyl-L-cysteine (ACS), which has been reported to moderately increase nasal bioavailability (7.5 to 12.2%). These and other mucosal degrading or mucosal removing agents typically contact the nasal mucosa in a concentration range of about 0.2 to 20 mM with synchronous administration with the bioactive agent to polar polar viscosity of intranasal mucus. And / or reduce elasticity.

  Still other mucosal degrading or mucosal removing agents can be selected from various glycosidase enzymes capable of cleaving glycosidic bonds within mucus glycoproteins. α-amylase and β-amylase represent the above classes of enzymes, although their mucosal degradation may be limited. In contrast, bacterial glycosidases allow these microorganisms to penetrate the host mucosal layer.

  In use in combination with most bioactive agents of the present invention, including peptide and protein therapeutics, generally non-ionogenic surfactants are also useful as mucosal degradation agents or mucosal removal agents. These agents typically do not alter or substantially impair the activity of the therapeutic polypeptide.

Viscosity-increasing agents Viscosity-increasing or suspending agents can affect the rate and absorption of drug release from the dosage formulation. As a result, viscosity increasing agents can be used to change the permeability of some glucose regulatory proteins. Examples of substances that can be used as pharmaceutically acceptable viscosity increasing agents include: methylcellulose (MC); hydroxypropylmethylcellulose (HPMC); carboxymethylcellulose (CMC); cellulose; gelatin; starch; hetastarch; poloxamer; Sorbitol, gum arabic, povidone, carbopol, polycarbophil, chitosan, chitosan microspheres, alginate microspheres, chitosan glutamate, amberlite resin, hyaluronan, ethyl cellulose, maltodextrin DE; Degradable starch microspheres (DSM); deoxyglycolate (GDC); hydroxyethyl cellulose HEC); hydroxypropyl cellulose (HPC); microcrystalline cellulose (MCC); polymethacrylic acid and polyethylene glycol; sulfobutyl ether B cyclodextrin; cross-linked eldexomer starch biosphere; sodium taurodihydrofusidate ( STDHF); N-trimethylchitosan chloride (TMC); degraded starch microspheres; amberlite resin; chitosan nanoparticles; spray-dried crospovidone; spray-dried dextran microspheres; spray-dried microcrystalline cellulose And cross-linked eldeoxymer starch microspheres.

Ciliary stasis and ciliary stasis methods Since the self-purifying ability of specific mucosal tissues (eg, nasal mucosal tissue) by mucociliary clearance is required as a protective function (removal of dust, allergens and bacteria), It is believed that this function should not be substantially impaired by mucosal drugs. Mucociliary transport within the airways is a particularly important defense mechanism against infection. To achieve this function, cilia pulsating in the nasal and respiratory tracts move the mucus layer along the mucosa to remove inhaled particles and microorganisms.

  Ciliary resting agents reduce the residence time of GRP, analogs and mimetics, and other bioactive agents described herein administered in mucosal (eg, intranasally) in the methods and compositions of the invention. Has an increasing use. In particular, the delivery of these agents in the methods and compositions of the present invention is markedly achieved by synchronous administration or combination preparations with one or more cilia quiescent agents that function to reversibly inhibit mucosal cell cilia activity. The residence time of the active agent administered accelerated and mucosally will be temporarily reversibly increased. When used in these aspects of the present invention, all of the aforementioned ciliary stasis factors, whether specific or indirect to cilia activity, cause unacceptable adverse side effects and are not mucosal cilia at the site of mucosal administration. Appropriate amounts (delivery) such that clearance is temporarily reduced (ie, reversibly) to facilitate delivery of GRP, analogs and mimetics, and other bioactive agents described herein. (Depending on the concentration, time and method) of the cilia.

  A variety of bacterial cilia resting factors that have been isolated and characterized in the literature can be used in embodiments of the present invention. Ciliary resting factor derived from bacteria of Pseudomonas aeruginosa includes phenazine derivatives, pyo compounds (2-alkali-4-hydroxyquinoline) and rhamnolipid (also known as hemolysin) It is. The pio compound produced cilia quiescence at a concentration of 50 μg / ml without clearly damaging the ultrastructure. The phenazine derivative also inhibited ciliary movement, but at a sufficiently higher concentration of 400 μg / ml than the pio compound, some membrane destruction occurred. Limited exposure of tracheal explants to rhamnolipid resulted in cilia quiescence with changes in the ciliary membrane. Exposure to larger amounts of rhamnolipid is accompanied by removal of dynein arms from the axon.

Surfactants and surfactant methods In more detailed aspects of the invention, one or more membrane permeation enhancers are used in the epithelial delivery methods or formulations of the invention to provide the GRP, analogs and The epithelial delivery of mimetics as well as other bioactive agents can be facilitated. Biomembrane permeation enhancers in this context can be selected from: (i) surfactants, (ii) bile salts, (iii) phospholipid additives, mixed micelles, liposomes or carriers, (iv) Alcohol, (v) enamine, (vi) NO donor compound, (vii) long chain amphiphilic molecule (viii) small hydrophobic permeation enhancer; (ix) sodium or salicylic acid derivative; (x) glycerol ester of acetoacetic acid (Xi) cyclodextrin or β-cyclodextrin derivative, (xii) medium chain fatty acid, (xiii) chelating agent, (xiv) amino acid or salt thereof, (xv) N-acetylamino acid or salt thereof, (xvi) selected An enzymatic degradation agent for membrane components, (xvii) an inhibitor of fatty acid synthesis, (xviii) an inhibitor of cholesterol synthesis; Is a combination of any of the membrane permeation enhancers described in (xix) (i)-(xviii).

  Certain surfactants (surfactants) are readily incorporated into the epithelial delivery formulations and methods of the present invention as epithelial absorption enhancers. These agents that can be formulated in conjunction with or combined with the GRPs, analogs and mimetics described herein, and other bioactive agents can be selected from a wide range of known surfactants. Surfactants are generally divided into two classes: (1) nonionic polyoxyethylene ethers; (2) biles such as sodium glycocholate (SGC) and deoxycholate (DOC). Acid salts; and (3) fusidic acid derivatives such as sodium taurodihydrofusidate (STDHF). The mechanism of action of these various classes of surfactants typically includes solubilization of bioactive agents. In the case of proteins and peptides that often form aggregates, the surface-active properties of these absorption enhancers allow smaller units, such as surfactant-coated monomers, to be more rapidly maintained in solution. Interaction with various proteins. Examples of other surfactants include L-α-phosphatidylcholine didecanoyl (DDPC) polysorbate 80 and polysorbate 20. These monomers are probably units that are easier to transport than aggregates. A second potential mechanism is the protection of peptides or proteins from proteolysis by proteases in the mucosal environment. Both bile salts and certain fusidic acid derivatives have been reported to inhibit protein proteolysis by nasal homogenates at concentrations below or similar to those required to promote protein absorption. This protease inhibition will be particularly important for peptides with a short biological half-life.

Degrading enzymes and inhibitors of fatty acid and cholesterol synthesis In suitable aspects of the invention, the GRP, analogs and mimetics, and other bioactive agents described herein for biomembrane administration are degrading enzymes or metabolites. Formulated or co-administered with a permeation enhancer selected from stimulants or fatty acid synthesis inhibitors, sterols or other selected epithelial barrier components, US Pat. No. 6,190,894. For example, by using degrading enzymes such as phospholipase, hyaluronidase, neuraminidase and chondroitinase, mucosal penetration of GRP, analogs and mimetics, and other bioactive agents without irreversible damage to the mucosal barrier Can be promoted. In one aspect, chondroitinase is used in a method or composition provided herein to alter a glycoprotein or glycolipid component of a mucosal osmotic barrier, thereby altering the GRP, analogs described herein. Mucosal absorption of the body and mimetics and other bioactive agents is facilitated.

  With regard to inhibitors of synthesis of mucosal barrier components, it is noted that free fatty acids account for 20-25% by weight of epithelial lipids. Two rate-limiting enzymes in the biosynthesis of free fatty acids are acetyl CoA carboxylase and fatty acid synthase. Free fatty acids are metabolized into phospholipids through a series of steps. Accordingly, inhibitors of free fatty acid synthesis and metabolism for use in the methods and compositions of the present invention include inhibitors of acetyl CoA carboxylase such as 5-tetradecyloxy-2-furancarboxylic acid (TOFA); fatty acid synthesis Inhibitors of enzymes; Phospholipase A such as trashin A, 2- (p-amylcinamyl) amino-4-chlorobenzoic acid, bromophenacyl bromide, monoalide, 7,7-dimethyl-5,8-eicosadienoic acid, Nicergoline, cephalanthin, nicardipine, quercetin, dibutyryl-cyclic AMP, R-24571, N-oleylethanolamine, N- (7-nitro-2,1,3-benzoxadiazol-4-yl) phosphatidylserine, cyclosporine A, dibucaine, prenylamine, all trans and 13-cis-retinoic acid, etc. Local anesthetics containing retinoids, inhibitors of W-7, trifluoperazine, R-24571 (calmidazolium), 1-hexadosyl-3-trifluoroethylglycero-sn-2-phosphomenthol (MJ33); nicardipine Channel blockers, including quinacrine, mepacrine, chloroquine and hydroxychloroquine; β-blockers including propanalol and labetalol; calmodulin antagonists; EGTA; timmersol; dexamethasone and prednisolone Corticosteroids including: non-steroidal anti-inflammatory drugs including but not limited to indomethacin and naproxen Not.

  Free sterols, primarily cholesterol, represent 20-25 weight percent of epithelial lipids. The rate-limiting enzyme in cholesterol biosynthesis is 3-hydroxy-3-methylglutaryl (HMG) CoA reductase. Inhibitors of cholesterol inhibition for use in the methods and compositions of the present invention include competitive inhibitors of (HMG) CoA reductase such as simvastatin, lovastatin, fluindostatin (fluvastatin), pravastatin, mevastatin, and cholesterol oleate Competitive inhibitors of other HMGCoA reductases such as cholesterol sulfate and cholesterol phosphate and oxidized sterols such as 25-OH-; and 26-OH-cholesterol; inhibitors of squalene synthase; squalene epoxidase; -Inhibitors of DELTA7 or DELTA24 reductase such as, but not limited to, diazacholesterol, 20,25-diazacholstenol, AY9944, and triparanol.

  Each of the inhibitors of fatty acid synthesis or sterol synthesis is co-administered or combined with one or more GRPs, analogs and mimetics as described herein, and other bioactive agents. Formulation can promote epithelial penetration of the active agent. The effective concentration range of a sterol inhibitor in a therapeutic or ancillary formulation for mucosal delivery is generally about 0.0001% to about 20% by weight of the total, more typically about 0.00%. 01% to about 5%.

Nitric Oxide Donor and Nitric Oxide Donation Method In another suitable aspect of the invention, the nitric oxide (NO) donor is one or more of the GRP, analogs and mimetics described herein. As well as biomembrane permeation enhancers to facilitate epithelial delivery of other bioactive agents. A variety of NO donors are known in the art and are useful at concentrations effective in the methods and compositions of the present invention. Representative NO donors include nitroglycerin, nitroprusside, NOC5 [3- (2-hydroxy-1- (methylethyl) -2-nitrosohydrazino) -1-propanamide], NOC12 [N-ethyl-2 -(1-ethylhydroxy-2-nitrosohydrazino) ethanamine], SNAP [S-nitroso-N-acetyl-DL-penicillamine], NORI and NOR4, but are not limited to these. In the methods and compositions of the present invention, an effective amount of the selected NO donor, along with one or more GRPs, analogs and mimetics described herein, and other bioactive agents, is epithelial. They can be administered synchronously or in combination, either inwardly or via the epithelium.

Agents for Modulating Epithelial Junction Structures and / or Physiological Functions The present invention is directed to one or more GRPs, analogs and mimetics, and / or other organisms in conjunction with the epithelial delivery enhancers disclosed herein. A pharmaceutical composition comprising an active agent and formulated into a pharmaceutical formulation for epithelial delivery is provided.

  The permeabilization agent typically reversibly promotes mucosal epithelial paracellular transport by modulating epithelial junction structures and / or mucosal epithelial surface physiology within the subject. This effect is typically accompanied by inhibition of homotypic or heterotypic binding between the epithelial adhesion proteins of neighboring epithelial cells by the permeabilizing agent. The target protein for blocking this homotypic or heterotypic binding can be selected from a variety of suitable junction adhesion molecules (JAM), occludin, or claudin. Examples include antibodies, antibody fragments or single chain antibodies that bind to the extracellular domain of these proteins.

  In a further detailed aspect, the present invention provides permeabilizing peptides and peptide analogs and mimetics for promoting mucosal epithelial paracellular transport. The subject peptides and peptide analogs and mimetics typically function in the compositions and methods of the invention by modulating the structure and / or physiology of epithelial junctions within the subject mammal. In certain embodiments, peptides and peptide analogs and mimetics effectively inhibit homotypic and / or heterotypic binding of an epithelial adhesion protein selected from junction adhesion molecules (JAM), occludin or claudin.

  One such well-studied drug is a bacterial toxin derived from Vibrio cholerae known as “zonula occludens toxin” (ZOT). The toxin mediates increased intestinal mucosal permeability within the infected subject, causing disease symptoms including diarrhea. Fasano, et al Proc. Nat. Acad. ScI, USA 5: 5242-5246, 1991. When tested on rabbit ileal mucosa, ZOT increased intestinal permeability by modulating the structure of the intercellular tight junction. More recently, it has been found that ZOT can reversibly open tight junctions in the intestinal mucosa. It has also been reported that ZOT can reversibly open tight junctions in the nasal mucosa. U.S. Patent No. 5,908,825.

  In the methods and compositions of the present invention, ZOT and various analogs and mimetics of ZOT that function as agonists or antagonists of ZOT activity increase paracellular absorption into or between nasal mucosa. Useful to facilitate intranasal delivery of bioactive agents. In this respect, ZOT typically acts by causing a restructuring of the structure of the tight junction, which is characterized by a change in the position of the junction protein ZO1. In these aspects of the invention, ZOT can be co-administered or formulated with an effective amount of a bioactive agent to reversibly permeabilize the nasal mucosa without causing substantial adverse side effects. Increase the absorption of the active agent significantly.

Vasodilators and vasodilator methods Another class of absorption enhancers that exhibit beneficial utility in synchronized administration and combination formulations according to the methods and compositions of the present invention are vasoactive compounds, more specifically. Is a vasodilator. These compounds, in the present invention, modulate the structure and physiology of the submucosal vasculature to enter or through the epithelium and / or to specific target tissues or compartments (eg, systemic circulation or central It has the function of increasing the transport rate of GRP, analogs and mimetics, and other bioactive agents to the nervous system.

  Vasodilators for use in the present invention are typically mucosal either by reducing cytoplasmic calcium, increasing nitric oxide (NO), or inhibiting myosin light chain kinase. Relax the underlying blood vessels. These vasodilators fall into the following nine classes: calcium antagonists, potassium channel openers, ACE inhibitors, angiotensin II receptor antagonists, α-adrenergic and imidazole receptor antagonists, β1-adrenergic Agonists, phosphodiesterase inhibitors, eicosanoids and NO donors.

  Despite chemical differences, the pharmacokinetic properties of calcium antagonists are similar. The rate of absorption into the systemic circulation is high, so these drugs will undergo substantial first-pass metabolism by the liver, resulting in individual pharmacokinetic differences. With the exception of new drugs of the dihydropyridine type (amlodipine, felodipine, isradipine, nilvadipine, nisoldipine and nitrendipine), the half-life of calcium antagonists is short. Thus, maintaining these many effective drug concentrations may require multiple doses as described elsewhere herein, or delivery by sustained release formulations. Treatment with the potassium channel opener minoxidil may limit the level and method of administration due to its potentially harmful side effects.

  AEC inhibitors prevent the conversion of angiotensin-I to angiotensin-II, and are most effective when renin production is increased. Since ACE is identical to kininase II, which inactivates bradykinin, a potent endogenous vasodilator, inhibition of ACE reduces bradykinin degradation. ACE inhibitors provide additional benefits of cardioprotection and cardiac recovery by preventing cardiac fibrosis and ventricular hypertrophy and reversing progression in model animals. The main excretion route for most ACE inhibitors is via renal excretion. Therefore, kidney damage is associated with a low excretion rate and it is recommended to reduce the dose by 25 to 50% in patients suffering from moderate to severe kidney damage.

  For NO donors, these compounds are particularly useful in the present invention because of their further effect on mucosal permeability. In addition to the NO donors described above, NO / nucleophiles or NO complexes with nucleophiles called NONOates, when dissolved in an aqueous solution at physiological pH, naturally non-enzymatically convert NO. discharge. In contrast, nitrovasodilators such as nitroglycerin require special enzyme activity for NO release. NONOate releases NO at a specific stoichiometry at a predictable rate in the range of less than 3 minutes for diethylamine / NO to about 20 hours for diethylenetriamine / NO (DETANO).

  In certain methods and compositions of the invention, selected vasodilators, along with one or more GRP, analogs and mimetics, and other bioactive agents, promote mucosal absorption of the active agent. Administered in an effective amount (e.g., systemically or intranasally, simultaneously or in combination with effective temporal relevance) or formulated in combination to target tissue in a subject Or it reaches the compartment (eg liver, hilar vein, CNS tissue or fluid, or plasma).

Selective transport enhancers and transport facilitating methods The compositions and delivery methods of the present invention may optionally incorporate selective transport facilitators that facilitate transport of one or more bioactive agents. One or more additional bioactive agents using these transport enhancers in combination formulations or synchronized administration protocols using one or more of the GRPs, analogs and mimetics described herein Synchronously facilitates delivery between multiple biomembrane transport barriers and facilitates epithelial delivery of active agents to target tissues or compartments within a subject (eg, mucosal epithelium, liver, CNS tissue or fluid, or plasma) Can be reached. Alternatively, transport enhancers are used in combination formulations or synchronized administration protocols to facilitate or without further delivery of additional bioactive agents, one or more GRPs, analogs and mimetics Can also directly facilitate epithelial delivery.

  Representative selective transport enhancers for use in the above aspects of the present invention include binders such as lectins that are known to specifically interact with glycosides, sugar-containing molecules, and epithelial transport barrier components. Including, but not limited to. For example, certain “bioadhesive” ligands, including lectins from various plants and bacteria, that bind to cell surface sugar moieties by receptor-mediated interactions, can be applied to the mucosa of bioactive agents within the scope of the present invention, For example, it can be used as a carrier or conjugated transport vehicle to facilitate nasal delivery. Certain bioadhesive ligands for use in the present invention are directed to epithelial target cells of biological signals that initiate selective uptake of the adhesive ligand by a special cell transport process (endocytosis or transcytosis). Will mediate the transmission of Thus, using these transport media as a “carrier system”, selective uptake of one or more GRPs, analogs and mimetics, and other bioactive agents through and / or through the mucosal epithelium. Can be stimulated or controlled. These and other selective transport enhancers significantly facilitate epithelial delivery of macromolecular biopharmaceuticals (particularly peptides, proteins, oligonucleotides and polynucleotide vectors) within the scope of the present invention. Lectins are plant proteins that bind to specific sugars found on the surface of eukaryotic glycoproteins and glycolipids. Concentrated solutions of lectins exhibit a “mucotractive” action, and various studies have shown that rapid mediated lectin and lectin conjugates between mucosal surfaces (eg, concanavalin A conjugated with colloidal gold particles). Sexual endocytosis (RME) has been demonstrated. Further studies have reported that the lectin uptake mechanism can be used for intestinal drug targeting in vivo. Certainly from these studies, polystyrene nanoparticles (500 nm) were covalently bound to tomato lectin, and it was reported that systemic uptake was improved after oral administration to rats.

  In addition to plant lectins, bacterial-derived adhesion and invasion factors also provide a rich source of candidate substances for use as adhesive / selective transport carriers in the mucosal delivery methods and compositions of the present invention. Bacterial adhesion processes require two types of components: bacterial-derived “adhesins” (adhesion or colonization factors) and receptors on the host cell surface. Bacteria causing mucosal infection need to penetrate the mucus layer before attaching to the epithelial surface. The attachment is usually mediated by bacterial cilia or hairy structures, although other components of the cell surface will also contribute to this process. Adherent bacteria settle in the mucosal epithelium by proliferation and initiation of a series of biochemical reactions (with or without the help of toxins) within the target cell. Various bioadhesive proteins (for example, invasin and internalin) originally produced from various bacteria and viruses are known as relating to these invasion mechanisms. These bioadhesive proteins allow such microorganisms to adhere extracellularly to host species and even specific target tissues with surprising selectivity. The signal transmitted by such receptor-ligand interactions is the transport of intact living microorganisms into and ultimately through the process of endocytosis and transcytosis. To start. Such phenomena occurring in nature can be used to facilitate delivery of bioactive compounds into or between biological membranes and / or other specified target sites of drug action, according to the teachings herein. (Eg, by conjugating a bioactive agent such as GRP with an adhesin).

  Various bacterial and plant-derived toxins that bind to epithelial surfaces in a specific, lectin-like manner are also useful in the methods and compositions of the invention. For example, diphtheria toxin (DT) rapidly enters host cells by RME. Similarly, the B subunit of E. coli heat labile toxin binds to the brush border of intestinal epithelial cells in a very specific lectin-like manner. Uptake and transcytosis of the toxin outside the basolateral side of enterocytes has been reported in vivo and in vitro. Other researchers expressed the transmembrane domain of diphtheria toxin in E. coli as a maltose binding fusion protein and chemically conjugated it to poly-L-lysine with a high molecular weight. The resulting complex succeeded in mediating reporter gene internalization in vitro. In addition to the above examples, Staphylococcus aureus is a series of proteins that act as both superantigens and toxins (eg, Staphylococcus enterotoxin A (SEA), SEB, Toxic shock syndrome toxin I (TSST- I)) is produced. Studies on these proteins have reported enhanced dose-dependent transcytosis of SEB and TSST-I in Caco-2 cells.

  Viral hemagglutinin constitutes another type of transport agent to facilitate mucosal delivery of bioactive agents within the methods and compositions of the invention. The first step in many viral infections is the binding of surface proteins (hemagglutinin) to mucosal cells. These binding proteins have been identified for most viruses, including rotavirus, varicella-zoster virus, Semliki Forest virus, adenovirus, potato cigar virus, and reovirus. These and other representative virus-derived hemagglutinins can be combined with one or more GRP, analogs and mimetics described herein in combination (eg, mixed or conjugated) or synchronized administration protocols. Used to synchronously facilitate mucosal delivery of one or more additional bioactive agents. Alternatively, virus-derived haemagglutinin is used in combination formulations or synchronous administration protocols to facilitate or without further delivery of additional bioactive agents, one or more GRPs, analogs, and Can also directly facilitate mucosal delivery of mimetics

  A variety of endogenous, selective transport mediators can also be utilized in the present invention. Mammalian cells have developed various mechanisms that promote the internalization of specific substances and target these substances to specific compartments. Collectively, these membrane deformation processes are termed “endocytosis” and include phagocytosis, phagocytosis, receptor-mediated endocytosis (clathrin-mediated RME), and protocytosis (non-clathrin). Mediated RME). REM is a highly specialized cellular biological process that, as its name suggests, various ligands bind to cell surface receptors and are subsequently transported into cells. It occurs in. In many cells, the endocytosis process is active enough that in less than 30 minutes all membrane surfaces are internalized and replaced. Based on its orientation in the cell membrane, two classes of receptors have been proposed; the amino terminus of type I receptors are located outside the membrane, whereas type II receptors have the same protein tail, the intracellular environment. Have.

  In a further aspect of the invention, transferrin is utilized as a carrier for epithelially delivered bioactive agents or as an RME stimulator. Transferrin, an 80 kDa iron transport glycoprotein, is efficiently taken up into cells by RME. Transferrin receptors can be found on the surface of most proliferating cells, and the number is increased on the surface of erythroblasts and many types of tumors. It has been reported that transcytosis of transferrin (Tf) and transferrin conjugates is increased by the presence of brefeldin A (BFA), a fungal metabolite. Another study reports that BFA treatment rapidly increases both lysine and HRP endocytosis at the apex of MDCK cells. Thus, in the methods of the present invention, BFA and other agents that promote receptor-mediated transport are used as agents that are formulated (eg, conjugated) and / or administered synchronously in combination. Can facilitate receptor-mediated transport of bioactive agents, including analogs and mimetics.

Polymer Delivery Excipients and Methods Within the scope of certain aspects of the invention, the GRP, analogs and mimetics disclosed herein, other bioactive substances, and delivery enhancements as described above The agents are incorporated individually or in combination within a formulation that is administered transepithelially (eg, nasally) containing a biocompatible polymer that functions as a carrier or base. Such polymer carriers include polymer powders, matrices, or particulate delivery excipients, among other polymer forms. The polymer may be of plant, animal or synthetic origin. The polymer is often crosslinked. Furthermore, in these delivery systems, GRP, analogs, or mimetics can be functionalized in a manner that can be covalently bound to the polymer and made inseparable from the polymer by simply being ished. In other embodiments, the polymer is chemically modified with inhibitors of enzymes or other drugs that can degrade or inactivate bioactive agents and / or delivery enhancers. In certain formulations, the polymer is a partially or completely water insoluble but water swellable polymer such as, for example, a hydrogel. The polymers useful in this aspect of the invention are inherently water-interacting and / or hydrophilic and desirably absorb large amounts of water, for a period of time sufficient to reach equilibrium with water. Often forms a hydrogel when placed in contact with an aqueous medium. In a more detailed aspect, the polymer is a hydrogel that, when placed in contact with excess water, absorbs at least twice its weight of water at equilibrium when exposed to water at room temperature (US Pat. No. 6 , 004,583).

    Drug delivery systems based on biodegradable polymers are preferred in many biomedical applications because such systems are degraded to non-toxic molecules either by hydrolysis or enzymatic reactions. The degradation rate is controlled by manipulating the composition of the biodegradable polymer matrix. Thus, this type of system can be used in specific situations for long-term release of bioactive substances. Biodegradable polymers such as poly (glycolic acid) (PGA), poly- (lactic acid) (PLA), and poly (D, L-lactic acid-co-glycolic acid) (PLGA) are the degradation products of these polymers. Has been attracting great attention as a possible drug delivery carrier. During the body's normal metabolic function, these polymers break down into carbon dioxide and water. These polymers also show excellent biocompatibility.

  In order to prolong the biological activity of the GRP, analogs, mimetics, and other bioactive substances and any delivery enhancing agents disclosed herein, these agents may be used, for example, polyorthoesters, polyanhydrides. Or may be incorporated into a polymer matrix such as polyester. This maintained the activity and release of the active substance as measured, for example, by degradation of the polymer matrix. Despite the ability to stabilize these molecules during storage and delivery by encapsulating biotherapeutic molecules within a synthetic polymer, the biggest obstacles to polymer-based release technology are often thermal, sonication, Or the loss of activity of the therapeutic molecule during the formulation process involving organic solvents.

  Absorption-promoting polymers intended for use within the scope of the present invention are desirable for useful applications such as modification, modification, or blending, such as water absorption, hydrogel formation, and / or chemical stability. In addition to other natural or synthetic polymers, rubbers, resins, and other drugs and blends of these materials with each other or with other polymers, as long as they do not adversely affect the properties of And chemically or physically modified versions. In a more detailed aspect of the present invention, polymers such as nylon, acrilan, and other normally hydrophobic synthetic polymers are fully modified by reaction, rendered water swellable, and / or stable in aqueous media. A gel may be formed.

  Absorption promoting polymers of the present invention include acrylic and methacrylic acid, acrylamide, methacrylamide, hydroxyethyl acrylate or methacrylate, vinyl monomers such as vinyl pyrrolidone, polyvinyl alcohol and its copolymers and terpolymers, polyvinyl acetate and the above listed. Mention may be made of copolymers and terpolymers with monomers and polymers from the group of homopolymers and copolymers based on various combinations of 2-acrylamido-2-methyl-propanesulfonic acid (AMPS®). Acrylic or methacrylamide acrylate or methacrylate derived from the above-listed monomers and a linear or branched alkyl or aryl group having an aromatic ring of 4 or less whose ester group may contain an alkyl substituent of 1 to 6 carbon atoms Copolymerizable functional monomers such as esters, N, N-dimethylaminoalkyl (meth) acrylamide, dimethylaminoalkyl (meth) acrylate, (meth) acryloxyalkyltrimethylammonium chloride, (meth) acryloxyalkyldimethyl Very useful are steroidal compounds such as benzylammonium chloride, sulfuric acid, phosphoric acid, or copolymers with cationic monomers.

  Further absorption-promoting polymers used within the scope of the present invention are polymers derived from dextran, polymers classified as dextrin, materials classified as natural rubber and resins, or processed collagen, chitin, chitosan, pullulan, zoolan. (Zooglan), alginate, and modified alginate such as “Kelcoloid” (polypropylene glycol modified alginate), gellan gum such as “Kelocogen”, xanthan gum such as “Keltrol” , Polymers derived from natural polymer classes such as estastine, α-hydroxybutyrate and its copolymers, hyaluronic acid and its derivatives, polylactic acid and glycolic acid.

  A very useful class of polymers applicable within the scope of the present invention is an olefinically unsaturated carboxylic acid containing at least one activated carbon-carbon olefinic carbon double bond and at least one carboxyl group, i. An acid or functional group that is easily converted to an acid containing an olefinic double bond that functions easily in polymerization because it is present in either monomer molecule at the β-position or as part of the terminal methylene group . As this type of olefinically unsaturated acid, acrylic acid itself, α-cyanoacrylic acid, β-methylacrylic acid (crotonic acid), acrylic acid represented by α-phenylacrylic acid, β-acryloxypropionic acid, cinnamon Represented by acids, p-chlorocinnamic acid, 1-carboxy-4-phenylbutadiene-1,3-itaconic acid, citraconic acid, mesaconic acid, glutaconic acid, aconitic acid, maleic acid, fumaric acid, and tricarboxyethylene Examples include materials such as acrylic acid. As used herein, the term “carboxylic acid” is formed by the elimination of one water molecule from two carboxyl groups where the polycarboxylic acid and anhydride groups are located on the same carboxylic acid molecule. These anhydrides, such as maleic anhydride.

  Typical acrylates useful as absorption promoters within the scope of the present invention include methyl acrylate, ethyl acrylate, propyl acrylate, isopropyl acrylate, butyl acrylate, isobutyl acrylate, methyl methacrylate, ethacrylyl. Examples include methyl acid, ethyl methacrylate, octyl acrylate, heptyl acrylate, octyl methacrylate, isopropyl methacrylate, 2-ethylhexyl methacrylate, nonyl acrylate, hexyl acrylate, and n-hexyl methacrylate. Higher alkyl acrylates are decyl acrylate, isodecyl methacrylate, lauryl acrylate, stearyl acrylate, behenyl acrylate, and melyl acrylate, and their methacrylic acid versions. A mixture of 2 or 3 or 4 or more long chain acrylates can be successfully polymerized with one carboxylic acid monomer. Other comonomers include olefins including alpha olefins, vinyl ethers, vinyl esters, and mixtures thereof.

  Other vinylidene monomers, including acrylonitrile, are also used as absorption enhancers within the methods and compositions of the present invention to target tissue or compartments within the subject (eg, liver, hepatic vein, CNS tissue or fluid). Or enhanced delivery of one or more GRP, analogs and mimetics, as well as other bioactive substances, including enhanced delivery of the active substance to plasma). Useful α-, β-olefinically unsaturated nitriles are preferably monoolefinically unsaturated nitriles having 3 to 10 carbon atoms such as acrylonitrile, methacrylonitrile and the like. Most preferred are acrylonitrile and methacrylonitrile. C3-C35 acrylic amides including monoolefinically unsaturated amides may be used. Representative amides include acrylamide, methacrylamide, Nt-butylacrylamide, N-cyclohexylacrylamide, higher alkylamides having 8 to 32 alkyl groups on the nitrogen, N-methylolacrylamide, N-propanol. Α, β-olefinic unsaturation, including those of 4 to 10 carbon atoms such as acrylamide, N-methylol methacrylamide, N-methylol maleimide, N-methylol maleate, N-methylol-p-vinylbenzamide and the like Examples include acrylic acid amides including N-alkylolamides of carboxylic acids.

  Further useful absorption enhancers are C2-C18, more preferably C2-C8 α-olefins; C4-C10 dienes; vinyl esters such as vinyl acetate and allyl esters; Vinyl aromatics such as styrene, methylstyrene, and chlorostyrene; vinyl and allyl ethers and ketones such as vinyl methyl ether and methyl vinyl ketone; chloroacrylates; α-cyanomethyl acrylate and α-, β Cyanoalkyl acrylates such as-and γ-cyanopropyl acrylate; alkoxy acrylates such as methoxyethyl acrylate; haloacrylates such as chloroethyl acrylate; vinyl halides and vinyl chloride, vinylidene chloride and the like; divinyl ether Divinyls such as diethylene glycol diacrylate, ethylene glycol dimethacrylate, methylene-bis-acrylamide, allylpentaerythritol, diacrylates, and other polyfunctional monomers; and bis (β-chloroethyl) vinylphosphonate Bis (β-haloalkyl) alkenylphosphonates, which are known to those skilled in the art. Copolymers in which the carboxy-containing monomer is a minor component and other vinylidene monomers present as a major component are readily prepared according to the methods disclosed herein.

When hydrogels are used as absorption promoters within the scope of the present invention, these are acrylic and methacrylic groups, acrylamide groups, methacrylamide groups, hydroxyethyl acrylate (HEA) which exhibit water interaction and water swellability. ) Groups or hydroxyethyl methacrylate (HEMA) groups, as well as synthetic copolymers derived from vinylpyrrolidone groups. Specific examples of polymers particularly useful for peptide or protein delivery are the following types of polymers: (meth) acrylamide and 0.1 to 99% by weight (meth) acrylic acid; (meth) acrylamides and 0.1 to 75% by weight (meth) acryloxyethyltrimethylammonium chloride; (meth) acrylamide and 0.1 to 75% by weight (meth) acrylamide; acrylic acid and 0.1 to 75% by weight alkyl (meth) ) Acrylates; (meth) acrylamide and 0.1 to 75 wt% AMPS.RTM. (Trademark of Lubrizol); (meth) acrylamide and 0 to 30 wt% alkyl (meth) acrylamides, and 0.1 To 75% by weight of AMPS.RTM .; (meth) acrylamide and 0.1 99% by weight HEMA; (meth) acrylamide and 0.1 to 75% by weight HEMA, and 0.1 to 99% by weight (meth) acrylic acid; (meth) acrylic acid and 0.1 to 99% by weight HEMA; 50 mol% vinyl ether and 50 mol% maleic anhydride; (meth) acrylamide and 0.1 to 75 wt% (meth) acryloxyalkyldimethylbenzylammonium chloride; (meth) acrylamide and 0.1 to 99% by weight vinyl pyrrolidone; (meth) acrylamide and 50% by weight vinyl pyrrolidone, and 0.1 to 99.9% by weight (meth) acrylic acid; (meth) acrylic acid and 0.1 to 75% by weight AMPS.RTM. And 0.1 to 75% by weight of alkyl (meth) acrylamide. In the above examples, alkyl means C ₁ to C ₃₀ , preferably C ₁ to C ₂₂ straight and branched chain and C ₄ to C ₁₆ cyclic, and when (meth) is used, a methyl group is It means that monomers are included and not included. Other very useful hydrogel polymers are the swellable but insoluble versions of poly (vinyl pyrrolidone) starch, carboxymethylcellulose, and polyvinyl alcohol.

  Additional polymerized hydrogel materials useful within the scope of the present invention include (poly) hydroxyalkyl (meth) acrylates: anionic and cationic hydrogels: poly (electrolyte) complexes; poly (vinyl alcohols) with low acetic acid residues: cross-linking Swellable mixture of agar and crosslinked carboxymethylcellulose: a swellable composition comprising methylcellulose mixed with microcrosslinked agar; water swellability produced by dispersion of a finely divided copolymer of maleic anhydride and styrene, ethylene, propylene, or isobutylene Copolymers; Water-swellable polymers of N-vinyl lactams; and swellable sodium salts of carboxymethyl cellulose.

  Other gelling fluid water absorption and retention polymers useful for the formation of hydrophilic hydrogels for mucosal delivery of bioactive substances within the scope of the present invention include pectin; agar, gum arabic, karaya, tragacenth, algin , And guar, and polysaccharides such as crosslinked versions thereof; acrylic acid polymers, copolymers, and salt derivatives, polyacrylamides; water swellable indene maleic anhydride polymers; starch graft copolymers; about 2 to 400 times the original weight Water-absorbing acrylate-type polymers and copolymers; polyglucan diesters; a mixture of cross-linked poly (vinyl alcohol) and poly (N-vinyl-2-pyrrolidone); polyoxybutylene-polyethylene block copolymer gel; carob bean gum; Polyester ; Polyurea gel; polyether gel; polyamide gel; polyimide gel; polypeptide gel; polyamino acid gel; poly cellulose gel; crosslinked indene - acrylate maleic anhydride polymers; and polysaccharides.

  Synthetic hydrogel polymers used within the scope of the present invention can be made by myriad combinations of several monomers in several ratios. The hydrogel may crosslink, which generally has the ability to assimilate and absorb fluids and swell or expand to an expanded equilibrium. Hydrogels usually swell or expand upon delivery to the nasal mucosal surface and absorb water about 2 to 5 times, 5 to 10 times, 10 to 50 times, 50 to 100 times, or more of its weight. . For various bioactive substances, the molecular weight, size, solubility, and diffusion properties of the active substance that is transported by or trapped within or encapsulated within the polymer, as well as the specific spacing and cooperation associated with each polymer Depending on factors such as chain movement, the optimal degree of swelling for a given hydrogel is determined.

  Hydrophilic polymers useful within the scope of the present invention are water insoluble but water swellable. Such a polymer swollen with water is usually referred to as a hydrogel or gel. Such gels can be conveniently generated from water soluble polymers by the process of cross-linking the polymer with a suitable cross-linking agent. However, stable hydrogels can also be formed from certain polymers under defined conditions of pH, temperature, and / or ionic concentration, according to methods known in the art. In general, polymers that are crosslinked, i.e., crosslinked to such an extent that the polymer has good hydrophilicity, have improved physical integrity (as compared to non-crosslinked polymers of the same or similar type) and are suitable in position. And the ability to retain within the gel network together with the desired biologically active substance and additional compounds for co-administration with it, such as cytokines or enzyme inhibitors, while retaining the ability to release the active substance over time. .

  In general, the hydrogel polymers used within the scope of the present invention are from 0.01 to 25% by weight, more preferably from 0.1 to 20% by weight, more frequently from 0. Crosslink with a bifunctional crosslinker in an amount of 1 to 15% by weight. Another useful amount of cross-linking agent is 0.1 to 10% by weight. Trifunctional, tetrafunctional, or higher polyfunctional crosslinking agents may be used. When using such reagents, small amounts may be required to obtain equivalent crosslink density, i.e., degree of crosslinking, or network properties sufficient to efficiently contain a bioactive substance.

  Crosslinking may be a covalent bond, an ionic bond, or a hydrogen bond with a polymer that has the ability to swell in the presence of a hydrous fluid. Such cross-linking agents and reactions are known to those skilled in the art and often depend on the polymer system. Therefore, a crosslinked network can be formed by free radical copolymerization of unsaturated monomers. Polymer hydrogels are formed by cross-linking of preformed polymers by reaction of functional groups found on polymers such as alcohols, acids and amines with groups such as glyoxal, formaldehyde or glutaraldehyde, dianhydrides. Also good.

The polymer can be any polyene such as, for example, decadiene or trivinylcyclohexane; an acrylamide such as N, N-methylene-bis (acrylamide); a multifunctional acrylate such as trimethylolpropane triacrylate; or, for example, divinyl It may be cross-linked with a multifunctional vinylidene monomer containing at least two terminal CH ₂ groups, including benzene, divinylnaphthalene, allyl acrylate, and the like. In certain embodiments, the crosslinking monomer for use in preparing the copolymer is made, for example, by etherification of a polyhydric alcohol containing at least 2 carbon atoms and at least 2 hydroxyl groups, Polyalkenyl polyethers having more than one alkenyl ether group per molecule, where the double bond may have an alkenyl group present attached to a terminal methylene group. This type of compound can be produced by reaction of an alkenyl halide such as allyl chloride or allyl bromide with a strong alkaline aqueous solution of one or more polyhydric alcohols. The product may be a complex mixture of polyethers and various numbers of ether groups. The efficiency of the polyether crosslinker increases with the number of potentially polymerizable groups on the molecule. Usually, polyethers containing an average of 2 or 3 or more alkenyl ether groups per molecule are used. Other crosslinking monomers include, for example, diallyl esters, dimethallyl ethers, allyl or methallyl acrylates and acrylamides, tetravinylsilane, polyalkenylmethanes, diacrylates, and dimethacrylates, divinylbenzene. And divinyl compounds, polyallyl phosphates, diallyloxy compounds, and phosphites. Typical drugs are allyl pentaerythritol, allyl sucrose, trimethylol propane triacrylate, 1,6-hexanediol diacrylate, trimethylol propane diallyl ether, pentaerythritol triacrylate, tetramethylene dimethacrylate, ethylene diacrylate, ethylene diacrylate. Methacrylate, triethylene glycol dimethacrylate and the like. Allyl pentaerythritol, trimethylol propane diallyl ether, and allyl sucrose provide suitable polymers. When a crosslinker is present, the polymer mixture is typically about 0.01 to 20% by weight, such as 1%, 5%, or 10%, or more, based on total carboxylic acid monomer. It contains crosslinking monomers and other monomers.

  In a more detailed aspect of the present invention, epithelial delivery of GRP, analogs and mimetics, and other bioactive substances disclosed herein, for example, of hydrogels that protect active substances from the action of degrading enzymes. Such sustained release may be facilitated by retaining the active substance in an enzymatic or physiological protective carrier or excipient. In certain embodiments, the active agent is conjugated to the carrier or excipient by chemical means, which may be mixed or conjugated with additional agents such as enzyme inhibitors, cytokines, and the like. Alternatively, the active agent may be immobilized through sufficient physical capture, for example, in a carrier or excipient such as a polymer matrix.

  Polymers such as hydrogels useful within the scope of the present invention incorporate functional binders such as glycosides that are chemically incorporated into the polymer to enhance the intranasal bioavailability of the active agent formulated therewith. It's okay. Examples of such glycosides are glucosides, fructosides, galactosides, arabinosides, mannosides, and alkyl-substituted derivatives thereof, and natural glycosides such as arbutin, phlorizin, amygdalin, digitonin, saponin, and indican. . There are several ways in which a typical glycoside can be attached to a polymer. For example, the hydrogen of the hydroxyl group of a glycoside or other similar carbohydrate can be replaced with an alkyl group derived from a hydrogel polymer to form an ether. Moreover, the hydroxyl group of glycoside can be made to react, esterify the carboxyl group of polymer hydrogel, and a polymer ester can be formed in situ. Another approach is to use condensation of cholest-5-en-3β-ol on a copolymer of acetobromoglucose and maleic acid. The following N-substituted polyacrylamides can be synthesized by reaction of activated polymers with ω-aminoalkylglycosides: (1) (carbohydrate spacer) (n) -polyacrylamide, “pseudopolysaccharides” (2) (carbohydrate spacer) (n) -phosphatidylethanolamine (m) -polyacrylamide, neoglycolipids, phosphatidylethanolamine derivatives; (3) (carbohydrate spacer) (n) -biotin (m)- Polyacrylamide. These biotinylated derivatives adhere to lectins on the mucosal surface and can promote absorption of physiologically active substances such as polymer-encapsulated GRP.

  Within the more detailed aspects of the present invention, one or two disclosed herein, optionally comprising secondary active substances such as protease inhibitors, cytokines, additional modulators of intercellular binding physiology, etc. The above GRP, analogs and mimetics, and / or other bioactive substances are modified and bound to a polymer carrier or matrix. This can be achieved, for example, by chemical bonding of peptide or protein actives with other actives in a crosslinked polymer network. It is also possible to chemically modify the polymer individually with an interacting agent such as a glycoside-containing molecule. In certain aspects, the bioactive agent and any secondary active agent may be functionalized, i.e., identify appropriate reactive groups, or be chemically added to the active agent. In most cases, an ethylene polymerizing group is added and then the functionalized active substance is monomerized using standard polymerization methods such as solution polymerization (usually in water), emulsion polymerization, suspension polymerization, or dispersion polymerization. And copolymerize with the crosslinking agent. In many cases, the functionalizing agent comprises a sufficient concentration of functional or polymeric groups to ensure that some sites on the active material are functionalized. For example, in a polypeptide comprising 16 amine sites, it is generally desirable to functionalize at least 2, 4, 5, 7, and 8 or 9 or more sites.

  After functionalization, the functionalized active material is mixed with a crosslinker that includes a monomer and a reagent from which the polymer of interest is formed. Polymerization is then induced in this medium to produce a polymer containing the bound active substance. The polymer is then combined with water or other suitable solvent and otherwise purified to remove traces of unreacted impurities, stirring, pressing into mesh, sonication, or desired particles as required. Grind or disintegrate by physical means such as other suitable means for diametering. The solvent (usually water) is then removed in such a way that the active substance does not denature or otherwise decompose. One desired method is freeze-drying (freeze drying), but other methods such as vacuum drying, air drying, spray drying, and the like are also available and can be used.

  Electrophiles containing amino groups, hydroxyl groups, thiol groups, and other reactive groups and unsaturated groups that can be used to introduce polymeric groups into peptides, proteins, and other actives within the scope of the present invention. It is possible to react with the agent. For example, N-hydroxysuccinimidyl group, activated carbon salts such as p-nitrophenyl carbonate, trichlorophenyl carbonate, tresylate, oxycarbonylimidazoles, epoxides, isocyanates and aldehydes, and unsaturated carboxymethyl azides and Unsaturated monomers containing unsaturated orthopyridyl-disulfides belong to this reagent class. Illustrative examples of unsaturated reagents are allyl glycidyl ether, allyl chloride, allyl bromide, allyl iodide, acryloyl chloride, allyl isocyanate, allylsulfonyl chloride, maleic anhydride, and copolymers of maleic anhydride and allyl ether. .

  All lysine-active derivatives except aldehydes generally react with other amino acids such as the imidazole group of histidine, the hydroxyl group of tyrosine, and the thiol group of cystine when the local environment enhances the nucleophilicity of these groups be able to. Aldehyde-containing functionalizing reagents are lysine specific. Reactions of these types with available groups derived from lysine, cysteine, tyrosine have been extensively described in the literature and are known to those skilled in the art.

  In the case of a physiologically active substance containing an amine group, it is convenient to react such group with acyloyl chloride such as acryloyl chloride and introduce a polymerizable acrylic group into the reacted drug. The functionalized activator is then attached and bonded to the polymer via the acrylic group during the preparation of the polymer, such as during cross-linking of the copolymer of acrylamide and acrylic acid.

  In a further aspect of the present invention, bioactive substances including peptides, proteins, nucleosides, and other molecules that are biologically active in vivo include one or more active substances and, for example, linear polyalkylene glycols, etc. Conjugated and stabilized by covalent attachment to a polymer that is incorporated as a composite part of both the hydrophilic and lipophilic parts of (see, eg, US Pat. No. 5,681,811). In one aspect, the bioactive substance is covalently bonded to a polymer comprising (i) a linear polyalkylene glycol moiety and (ii) a lipophilic moiety, wherein the active substance, the linear polyalkylene glycol moiety, and the lipophilic moiety are , (Ie, compared to stability under similar conditions in an unconjugated form that lacks the polymer attached to it) and are conformationally related so that the active therapeutic agent enhances in vivo resistance to enzymatic degradation. Arranged. In another aspect, the conjugated stabilized formulation has a three-dimensional conformation comprising a bioactive agent covalently linked to a polysorbate complex comprising (i) a linear polyalkylene glycol moiety and (ii) a lipophilic moiety, Wherein the active substance, the linear polyalkylene glycol moiety, and the lipophilic part are: (a) the lipophilic part is available outside the three-dimensional higher order structure; and (b) the enzyme of the active substance in the composition They are arranged in a structurally interrelated manner so that in vivo resistance to degradation is enhanced.

  In a further related aspect, the bioactive substance covalently bonded to the triglyceride skeleton moiety via a polyalkylene glycol spacer group bonded at the carbon atom of the triglyceride skeleton moiety and the covalently bonded or polyalkylene to the carbon atom of the triglyceride skeleton moiety Provided are multi-ligand conjugate conjugates comprising at least one fatty acid moiety covalently linked through a glycol spacer moiety (see, eg, US Pat. No. 5,681,811). In such multi-ligand conjugated therapeutic agent conjugates, the α ′ and β carbon atoms of the triglyceride bioactive moiety have a fatty acid moiety attached by either a direct covalent bond or an indirect covalent bond through a polyalkylene glycol spacer moiety. You can do it. Alternatively, the fatty acid moiety may be covalently bonded to the α and α ′ carbons of the triglyceride backbone moiety directly or via a polyalkylene glycol spacer moiety and the bioactive therapeutic agent is directly covalently bonded or poly It is covalently bonded to the γ-carbon of the triglyceride backbone that is indirectly bonded via an alkylene glycol spacer moiety. Within the scope of the present invention, it is recognized that a wide variety of structural, compositional and conformational forms are possible for multi-ligand conjugated therapeutic agent conjugates containing a triglyceride backbone moiety. Within the scope of the present invention, in such multi-ligand conjugated therapeutic agent conjugates, it is advantageous if the bioactive agent is covalently linked to the triglyceride modified backbone moiety via an alkyl spacer group or other acceptable spacer group. Is further described. When used in such circumstances, the tolerance of the spacer group refers to steric, compositional, and end-use specific tolerances.

  In a further aspect of the invention, the α, α ′, and β carbon atoms are shared by (i) a fatty acid group and (ii) a physiologically active substance or an appropriate functional group such as a polyethylene glycol group. Provided is a conjugated stabilizing complex comprising a polysorbate complex comprising a polysorbate moiety comprising a triglyceride backbone covalently linked to a functional group comprising a polyethylene glycol group having a linked moiety. Such a covalent bond may be, for example, a direct covalent bond with a hydroxyl-terminated functional group of a polyethylene glycol group, or, for example, the obtained capping polyethylene glycol group may be covalently bonded to a physiologically active substance or moiety. To have a good terminal carboxy functional group, it may be an indirect covalent bond, such as by reactive capping of the hydroxyl end with a terminal carboxy functional spacer group of the polyethylene glycol group.

  In a further aspect of the present invention, one or more GRP, analogs, and mimetics disclosed herein covalently attached to a glycolipid moiety modified with physiologically compatible polyethylene glycol (PEG). The present invention provides a stable water-soluble conjugate-stabilized complex comprising the body and / or other physiologically active substance. In such conjugates, the bioactive agent may be covalently linked to a physiologically compatible PEG-modified glycolipid moiety by a labile covalent bond at the free amino acid group of the active agent, where the labile Covalent bonds can be cleaved in vivo by biochemical hydrolysis and / or proteolysis. Physiologically compatible PEG-modified glycolipid moieties consist of, for example, monopalmitate, dipalmitate, monolaurate, dilaurate, trilaurate, monooleate, dioleate, trioleate, monostearate, distearate, and tristearate It is advantageous if it contains a polysorbate polymer such as a polysorbate polymer containing a fatty acid ester group selected from the group. In such conjugates, the physiologically compatible PEG-modified glycolipid moiety may suitably comprise a polymer selected from the group consisting of polyethylene glycol ethers of fatty acids and polyethylene glycol esters of fatty acids, wherein The fatty acid includes, for example, a fatty acid selected from the group consisting of lauric acid, palmitic acid, oleic acid, and stearic acid.

Storage of Materials In certain aspects of the present invention, the combination formulations and / or coordinated administration methods herein may adhere to an effective amount of charged glass, thereby reducing the effective concentration in the container. Incorporates certain peptides and proteins. A silanized container, such as a silanized glass container, is used to store the final product and reduce the adsorption of polypeptides or proteins to the glass container.

  In a further aspect of the invention, a therapeutic kit for a mammalian subject comprises a stable pharmaceutical composition of one or more GRP compounds formulated for mucosal delivery of the mammalian subject, the composition comprising: One or more symptoms of diabetes, obesity, cancer, hyperglycemia, dyslipidemia, metabolic syndrome, coronary syndrome, myocardial infarction, or neurological disease without unacceptable adverse side effects It is effective in mitigating The kit further includes a pharmaceutical reagent vial for containing one or more GRP compounds. Pharmaceutical reagent vials are composed of pharmaceutical grade polymer, glass, or other suitable material. The pharmaceutical reagent vial is, for example, a silanized glass vial. The kit further includes an opening for delivering the composition to the nasal mucosal surface of the subject. The delivery opening is composed of a pharmaceutical grade polymer, glass, or other suitable material. The delivery opening is, for example, silanized glass.

  The silanization technique combines a special cleaning technique for the surface to be silanized and a silanization process at low pressure. Silane is in the gas phase and the surface is silanized at high temperatures. This method results in a reproducible surface with a stable and homogeneous functional silane layer having the characteristics of a single layer. The silanized surface prevents binding of the polypeptide or mucosal delivery enhancer of the present invention to the glass.

This procedure is useful for preparing silanized pharmaceutical reagent vials to hold the GRP compositions of the present invention. Clean glass trays by rinsing with double distilled water (ddH ₂ O) before use. The silane tray is then rinsed with 95% EtOH and the acetone tray is rinsed with acetone. The pharmaceutical reagent vial is sonicated in acetone for 10 minutes. After acetone sonication, the reagent vial is immersed in the ddH ₂ O tray at least twice. Reagent vials are sonicated in 0.1 M NaOH for 10 minutes. Resonate vials in NaOH and make silane solution in draft. (Silane solution: 800 mL 95% ethanol; 96 L glacial acetic acid; 25 mL glycidoxypropyltrimethoxysilane). After NaOH sonication, dip reagent vials in ddH ₂ O tray at least twice. The reagent vial is silanized in the silane solution for 3 to 5 minutes. The reagent vial is immersed in a 100% EtOH tray. Reagent vials are dried with pre-purified N ₂ gas and stored in an oven at 100 ° C. for at least 2 hours prior to use.

Bioadhesive delivery excipients and methods In certain aspects of the present invention, the combination formulations and / or coordinated administration methods herein provide an aid to enhance epithelial delivery of one or more bioactive agents. An effective amount of a non-toxic bioadhesive is incorporated as a compound or carrier. The bioadhesive in this context exhibits general or specific adhesion to the surface of one or more components or target biomembrane. Bioadhesives retain the desired concentration gradient of bioactive substance in or across the mucosa and ensure even the penetration of macromolecules (eg, peptides and proteins) in or through the epithelium. Typically, the use of bioadhesive agents within the methods and compositions of the present invention results in 2 to 5 times, often 5 to 10 times the permeability of peptides and proteins in or through the epithelium. increase. This enhanced epithelial penetration often effectively transmucosally delivers macromolecules into, for example, the basal portion of the nasal epithelium or adjacent extracellular compartment or plasma or CNS tissue or fluid.

  The enhanced delivery greatly improves the delivery efficiency of bioactive peptides, proteins, and other macromolecular therapeutic species. These results depend in part on the hydrophilicity of the compound, thereby providing a higher permeability using hydrophilic species compared to water-insoluble compounds. In addition to these effects, the use of bioadhesives to enhance drug persistence at the surface of the biomembrane can trigger a reservoir mechanism for sustained drug delivery, which allows the compound to In addition to permeating through the membrane, when the material on the surface is depleted, it diffuses back to the surface.

  In the field of oral administration, various suitable bioadhesives are described in U.S. Pat. Nos. 3,972,995; 4,259,314; 4,680,323; 4,740,365. No. 4,573,996; No. 4,292,299; No. 4,715,369; No. 4,876,092; No. 4,855,142; No. 4,855,142; No. 4,226,848; U.S. Pat. No. 4,948,580; US Reissue Pat. No. 33,093, which are used in the novel methods and compositions of the present invention. Has been. The ability of various bioadhesive polymers as mucosal delivery platforms, such as nasal mucosa, within the methods and compositions of the present invention, measures the ability to retain and release GRP, and incorporates active substances therein. It can be easily assessed by its ability to interact with the mucosal surface afterwards. In addition, known methods are applied to determine the biocompatibility of the selected polymer with the tissue at the site of mucosal administration. When the target mucous membrane is covered with mucus (ie, no mucolytic treatment or mucus removal treatment is performed), it can function as a connection that leads to the underlying mucosal epithelium. Thus, as used herein, the term “bioadhesive” is also directed to mucoadhesive compounds useful for facilitating mucosal delivery of bioactive substances within the scope of the present invention. However, adhesive contact to mucosal tissue mediated through adhesion to the mucus gel layer is incomplete or incomplete between the mucus layer where rapid mucus clearance occurs and the underlying tissue (especially the nasal surface). May be limited by excessive adhesion. In this regard, mucin glycoprotein is continuously secreted and a viscoelastic gel is formed immediately after its release from the cell or gland. However, the luminal surface of the adhesive gel layer is continuously eroded by mechanical, enzymatic and / or ciliary effects. If such activity is more prominent or longer adhesion times are desired, the coordinated administration method and combination formulation method of the present invention can be the mucolytic and / or ciliastatic method disclosed herein above or Additional drugs may be incorporated.

  Typically, the mucoadhesive polymers used within the scope of the present invention are natural or synthetic polymers that adhere to wet mucosal tissue surfaces by a complex but non-specific mechanism. In addition to these mucoadhesive polymers, the present invention also includes methods and compositions that incorporate bioadhesives that adhere directly to cell surfaces rather than mucus using specific interactions, including those mediated by receptors. I will provide a. An example of a bioadhesive that functions in this specific manner is a group of compounds known as lectins. These are, for example, glycoproteins that have the ability to specifically recognize and bind to glycomolecules such as glycoproteins or glycolipids, which form part of the intranasal epithelial cell membrane and are considered “lectin receptors” be able to.

  In a particular aspect of the invention, the bioadhesive material for facilitating nasal delivery of a bioactive substance can adhere to a wet mucosal surface, for example, a hydrophilic polymer such as water soluble or water swellable or Contains a matrix of polymer blend. These adhesives can be formulated as ointments, hydrogels (see above), thin films, and other forms of application. In many cases, these adhesives have a bioactive substance mixed with it to achieve delayed release or local delivery of the active substance. For example, some have been formulated with additional ingredients to facilitate penetration of the active substance through the nasal mucosa into the individual's circulatory system.

  Various polymers (both natural and synthetic) show significant binding to mucus and / or mucosal epithelial surfaces under physiological conditions. The strength of this interaction can be easily measured by a mechanical peel test or a shear test. When applied to moist mucosal surfaces, many dry materials adhere at least slightly spontaneously. After such initial contact, if some hydrophilic material begins to draw water by adsorption, swelling, or capillary force, and this water is absorbed from the underlying substrate or polymer tissue interface, adhesion is bioactive It may be sufficient to achieve the purpose of promoting mucosal absorption of the substance. Such “hydration adhesion” can be very strong, but formulations adapted to use this mechanism must contribute to the continued swelling of the dosage to convert it into hydrated mucus. . This is planned for a number of hydrocolloids useful within the scope of the present invention that are generally non-adhesive when applied in a pre-hydrated state, particularly some cellulose derivatives. Nevertheless, bioadhesive delivery systems for epithelial administration are useful within the scope of the present invention when such materials are applied in dry polymer powder, microparticle, or film type delivery forms.

  Other polymers adhere to the epithelial surface not only when applied in a dry state, but also in a fully hydrated state and in the presence of excess water. Therefore, the selection of mucoadhesive agents requires careful consideration of the physiological and physicochemical conditions that form and maintain contact with the tissue. In particular, the amount of water or moisture normally present at the intended adhesion site and the general pH are known to have a significant effect on the mucoadhesive bond strength of various polymers.

  Some polymer bioadhesive delivery systems have been made and studied over the last 20 years, but have not always been successful. However, a variety of such carriers are currently used in clinical applications involving use in dentistry, orthopedics, ophthalmology, and surgery. For example, acrylic hydrogels are widely used for bioadhesive devices. Acrylic hydrogels are well-suited as bioadhesives due to their partially swollen flexibility and non-abrasion properties that reduce friction to the injured contact tissue. Furthermore, its high permeability in the swollen state allows unreacted monomers, uncrosslinked polymer chains, and initiators to be immersed from the matrix after polymerization, which is within the scope of the present invention. This is an important feature for the selection of the bioadhesive material used. Acrylic polymer devices exhibit very high adhesive bond strength. In order to control epithelial delivery of peptide and protein drugs, the methods and compositions of the invention optionally function in part to protect the bioactive agent from proteolytic disruption, eg, polymer delivery enhancement. Including the use of a carrier such as a dosage form, while enhancing the penetration of peptides or proteins in or through the nasal mucosa. In this situation, bioadhesive polymers have proven sufficient ability to facilitate oral drug delivery. As an example, 9-desglycinamide, 8-arginine vasopressin (DGAVP) administered intraduodenally to rats with 1% (w / v) saline dispersion of polycarbophil, a mucoadhesive poly (acrylic acid) derivative The bioavailability of is increased 3 to 5 times compared to aqueous solutions of peptide drugs that do not contain this polymer.

  Poly (acrylic acid) type mucoadhesive polymers are potent inhibitors of several intestinal proteases. The mechanism of enzyme inhibition is explained by the strong affinity of this class of polymers for divalent cations such as calcium and zinc, which are essential cofactors for metalloproteases such as trypsin and chymotrypsin. Depletion of protease cofactors by poly (acrylic acid) has been reported to induce irreversible structural changes in enzyme proteins that are achieved by loss of enzyme activity. At the same time, other mucoadhesive polymers (eg, some cellulose derivatives and chitosan) are unable to inhibit proteolytic enzymes under certain conditions. In contrast to other enzyme inhibitors that are relatively small molecules intended for use within the scope of the present invention, such as aprotinin, bestatin, etc., nasal absorption of inhibitory polymers is the size of these molecules. From the point of view, it is likely to be negligible, thereby eliminating the possibility of harmful side effects. Thus, in particular, poly (acrylic acid) type mucoadhesive polymers function as both absorption-promoting adhesives and enzyme protectants, and in particular, controlled delivery of peptide and protein drugs when safety concerns are considered Can be promoted.

  In addition to protection against enzymatic degradation, bioadhesives and other polymeric or non-polymeric absorption enhancers used within the scope of the present invention can directly increase epithelial permeability to bioactive substances. To facilitate transport of large and hydrophilic molecules across the nasal epithelial barrier, such as peptides and proteins, mucoadhesive polymers and other drugs are described by extending the premucosal residence time of the delivery system. It has been thought to bring about an increase in the transmission effect over the effect. Reports have suggested that bioadhesive microparticles rapidly but transiently increase insulin permeability through the nasal mucosa with changes in drug plasma concentration over time. For example, other mucoadhesive polymers used within the scope of the present invention, such as chitosan, reportedly enhance the permeability of certain mucosal epithelia, even when applied as an aqueous solution or gel. Another mucoadhesive polymer that has been reported to directly affect epithelial permeability is hyaluronic acid and its ester derivatives. Particularly useful bioadhesives within the scope of the coordinated administration and / or combination formulation methods and compositions of the present invention are chitosan and analogs and derivatives thereof. Chitosan is a non-toxic, biocompatible and biodegradable polymer that is widely used for pharmaceutical and medical applications because of its favorable properties that exhibit low toxicity and good biocompatibility. Chitosan is a natural aminopolysaccharide prepared from chitin by N deacetylation with alkali. When used within the methods and compositions of the present invention, chitosan reduces the retention of the GRP, analogs and mimetics disclosed herein, and other bioactive substances at the applied mucosal site. increase. This mode of administration can also improve patient compliance and tolerance. As further provided herein, the methods and compositions of the present invention optionally include novel chitosan derivatives or chemically modified forms of chitosan. One such novel derivative used within the scope of the present invention is designated β- [1 → 4] -2-guanidino-2-deoxy-D-glucose polymer (poly-GuD). Chitosan is a naturally occurring polymer that is an N-deacetylated product of chitin and is widely used to prepare microparticles for oral and nasal formulations. Chitosan polymers have also been proposed as soluble carriers for parenteral drug delivery. Within one aspect of the invention, o-methylisourea is used to convert chitosanamine to its guanidinium moiety. The guanidinium compound is prepared, for example, by reaction of an equi-normal solution of chitosan at a pH exceeding 8.0 with o-methylisourea.

  Additional compounds classified as bioadhesive agents for use within the scope of the present invention are usually classified as “receptor-ligand interactions” between the complementary structure of the bioadhesive compound and the mucosal epithelial surface components. It acts by mediating specific interactions. Many natural product examples describe specific binding bioadhesive forms, as exemplified by lectin-sugar interactions. Lectins are non-immunogenic (glyco) proteins that bind to polysaccharides or glycoconjugates.

  Several plant lectins have been investigated as possible pharmaceutical absorption enhancers. One plant lectin, kidney bean hemagglutinin (PHA), exhibits high oral bioavailability of greater than 10% after feeding rats. Tomato (Lycopersicon esculentum) lectin (TL) appears to be safe in various modes of administration.

  In summary, the bioadhesives optionally extend the persistence or otherwise increase epithelial absorption of one or more GRP, analogs, and mimetics, and other bioactive agents. Are useful in the combination formulations and coordinated administration methods of the present invention, which incorporate an effective amount and form of the bioadhesive. The bioadhesive may be coordinately administered as an auxiliary compound or additive in the combination formulation of the present invention. In certain embodiments, the bioadhesive acts as a “pharmaceutical glue”, while in other embodiments, in some cases, promotes specific receptor-ligand interactions with epithelial cell “receptors”. In other cases, the bioadhesive aid is increased by increasing epithelial permeability to significantly increase the drug concentration gradient measured at the delivery target site (eg, liver, plasma, or CNS tissue or fluid). The delivery or combination formulation functions to enhance contact between the bioactive substance and the nasal mucosa. Additional bioadhesives used within the scope of the invention are enzymes (eg, proteases) that are delivered in concert with the bioadhesive or enhance the stability of the mucosally administered biotherapeutic agent in a combination formulation. Acts as an inhibitor.

Liposome and micelle delivery excipients The coordinated delivery methods and combination formulations of the present invention incorporate effective lipid or fatty acid based carriers, processing aids, or delivery excipients as desired, GRPs, analogs and mimetics. As well as improved formulations for epithelial delivery of other bioactive substances. For example, various for mucosal delivery containing one or more of these active agents, such as peptides or proteins, mixed or encapsulated with liposomes, mixed micelle carriers, or emulsions or co-administered together Formulations and methods for enhancing the chemical and physical stability of bioactive substances and increasing half-life upon mucosal delivery (eg, by reducing sensitivity to proteolysis, chemical modification, and / or denaturation) .

  Within the scope of certain aspects of the present invention, bioactive agent-specific delivery systems include lipid vesicles known as liposomes. They are usually made from natural, biodegradable, non-toxic, and non-immunogenic lipid molecules and can efficiently capture or bind drug molecules, including peptides and proteins, in or on the membrane. . Peptides and proteins within the scope of the present invention by the fact that the encapsulated protein can remain in its preferred aqueous environment within the vesicle, while the liposome membrane protects the protein from proteolytic and other destabilizing factors The appeal of liposomes as a delivery system is increased. Even though not all known liposome preparation methods are feasible to encapsulate peptides and proteins due to their inherent physical and chemical properties, these methods can be used without substantial inactivation by several methods. Can be encapsulated.

  Various methods such as US Pat. Nos. 4,235,871, 4,501,728, and 4,837,028 are available for the preparation of liposomes for use within the scope of the present invention. It is. For use with liposome delivery, the bioactive agent is usually entrapped within the liposome or lipid vesicle or bound to the outside of the vesicle.

  Like liposomes, unsaturated long-chain fatty acids that also have mucosal absorption enhancing activity can form closed vesicles with a bilayer-like structure (so-called “ufasome”). These can be formed, for example, using oleic acid to capture bioactive peptides and proteins for mucosal delivery, eg, intranasal, within the scope of the present invention.

  Other delivery systems used within the scope of the present invention combine polymers and liposomes, combining the advantageous properties of both vesicles, such as encapsulation within the natural polymer fibrin. Furthermore, the release of biotherapeutic compounds from this delivery system can be controlled by the use of covalent crosslinks and the addition of antifibrinolytic agents to the fibrin polymer.

  Simplified delivery systems used within the scope of the present invention are effectively used to obtain electrostatic interactions between lipid carriers and charged bioactive substances such as proteins and polyanionic nucleic acids. The use of cationic lipids as delivery vesicles or carriers that can be mentioned. This effectively packages the drug into a form suitable for mucosal administration and / or subsequent delivery to the systemic compartment.

  Additional delivery excipients used within the scope of the present invention include long and medium chain fatty acids and surfactant mixed micelles containing fatty acids. Most naturally occurring lipids in ester form have important implications for transport of the lipid itself across the mucosal surface. Monoglycerides combined with free fatty acids and polar groups have been shown to act in the intestinal barrier as permeation enhancers in the form of mixed micelles. This discovery of the barrier-modifying function of free fatty acids (carboxylic acids whose chain length varies between 12 and 20 carbon atoms) and their polar derivatives has prompted extensive research on the use of these agents as mucosal absorption enhancers. Yes.

  For use within the method of the present invention, long chain fatty acids, especially fusogenic lipids (unsaturated fatty acids and monoglycerides such as oleic acid, linoleic acid, linoleic acid, monoolein, etc.) Useful carriers for facilitating mucosal delivery of GRP, analogs and mimetics, and other bioactive substances disclosed in the specification are obtained. Medium chain fatty acids (C6 to C12) and monoglycerides also show enhanced intestinal drug absorption activity and can be adapted for use within the mucosal delivery formulations and methods of the invention. In addition, sodium salts of medium and long chain fatty acids are effective delivery excipients and absorption enhancers for epithelial delivery of bioactive substances within the scope of the present invention. Thus, the fatty acids can be used in the solubilized form of the sodium salt or by the addition of non-toxic surfactants such as, for example, polyoxyethylenated hydrogenated castor oil, sodium taurocholate. Other fatty acids and mixed micelles useful within the scope of the present invention include sodium caprylate (C8), sodium caprate (C10), optionally in combination with bile salts such as glycolate and taurocholate. , Sodium laurate (C12), or sodium oleate (C18).

Pegylation Additional methods and compositions provided within the scope of the present invention include bioactive peptides by covalent attachment of polymeric substances such as, for example, dextrans, polyvinylpyrrolidones, glycopeptides, polyethylene glycols, and polyamino acids and Includes chemical modification of proteins. The resulting conjugated peptides and proteins retain their bioactivity and solubility for epithelial administration. In another aspect, GRP, analogs and mimetics, and other bioactive peptides and proteins are conjugated to polyalkylene oxide polymers, particularly polyethylene glycol (PEG). U.S. Pat. No. 4,179,337.

  Amine reactive PEG polymers used within the scope of the present invention include SC-PEG, U-PEG-10000, NHS-PEG-3400-biotin, T- with molecular weights of 2000, 5000, 10000, 12000, and 20000. PEG-5000, T-PEG-12000, and TPC-PEG-5000. PEGylation of bioactive peptides and proteins can be performed by modification of the carboxy site (for example, aspartate group or glutamate group in addition to the carboxyl terminus). The utility of PEG-hydrazide in the selective modification of carbodiimide activated protein carboxyl groups under acidic conditions has been described. Alternatively, bifunctional PEG modifications of bioactive peptides and proteins can be used. In some procedures, charged amino acid residues, including lysine, aspartic acid, and glutamic acid, exhibit a pronounced solvent accessible tendency on the protein surface.

Other stabilizing modifications of the active substance In addition to pegylation, the physiologically active substances such as peptides and proteins used within the scope of the present invention are modified, such as one or more immunoglobulin chains. Protection of active substances through conjugation to other known protective or stabilizing compounds by making fusion proteins with active peptides, proteins, analogs or mimetics bound to one or more carrier proteins Can increase the circulation half-life.

Formulation Preparation, Manufacture and Administration The epithelial delivery formulations of the present invention typically comprise GRP, analogs and mimetics in combination with one or more pharmaceutically acceptable carriers and optionally other therapeutic ingredients. . The carrier must be “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Such carriers are described at the top of this specification or are well known to those of ordinary skill in the pharmacology arts. Desirably, the formulation should not contain substances such as enzymes or oxidants known to be incompatible with the physiologically active substance to be administered. The formulation can be prepared by any method known in the pharmaceutical arts.

  Within the scope of the compositions and methods of the present invention, the GRPs, analogs and mimetics disclosed herein, and other bioactive substances are oral, rectal, vaginal, intranasal, intrapulmonary, Alternatively, it may be administered to a subject by various modes of mucosal administration including transdermal delivery or topical delivery to the eye, ear, skin, or other mucosal surface. The compositions and methods of the present invention also include transdermal modes of administration. If desired, the GRP, analogs and mimetics disclosed herein, and other bioactive substances may be applied to skin patches, topical formulations applied to the skin, intramuscular, subcutaneous, intravenous, intraarterial, joints. It may be administered in a coordinated or adjunct way by non-mucosal routes including internal, intraperitoneal, or parenteral routes. In other alternative embodiments, the bioactive agent is a cell, tissue, or organ from a mammalian subject, eg, as a component of an ex vivo tissue or organ therapy formulation that contains the bioactive agent in a suitable liquid or solid carrier. May be administered ex vivo by direct exposure to.

  The compositions according to the invention are often administered in aqueous solution as a nasal or pulmonary spray and may be dispensed in spray form by various methods known to those skilled in the art. A preferred system for dispensing liquids as a nasal spray is disclosed in US Pat. No. 4,511,069. The formulation can be present in a multi-dose container such as, for example, a sealed dosing system disclosed in US Pat. No. 4,511,069. Additional aerosol delivery forms include, for example, compressed air, jets, ultrasound, and piezoelectric nebulizers that deliver bioactive substances dissolved or dispersed in pharmaceutical solvents such as water, ethanol, or mixtures thereof. It's okay.

  The nasal and pulmonary spray solutions of the present invention are typically drugs formulated with a surfactant, such as a nonionic surfactant (eg, polysorbate 80) and one or more buffers, if desired. Or contains the drug to be delivered. In some embodiments of the invention, the nasal spray solution further comprises a propellant. The pH of the nasal spray solution is optionally from about pH 2.0 to pH 8, preferably pH 4.5 ± 0.5. Suitable buffers for use within these compositions are as described above or are known in the art. Preservatives, surfactants, dispersants, or other ingredients including gases can be added to enhance or maintain chemical stability. Suitable preservatives include, but are not limited to, phenol, methylparaben, paraben, m-cresol, thiomersal, chlorobutanol, benzylalkonium chloride, sodium benzoate, ethanol, phenylethyl ether, benzyl alcohol, and the like. Suitable surfactants include, but are not limited to, oleic acid, sorbitan trioleate, polysorbates, lecithin, phosphatidylcholines, and various long chain diglycerides and phospholipids. Suitable dispersants include, but are not limited to, ethylenediaminetetraacetic acid. Suitable gases include, but are not limited to, nitrogen, helium, chlorofluorocarbon (CFC), hydrofluorocarbon (HFC), carbon dioxide, and air.

  Within an alternative embodiment, the mucosal formulation is administered as a dry powder formulation comprising a bioactive substance in a particle size suitable for nasal administration or in a dry (usually lyophilized) form within the appropriate particle size range. The dry formulation is also suitable for transdermal delivery. The minimum particle size suitable for deposition in the nasal or pulmonary route is often 0.5 μm median aerodynamic equivalent particle size (MMEAD), generally about 1 μMMEAD, more typically about 2 μM MEAD. A suitable maximum particle size for intranasal accumulation is often about 10 μM MEAD, generally about 8 μM MEAD, and more typically about 4 μM MEAD. Intranasal respiratory powders within these particle size ranges can be made by a variety of conventional techniques such as jet milling, spray drying, solvent precipitation, and supercritical fluid concentration. These dry powders of appropriate MMEAD may be administered to the patient via a conventional dry powder inhaler (DPI), which depends on the patient's breathing during pulmonary or nasal inhalation, Disperse the powder in aerosolized amount. Alternatively, the dry powder may be administered via an air-assisted device that uses an external power source to disperse the aerosolized amount of powder, such as, for example, a piston pump.

  Dry powder devices typically require powders in the mass range of about 1 mg to 20 mg to produce a single aerosolized dose (“puff”). If the required or desired dose of bioactive agent is less than this amount, the powdered active agent is usually combined with a pharmaceutically dry-filled powder to obtain the required total powder mass. Preferred dry filled powders include sucrose, lactose, dextrose, mannitol, glycine, trehalose, human serum albumin (HSA), and starch. Other suitable dry-filled powders include cellobiose, dextran, maltotriose, pectin, sodium citrate, sodium ascorbate, and the like.

  In order to formulate a composition for epithelial delivery within the scope of the present invention, the bioactive substance may be combined with various pharmaceutically acceptable additives as well as a base or carrier for the dispersion of the active substance. . Desired additives include, but are not limited to, pH regulators such as arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid, acetic acid. In addition, local anesthetics (eg benzyl alcohol), isotonic agents (eg sodium chloride, mannitol, sorbitol), adsorption inhibitors (eg Tween 80), solubility enhancers (eg cyclodextrin and its derivatives), stable An agent (eg, serum albumin) and a reducing agent (eg, glutathione) may be included. When the composition for epithelial delivery is a liquid, when the tonicity of the preparation is measured with reference to the tonicity of physiological saline used as a unit (0.9% (w / v)), it is usually at the site of administration. Adjust to a value that does not induce substantially irreversible tissue damage in the nasal mucosa. In general, the tonicity of the solution is adjusted to a value of about 1/3 to 3, more typically 1/2 to 2, and most often 3/4 to 1.7.

  The bioactive substance may be dispersed in a base or excipient that may include a hydrophilic compound having the ability to disperse the active substance and any desired additives. Bases include polycarboxylic acids or salts thereof, copolymers of carboxylic anhydrides (eg, maleic anhydride) and other monomers (eg, methyl (meth) acrylate, acrylic acid, etc.), polyvinyl acetate, polyvinyl alcohol, polyvinyl pyrrolidone These include hydrophilic vinyl polymers such as, cellulose derivatives such as hydroxymethylcellulose, hydroxypropylcellulose, and natural polymers such as chitosan, collagen, sodium alginate, gelatin, hyaluronic acid, and non-toxic metal salts thereof. You may choose from a wide range of suitable carriers that are not limited to: In many cases, biodegradable polymers such as, for example, polylactic acid, poly (lactic acid-glycolic acid) copolymer, polyhydroxybutyric acid, poly (hydroxybutyric acid-glycolic acid) copolymer, and mixtures thereof are selected as the base or carrier. To do. Alternatively or additionally, synthetic fatty acid esters such as polyglycerin fatty acid esters, sucrose fatty acid esters and the like may be used as the carrier. The hydrophilic polymer and other carriers may be used alone or in combination, and the structural integrity of the carrier can be enhanced by partial crystallization, ionic bonding, crosslinking, and the like. The carrier may be provided in a variety of forms for direct application to the nasal mucosa, including fluid or viscous solutions, gels, pastes, powders, microparticles, and films. The use of selected carriers in this situation can facilitate the absorption of bioactive substances.

  The bioactive substance can be combined with a base or carrier according to various methods, and the active substance can be released by diffusion, disintegration of the carrier, related formulations of the water channel. In some circumstances, for example, a biocompatible dispersion applied to the nasal mucosa by dispersing the active substance in microcapsules (microparticles) or nanocapsules (nanoparticles) prepared from a suitable polymer such as isobutyl 2-cyanoacylate. Dispersed in the medium, thereby sustaining delivery and biological activity over a long period of time.

  In order to further enhance the epithelial delivery of pharmaceuticals within the scope of the present invention, the formulation comprising the active substance may also comprise a hydrophilic low molecular weight compound as a base or excipient. Such a hydrophilic low molecular weight compound provides a pathway medium in which a water-soluble active substance such as a physiologically active peptide or protein diffuses to the body surface through which the active substance can be absorbed. If desired, the hydrophilic low molecular weight compound absorbs water from the mucosa or administration environment and dissolves the water-soluble active peptide. The molecular weight of the hydrophilic low molecular weight compound is generally 10,000 or less, preferably 3000 or less. Typical hydrophilic low molecular weight compounds include sucrose, mannitol, sorbitol, lactose, L-arabinose, D-erythrose, D-ribose, D-xylose, D-mannose, trehalose, D-galactose, lactulose, cellobiose, gentibiose And polyol compounds such as oligosaccharides such as glycerin, and polyethylene glycol, disaccharides, and monosaccharides. Other examples of hydrophilic low molecular weight compounds useful as carriers within the scope of the present invention include N-methylpyrrolidone and alcohols (eg, oligovinyl alcohol, ethanol, ethylene glycol, propylene glycol, etc.). These hydrophilic low molecular weight compounds can be used alone or in combination with each other or with other active or inactive substances of nasal preparations.

  Alternatively, the composition of the present invention may be prepared by using a pH adjusting agent, a buffering agent, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, as necessary. Pharmaceutically acceptable carrier materials may be included to approximate physiological conditions, such as tonicity adjusting agents and wetting agents. For solid compositions, conventional non-toxic pharmaceutically acceptable carriers including, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like May be used.

  Therapeutic compositions for administration of bioactive substances may be formulated as solutions, microemulsions, or other ordered structures suitable for high concentrations of active ingredients. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and lipid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the desired particle size in the case of dispersible preparation, and by the use of surfactants. In many cases, it will be desirable to include tonicity agents, such as, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. For example, the absorption of bioactive substances can be extended by including in the composition an agent that delays absorption, such as monostearate and gelatin.

  In certain aspects of the invention, the bioactive agent is administered in a sustained release formulation, such as, for example, a composition comprising a delayed release polymer. For example, the active agent may be prepared using carriers that prevent rapid release, such as polymers, microencapsulated delivery systems, or controlled release excipients such as bioadhesive gels. Delivery of active agents in the various compositions of the present invention can be extended by including agents that delay absorption in compositions such as, for example, aluminum monostearate, hydrogels, and gelatin. If a controlled release formulation of a bioactive substance is desired, a controlled release binder suitable for use in accordance with the present invention is any biocompatible that is inert to the active substance and can incorporate the bioactive substance. Controlled release substances. A number of such materials are known in the art. Useful controlled release binders are substances that are slowly metabolized under physiological conditions after nasal delivery (eg, at the nasal mucosal surface or in the presence of body fluids after transmucosal delivery). Suitable binders include, but are not limited to, biocompatible polymers and copolymers already used in the art for sustained release formulations. Such biocompatible compounds are non-toxic and inert to surrounding tissues and do not induce significant side effects such as nasal irritation, immune response, inflammation and the like. They are metabolized into metabolites that are also biocompatible and are easily excreted from the body.

  Exemplary polymeric materials used in this context include, but are not limited to, polymer matrices derived from copolymers and homopolymeric polyesters having hydrolysable ester linkages. Many of these are known in the art to yield degradation products that are biodegradable and have little or no toxicity. Typical polymers include polyglycolic acid (PGA) and polylactic acid (PLA), poly (DL-lactic acid-co-glycolic acid) (DL PLGA), poly (D-lactic acid-co-glycolic acid) (D PLGA) And poly (L-lactic acid-co-glycolic acid) (L PLGA). Other useful biodegradable or bioerodible polymers include poly (ε-caprolactone), poly (ε-aprolactone-CO-lactic acid), poly (ε-aprolactone-CO-glycolic acid), poly (β -Hydroxybutyric acid), poly (alkyl-2-cyanoacrylate), hydrogels such as poly (hydroxyethyl methacrylate), polyamides, poly (amino acids) (ie, L-leucine, glutamic acid, L-aspartic acid, etc.) , Polymers such as poly (ester urea), poly (2-hydroxyethyl DL-aspartamide), polyacetal polymers, polyorthoesters, polycarbonates, polymaleamides, polysaccharides, and copolymers thereof. Not. Many methods for preparing such formulations are generally known to those skilled in the art. Other useful formulations include, for example, controlled release compositions such as microcapsules (US Pat. Nos. 4,652,441 and 4,917,893), macrocapsules and other formulations useful. Lactic acid-glycolic acid copolymers (US Pat. Nos. 4,677,191 and 4,728,721) and sustained release compositions for water-soluble peptides (US Pat. No. 4,675,189). It is done.

  The manufacturing process for nasal spray products generally involves the preparation of a diluent for GRP nasal sprays that includes, in addition to less than 85% water, ingredients of a nasal spray formulation that does not contain GRP. The pH of the diluent is then measured and adjusted to the desired formulation pH with sodium hydroxide or hydrochloric acid as necessary. Water is used to obtain the final target volume of diluent. Prepare a GRP nasal spray by transferring aseptically less than 85% of the final target volume to the screw-mouth bottle. Add the appropriate amount of GRP and mix until completely dissolved. The pH is measured and adjusted to the desired formulation pH with sodium hydroxide or hydrochloric acid as necessary. A sufficient amount of diluent is added to make the final target volume. Fill screw cap jar and cap. The above description of the manufacturing process represents the method used to prepare the initial clinical batch of drug product. This method may be modified during the development process to optimize the manufacturing process.

  Currently sold GRP requires sterilized manufacturing conditions to comply with FDA regulations. Parenteral administration including GRP for injection or infusion requires a sterile (aseptic) manufacturing process. Standards for design and construction features (standards for design and construction features) (Federal Regulatory Standards Article 21 § 211.42 (April 1, 2005)) Approval or rejection of tests and ingredients, standards for drug product containers and seals, drug production containers, drug clauses (§ 211.84); control of microbiological contamination (§211.113); and other special tests And other special testing requirements (§211.167). Non-parental (non-sterile) products, such as the nasal administration products of the present invention, do not require these special sterile manufacturing conditions. As can be readily appreciated, the requirements of the sterilization manufacturing process are significantly higher and correspondingly more expensive than those required for the manufacturing process of non-sterile products. These costs include higher capitalization costs for equipment and higher manufacturing costs, ie additional equipment for sterilization manufacturing, including additional room and air conditioning, additional workforce, extensive quality control and quality Includes additional costs associated with sterilization manufacturing including warranty and administrative support. As a result, the manufacturing cost of a nasal GRP product, such as the product of the present invention, is significantly less than the manufacturing cost of a parenterally administered GRP product. The present invention satisfies the need for a non-sterile manufacturing process for GRP.

  The present invention includes GRP drug products without preservatives. Such formulations do not contain preservatives. In the absence of antimicrobial excipients, the formulation is filled into a preservative-free nasal spray device under sterile conditions or incorporated into a skin patch. The device can deliver an effective amount without contaminating the formulation within the delivery system. Such GRP drug products can be dispensed multiple times from the same container, thereby significantly reducing the cost of goods compared to disposable drug products. Advantages of multi-use preservative-free GRP formulations are improved stability, alternative means of preventing microbial contamination, and reduced product costs that can make product commercialization easier.

  Sterile solutions can be prepared by incorporating the required amount of the active compound into a suitable solvent having one or more of the above-listed ingredients and, if necessary, then filter sterilizing. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders, preparation methods include vacuum drying and freeze drying in which a prefilter sterilized solution provides a powder of any additional desired ingredients in addition to the active ingredient. For example, microbial action can be prevented by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal.

  If sufficient preventive measures are in place to control and monitor medication and side effects, the mucosal and dermal administration of the present invention provides an effective self-administration treatment by the patient. Mucosal and dermal administration also overcomes certain drawbacks of other dosage forms, such as injections, which are painful, expose the patient to the risk of infection, and where drug bioavailability problems may exist. Systems for controlling aerosol dosing of liquid therapeutics as a spray are well known for nasal and pulmonary delivery. In one embodiment, a metered amount of active substance is delivered using a specially constructed mechanical pump valve (US Pat. No. 4,511,069). In another aspect, the active substance is delivered by skin patch technology.

Dosage For prophylactic and therapeutic purposes, the bioactive agents disclosed herein can be delivered in a single bolus delivery over a prolonged sustained delivery (eg, sustained transdermal, mucosal, or intravenous delivery) or Subjects may be administered in a repeated dose protocol (eg, an hourly, daily, or weekly repeated dose protocol). In this situation, a therapeutically effective dose of GRP is a long-term prevention or treatment plan with clinically significant results that alleviates one or more symptoms or detectable conditions associated with the target disease or condition. Repeated administration within the range of may be included. The determination of effective dosage in this situation is usually based on animal model experiments and subsequent human clinical trials, with effective dosages and administration protocols that significantly reduce the onset or severity of the subject's target disease symptom or condition. Guided by decision. Suitable models in this regard include, for example, mice, rats, pigs, cats, non-human primates, and other acceptable animal model subjects known in the art. Alternatively, effective dosages may be determined using in vitro models (eg, immunoassays and histopathology assays). With such a model, only a general calculation and adjustment is required, and usually a therapeutically effective amount (eg, an amount effective nasally, transdermally, intravenously, or intramuscularly to elicit the desired response). The appropriate concentration and dose of the physiologically active substance are determined.

  In another aspect, the present invention provides compositions and methods for nasal delivery of GRP, each day to maintain an increasing or decreasing pulsatile concentration of GRP that is therapeutically effective during long-term administration. Alternatively, compositions and methods are provided for repeated administration of a GRP compound by a nasal effective dosing regimen comprising multiple administrations of GRP to a subject on a weekly schedule. Depending on the composition and method, it is self-administered by the subject as a nasal formulation once to six times daily to maintain a therapeutically effective pulsatile concentration of GRP over a period of 8 to 24 hours of administration. A GRP compound is obtained.

Kits The present invention also includes kits, packaging, and multi-container units comprising the above pharmaceutical compositions, active ingredients, and / or administration means thereof for use in the prevention and treatment of diseases and other conditions in mammalian subjects. . In summary, these kits comprise one or more GRP proteins, analogs or mimetics in combination with an epithelial delivery enhancer disclosed herein formulated in a medicament for epithelial delivery, And / or containers or formulations containing other physiologically active substances.

  The nasal formulations of the present invention may be administered using any spray bottle or syringe or by infusion. An example of a nasal spray bottle is “Nasal Spray Pump w / Safety Clip, Pfeiffer SAP # 60548” which delivers a dose of 0.1 mL per squirt and has a dip tube length of 36.05 mm. This can be purchased from Pfeiffer of America, Princeton, NJ.

Aerosol Nasal Administration of GRP We have found that the above GRP can be administered intranasally using nasal sprays or aerosols. This was surprising because many proteins and peptides have been shown to be shredded or denatured by the mechanical force generated by the actuator when creating a spray or aerosol. The following definitions are useful in this field.

1. Aerosol-A product containing a therapeutic active ingredient that is packaged under pressure and released by actuation of a suitable valve device.

2. Metered aerosol-A pressurized dosage form consisting of a metered dose valve that allows delivery of a certain amount of spray with each actuation.

3. Powder aerosol—a product that is packaged under pressure and contains the therapeutically active ingredient in the form of a powder that is released by actuation of a suitable valve device.

4). Spray aerosol-an aerosol product that utilizes a compressed gas as a propellant to provide the force necessary to fire the product as a wet spray; generally a solution of a drug in an aqueous solvent Can be used.

5). Spray-liquid finely separated by air or steam jets. The nasal spray drug product includes a therapeutic active ingredient dissolved or suspended in a solution or mixture of excipients in a non-pressurized dispenser.

6). Metered spray—a non-pressurized dosage form consisting of valves that allow dispensing of a specific amount of spray with each actuation.

7. Suspension spray-A liquid formulation containing solid particles dispersed in a liquid medium and in the form of droplets or finely separated solids.

  Hydrodynamic characterization of an aerosol spray released by a metered nasal spray pump as a drug delivery device (“DDD”). Spray characterization is an integral part of the documentation required for approval by the Food and Drug Administration ("FDA") on research and development, quality assurance and stability testing methods for new and existing nasal spray pumps. .

  A thorough characterization of the spray geometry has been found to be the best indicator of the overall performance of a nasal spray pump. In particular, measurement of spray divergence angle (plume geometry) as it exits the apparatus; spray cross-sectional ellipticity, uniformity, and particle / droplet distribution (spray pattern); The tracking spread has been found to be the most representative performance quantity in nasal spray pump characterization. During quality assurance and stability testing, plume geometry and spray pattern measurements are key identifiers to prove consistency and suitability with nasal spray pump approved data criteria. is there.

  Plume Height—The length from the tip of the actuator to the point where the plume angle becomes non-linear due to the collapse of the linear flow. Based on visual inspection of the digital image, a height of 30 mm was defined for this test to set a width measurement point that coincided with the farthest measurement point of the spray pattern.

  Longest axis-the longest string that can be drawn in an approximate spray pattern across COMw, measured in base units (mm)

  Short axis-the shortest string that can be drawn in an approximate spray pattern across COMw, measured in base units (mm)

  Ellipticity—the ratio of the major axis to the minor axis, preferably 1.0 to 1.5, and most preferably 1.0 to 1.3.

D _10-The diameter of the droplet, where 10% of the total liquid volume of the sample is composed of droplets having a smaller diameter (μm) than the droplet

D _50- the diameter of a droplet, also known as the mass median diameter, where 50% of the total liquid volume of the sample is composed of droplets with a smaller diameter (μm) than the droplet ing

D _90-The diameter of the droplet, where 90% of the total liquid volume of the sample is composed of droplets having a smaller diameter (μm) than the droplet

  The smaller the length of span-dispersion width, the smaller the variance. The span is calculated as

  RSD%-percentage of the relative standard deviation, the value obtained by dividing the standard deviation by the mean value of the series, multiplied by 100, also known as CV%.

  Volume—The volume of liquid or powder released from the delivery device with each actuation, preferably 0.01 mL to about 2.5 mL, most preferably 0.02 mL to 0.25 mL.

  The above disclosure generally describes the present invention, which is further illustrated by the following examples. These examples are described solely for purposes of illustration and are not intended to limit the scope of the invention. Although specific terms and values are used herein, it will be understood that these terms and values are also exemplary and not limiting the scope of the invention.

Example 1
Materials and Devices Used In this example, reagents and devices used in Examples 2 and later of this application, and their sources are illustrated. Table 1 exemplifies sample reagents used in Examples 2 and later.

  Table 2 illustrates the sources and components of the MatTek EpiAirway ™ System described in more detail in Example 2 of this application.

  Table 3 illustrates the sources and components of the LDH assay system described in more detail in Example 2 of this application.

  Table 4 illustrates the instruments and other related laboratory consumables and sources used here, respectively.

Example 2
In Vitro Permeation Kinetics of Glucagon-Like Peptide-1 (GLP-1) Pharmaceutical Formulations In this example, representative pharmaceuticals of the invention comprising the excipients DDPC, EDTA and M-β-CD alone or in combination. It demonstrates that the formulation promotes GLP-1 permeation between epithelial cell monolayers. Table 5 shows the in vitro EpiAirway Model System to determine which formulations achieved the highest GLP-1 tissue permeability and transepithelial resistance (TEER) reduction without causing significant cytotoxicity. Illustrates formulations screened by transepithelial resistance assay (TEER), cell viability assay (MTT), lactate dehydrogenase cell death assay (LDH) and tissue permeation assay.

Three overlapping system samples for each formulation and control were evaluated using an in vitro model tracheal / bronchial epithelial cell membrane insert, EpiAirway system (Catalog ## Air-100) from MatTek (Ashland, MA). did. EpiAirway ™ system was developed by MatTek (Ashland, Mass.) As a model of pseudostratified epithelium layered in the respiratory tract. When the epithelial cells are grown on a cell culture insert with a porous membrane at the bottom at the air-liquid interface, the cells differentiate into a highly polar form. The apical surface has cilia with microvilli ultrastructure, and the epithelium produces mucus (the presence of mucin was confirmed by immunoblotting). Cells are seeded on inserts at the factory about 3 weeks prior to shipment. The insert has a diameter of 0.875 cm and provides an area of 0.6 cm ² . Cells are seeded on inserts at the factory about 3 weeks before shipment.

EpiAirway ™ culture membranes were received one day before starting the experiment. These culture membranes were shipped in Dulbecco's Modified Eagle's Medium (DMEM) without phenol red and without hydrocortisone. Each tissue insert was placed in a well of a 6 well plate containing 0.9 ml serum free DMEM. Tissues were equilibrated by culturing the membranes at 37 ° C./5% CO ₂ for 24 hours. This DMEM-based medium is serum-free but is supplemented with epidermal growth factor and other factors.

  The medium was tested each time for any cytokine and growth factor endogenous levels considered for intranasal delivery, but none of the cytokines or factors studied so far. The volume is sufficient to provide contact with the basal portion at the height of the unit, but leave the apical surface of the epithelium in direct contact with air. Sterilized forceps are used to ensure that no air enters between the bottom of the unit and the medium in this step and all subsequent steps including transferring the unit to a liquid-containing well.

  The EpiAirway ™ model system was used to evaluate the effect of each GLP-1 containing formulation on TEER, cell viability (MTT assay), cytotoxicity (LDH assay) and permeation. These assays are described in detail below.

Transepithelial electrical resistance (TEER)
The TEER value was measured using a tissue resistance measuring chamber connected to an epithelial voltage ohmmeter having an induction electrode. Both the epithelial voltage ohmmeter and the tissue resistance measurement chamber were from World Precision Instruments. On the day the experiment was started, the background TEER was first measured for each insert. After measuring the TEER, 1 ml of fresh medium was poured into the bottom of each well of a 6-well plate. The inserts blotted excess water on a paper towel and the inserts were placed in new wells with fresh media, numbered to correspond with background TEER measurements. 100 μl of experimental formulation was added to each insert. The insert was left at 100 rpm for 1 hour in a 37 ° C. shaking incubator.

Prior to checking the calibration, the power was turned off and the electrode and tissue culture blank insert were allowed to equilibrate in fresh medium for at least 20 minutes. Background resistance was measured using 1.5 ml medium in an Endohm tissue chamber and 300 μl medium in a blank Millicell-CM insert. The tip electrode was immersed in the medium but adjusted so as not to contact the top surface of the insert membrane. The background resistance of the blank insert was about 5-20 ohms. In each TEER measurement, 300 μl of medium was added to the insert, followed by a 20 minute incubation at room temperature before standing in the Endohm chamber to measure the TEER. Resistance was expressed as (resistance measurement-blank) X 0.6 cm ² . All TEER values are reported as a function of tissue area. TEER was calculated by the following formula:

Here, R _I is the resistance of the insert with the membrane, R _b is the resistance of the blank insert, and A is the area of the membrane (0.6 cm ² ). A decrease in TEER value relative to the control value (control = approximately 1000 ohm-cm ² ; 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 insert was removed from the incubator. 200 μl of fresh medium was poured into each well of a 24-well plate and the tissue insert was transferred to the 24-well plate. 200 μl of fresh media was slowly added to each tissue insert. The TEER was measured again for each insert.

  After transferring the tissue culture insert from the 6-well plate to the 2-well plate, the basal medium was distributed into the three systems and stored in an Eppendorf tube. All three distributed media were left in -80 ° C until use.

Lactate dehydrogenase (LDH) assay The amount of cell death was assessed by measuring LDH release from the cells using the CytoTox 96 Cytotoxicity Assay Kit (Promega). This test was performed on three overlapping samples for each tissue culture insert. The recovered 50 μl of medium (stored at 4 ° C.) was injected into a 96-well plate in triplicate. Fresh, cell-free medium was used as a blank. 50 μl of substrate solution (12 ml of assay buffer was added to a new bottle of substrate mix made according to the kit) was added to each well and the plate was incubated in the dark at room temperature for 30 minutes. After incubation, 50 μl of stop solution was added to each well and the plate was read at 490 nm using KCJr software on a μQuant absorbance plate reader.

MTT assay The cell viability of each cell culture insert was tested by MTT assay. Cell viability was assessed using the MTT assay (MTT-100, MatTek kit). This kit measures the incorporation of tetrazolium salts and conversion to formazan dyes. The MTT concentrate was thawed and diluted with 2 ml of MTT: medium: 8 ml of medium. Diluted MTT concentrate was pipetted into a 24-well plate (300 μl). The tissue insert was gradually dried, placed in the wells of the plate, and incubated for 3 hours at 37 ° C. in the dark. After incubation, each insert was removed from the plate, gently absorbed and placed in a 24-well extraction plate. The cell culture insert was immersed in 2.0 ml of extraction solution per well (to completely cover the sample). The extraction plate was covered and sealed to reduce evaporation of the extraction solvent. After overnight incubation at room temperature in the dark, the liquid in each insert was decanted back into the well from which it was removed and the insert discarded. The extraction solution (50 μl) from each well was pipetted in triplicate into a 96-well microtiter plate with an extraction blank and diluted by adding 150 μl of fresh extraction solution. Sample absorbance was read at 550 nm using KCJr software on a μQuant absorbance plate reader.

Tissue Permeation Assay The amount of GLP-1 (7-36) that permeates from the apical surface of the EpiAirway epithelial cell monolayer to the basolateral surface was indicative of GLP-1 permeability. The amount of GLP-1 protein observed on the basolateral surface of the cultured cells was measured by ELISA. The GLP-1 (7-36) amide ELISA kit was purchased from Linco Research (St. Charles, Michigan). The ELISA assay was performed according to the manufacturer's protocol. The collected sample was diluted with the assay buffer provided in the kit. In vitro screening was repeated several times. Percent permeation was calculated by multiplying the measured amount of GLP-1 measured by ELISA on the basolateral surface of the cell divided by the total amount of GLP-1 starting material added to the apical portion of the cell, multiplied by 100. Initially, the experimental design (DOE) was tested with different amounts of excipients EDTA, M-β-CD and DDPC. The results are summarized in Table 5. All formulations containing one or more excipients showed improved permeability relative to the control without excipient (# 31).

  In addition, the intranasal pharmaceutical formulations tested in the in vitro TEER, MTT, LDH and Permeation% tests are shown in Table 6. The TEER, MMT, LDH and permeation results for these formulations are summarized in Table 7.

  A decrease in the measured TEER value relative to the control represents a decrease in cell membrane resistance, or in other words, the passage of ionic species from the apical to basolateral of the epithelial monolayer. The data shown in Table 7 shows that all accelerator formulations significantly reduced TEER compared to the control formulation.

  The MTT assay measured cell viability while the LDH assay measured cytotoxicity. These assays are used in combination to measure the effect of a pharmaceutical formulation on cell “health”. Both MTT and LDH results were expressed as percentages. The percent MTT was calculated by multiplying the measured MTT value for each formulation by 100 times the MTT value of the control formulation. Therefore, the MTT positive control was 100% and was used as a baseline comparison with all other formulations. An MTT value of less than 80% represents a negative effect on cell viability. Similarly, the percent LDH was calculated by multiplying the LDH value measured for each formulation divided by the LDH value of the control formulation multiplied by 100. Therefore, the LDH positive control was 100% and was used as a baseline comparison with all other formulations. The MTT assay results show that all formulations except one formulation (# 12 in Table 7) did not reduce cell viability below 80%. This data is further supported by the LDH cytotoxicity assay, which shows that the majority of formulations did not show significant levels of cytotoxicity.

  GLP-1 tissue permeability is expressed as% permeation and fold increase over the permeability of the control formulation (sample # 13). The fold increase over the control of the excipient-containing formulation promoted GLP-1 permeation by about 74 to 341 times over the control permeation. These data indicate that inclusion of excipients DDPC, EDTA and M-β-CD significantly promotes GLP-1 permeation between epithelial cell monolayers. Formulations # 1, # 2, # 6, and # 12 in Table 7 resulted in a permeation rate improvement of more than 200 times over the control without excipient (# 13 in Table 7).

  In summary, in vitro data show that a representative pharmaceutical formulation of the present invention (# 6 in Table 6) containing 2 mg / ml GLP-1, 10 mg / ml EDTA and 10 mM citrate buffer is most It showed good GLP-1 permeation promoting ability and TEER lowering ability while having a minimal negative effect on cell viability. This formulation therefore represents an ideal candidate for the delivery of GLP-1 between mucosal surfaces, for example intranasal (IN) drug administration, in the treatment of human diseases including obesity and diabetes. .

Example 3
Comparison of in vitro permeation kinetics of glucagon-like peptide-1 (GLP-1) pharmaceutical preparations containing EDTA, EDTA zinc salt or EDTA magnesium salt In this example, the in vitro permeation kinetics of GLP-1 pharmaceutical preparations Demonstrate sensitivity to the form of EDTA used therein. Table 8 below illustrates formulations screened by in vitro EpiAirway Model System for transepithelial resistance (TEER assay), cell viability (MTT assay), lactate dehydrogenase (LDH assay; cell death) and tissue permeation. Has been. All samples contained 2 mg / ml GLP-1 except for samples # 9 and # 10, which served as controls. Since the formulation was used within 24 hours of manufacturing, no preservative was added. Each formulation was made in a total volume of 0.5 ml and evaluated in 3 overlapping systems (n = 3). Each sample was evaluated according to the protocol detailed in Example 2.

Abbreviations M-β-CD = methyl-β-cyclodextrin, EDTA = disodium edetate, DDPC = L-α-phosphatidylcholine didecanoyl, CB = chlorobutanol, Mg EDTA = EDTA disodium magnesium salt; Zn EDTA = EDTA Disodium zinc salt; MTT = MTT assay; LDH = LDH assay; TEER = transepithelial resistance.

Results For example, the effect of a pharmaceutical preparation containing an intranasal delivery enhancer such as excipients DDPC, EDTA and M-β-CD on EpiAirway ™ Cell Membrane (mucosal epithelial cell layer) is shown. The permeation kinetic results are summarized in Table 9 and represent measurements made 1 hour after the cells were incubated with the formulations shown in Table 8.

  Previous data show that various EDTA salt forms can be used in pharmaceutical formulations to manipulate drug permeation kinetics.

Example 4
GLP-1 Stability In this example, it is shown that low molecular weight excipients such as M-β-CD, EDTA, and DDPC do not improve the physical stability of GLP-1 in pharmaceutical formulations. In this example, the stability of GLP-1 was evaluated using two formulations described in Table 10 below. The purpose of this example was to determine whether heating of GLP-1 would cause proteolysis.

  One type of differential scanning calorimetry (DSC) with rescan was performed on sample # 1 in Table 10; the sample was scanned from 5 ° C. to 100 ° C. at a scan rate of 60 ° C./hour. Data was graphed as Cp (cal / ° C.) vs. temperature (° C.). In the first thermal scan, a sharp transition peak is observed around 35 ° C. A large decrease in noise after the transition and heat capacity between 80 ° C. and 90 ° C. suggests the formation of aggregates. The solution was observed to be slightly cloudy after the scan, confirming the formation of a precipitate. The transition peak is much narrower than expected for peptide unfolding, which may be due to aggregation occurring immediately after peptide unfolding. These data indicate that GLP-1 initiates denaturation and / or aggregate formation from 35 ° C or around 35 ° C.

  DSC experiment with rescan was also performed on sample # 2; samples were scanned from 5 ° C. to 100 ° C. at a scan rate of 60 ° C./hour. Data was graphed as Cp (cal / ° C.) vs. temperature (° C.). In the first thermal scan, a very broad peak is observed around 46 ° C. The significant decrease in heat capacity between 80 ° C. and 90 ° C. may be due to the formation of aggregates. However, the solution did not appear cloudy after scanning, suggesting very small aggregates. These data indicate that GLP-1 initiates denaturation and / or aggregate formation from around 46 ° C. or around 46 ° C. in the formulation.

  These data indicate that adding the excipients listed in Table 10 to the GLP-1 formulation does not improve its physical stability.

Example 5
Pharmacokinetic (PK) assessment of glucagon-like peptide-1 (GLP-1) in selected pharmaceutical formulations in nasal and intravenous rabbits In this example, bioavailability of GLP-1 by intranasal administration Is significantly promoted by including dipeptidyl peptidase-IV (DPP-IV) such as, for example, Lys (4-nitro-Z) -pyrrolidide in the formulation. Rabbit pharmacokinetic (PK) studies were performed to assess the plasma pharmacokinetic properties of GLP-1 for various formulations administered by intranasal (IN) delivery versus intravenous (IV) injection. The overall experimental design is shown in Table 11. Formulations # 1 through # 4 represent IN formulations, while formulation # 5 represents IV injected formulations.

  In this study, New Zealand White rabbit (Hra: (NZW) SPF) was used as a test subject to evaluate the plasma pharmacokinetics of GLP-1 by intranasal administration and intravenous injection. Since the pharmacokinetic profile obtained from the drug administered to the rabbit closely resembles the PK profile of the same drug in the human body, the rabbit was selected as the target animal in this study.

  In this study, four intranasal and one intravenous formulations of GLP-1 were evaluated. The media composition of each formulation is presented in Table 12. GLP-1 intranasal and intravenous formulations were manufactured for final preparation and testing.

  For each dosing solution, the ingredients were provided in two parts, part A and part B (see Table 12). The final formulation for each intranasal group (Group 1-4) was created by mixing equal parts Part A (1 mL) and Part B (1 mL). For the intravenous group ("Group 5", 1.5 mL of Part A and 3.5 mL of Part B were mixed. The final dosing solution for all groups was used within 6 hours of preparation.

  In the four intranasal formulations, the concentration of GLP-1 was constant. Citric acid concentration and pH were also constant in the four formulations. The EDTA concentration was constant for formulations 1, 3 and 4, while formulation 2 contained the tight junction modulator PN159 previously shown to facilitate the delivery of peptides between epithelial cell layers, EDTA was not included. Formulation 5 contained citric acid, EDTA and sodium chloride, each at a concentration suitable for intravenous administration; the concentration of GLP-1 was such that the total dose of GLP-1 was 10% of intranasal administration. I reduced it to be.

  Lys (4-nitro-Z) -pyrrolizide is a major enzyme involved in the metabolism of active GLP-1 (7-36 amino acid fragment) into an inactive metabolite (9-36 amino acid fragment), dipeptidylamino It is a specific inhibitor of peptidase (DPP) IV. In the evaluation of this study, the concentration of Lys (4-nitro-Z) -pyrrolizide was different for each intranasal administration group except that the concentrations of Formulation 1 and Formulation 2 were the same. The total dose of Lys (4-nitro-Z) -pyrrolizide was about 0.04 mmol / kg in Groups 1 and 2 (intranasal group) and Group 5 (intravenous group).

GLP-1 Assay Method Test samples, standards and quality control samples were assayed using a glucagon-like peptide-1 (activity) ELISA kit (Linco Research, catalog # EGLP-35K). Each sample was analyzed in two overlapping systems.

  This assay involves the capture of active GLP-1 (7-36 and 7-36 amide fragments) by monoclonal antibodies (specific for the N-terminal region) immobilized in the wells of a 96-well microtiter plate, and anti-antibody It is based on detection with a GLP-1 alkaline phosphatase labeled secondary antibody. After washing, methyl umbelliferyl phosphate, which forms the fluorescent product umbelliferone in the presence of alkaline phosphatase, is added to each well. The amount of fluorescence produced is directly proportional to the concentration of active GLP-1 in the unknown sample, which is obtained by interpolation from a standard curve using a reference standard of active GLP-1 at a known concentration.

  Due to the species similarity between human and rabbit GLP-1, it was expected that the assay would detect endogenous (ie, rabbit) active GLP-1. The endogenous level of rabbit GLP-1 is measured at time O. The 9-36 GLP-1 fragment was not detected in this assay, regardless of its source.

Pharmacokinetic Evaluation Pharmacokinetic calculations were performed using WinNonlin software (Pharsight, version 4.0, Mountain View, Calif.) And a non-compartmental model of extravascular administration. The evaluation parameters are listed in Table 13.

Pharmacokinetic Parameter Analysis Mean GLP-1 pharmacokinetic data for both intranasal and intravenous groups are presented in Table 14.

  The pre-dose (baseline) concentration of endogenous GLP-1 in serum or plasma is generally less than 10 pg / mL. For some samples, the sample volume was limited, preventing repeated analysis to obtain accurate results. For these samples, a value of <4 pg / mL was reported. Data were baseline corrected where appropriate to calculate group means and assess pharmacokinetics. The result expressed as <NUMBER was set to "<NUMBER / 2" and by this method the value <4 pg / mL was also set to 2 pg / mL.

Following administration, plasma GLP-1 values in individual animals exceeded baseline 10 pg / mL. At the final time point (90 minutes) after intravenous administration, the plasma concentration of GLP-1 is at or around the baseline value, and the injection and drainage phases are within the sampling time frame used in this study. It was suggested that he was caught. After intranasal instillation, several animals showed GLP-1 plasma concentrations above the baseline concentration. AUC _inf was measured to assess the overall exposure profile of GLP-1.

Formulation 1 / Group 1
The peak concentration of GLP-1 (T _max ) occurred between 5 and 20 minutes after administration (11 minutes on average for the group). The average C _{max for the} group was 844.9 pg / mL. The plasma concentration remained above baseline at 90 minutes after administration, suggesting that elimination was not complete at this time. This is reflected in an AUC _last of 20,792.7 min * pg / mL and an AUC _inf of 32,415.9 min * pg / mL. The average terminal half-life (t _1/2 ) was estimated to be 55.5 minutes.

Formulation 2 / Group 2
The mean C _{max for} Group 2 was estimated to be 424.0 pg / mL. The average T _max was estimated to be 43 minutes; however, there was considerable variability between animals for this parameter. Examination of the concentration versus time profile of these animals showed an absorption (increase) and excretion (decrease) phase. The average AUC _{last for} Group 2 was 17,069.8 min * pg / mL. T _1/2 could not be estimated because there was no clear elimination phase in the two animals in the group. However, the average t _1/2 based on the data collected for the remaining animals in this group was 66.8 minutes. The average AUC _inf (3 animals that could measure Kel) was 33,324.4 min * pg / mL.

Formulation 3 / Group 3
The mean C _max , T _max , t _1/2 , and AUC _{last for} Group 3 were 283.9 pg / mL, 25 minutes, 39.3 minutes, and 9,331.5 minutes * pg / mL, respectively. One animal in this group showed a higher than expected value at 60 minutes, so an accurate Kel could not be measured and therefore t _1/2 could not be measured. Therefore, this value was not included in the group average t _1/2 of 39.3 minutes and the group average AUC _inf of 13,232.7 minutes * pg / mL.

Formulation 4 / Group 4
The average C _{max for} Group 4 was estimated to be 154.5 pg / mL. The average T _{max for the} group was 47 minutes, but two animals showed their highest measured plasma concentration of GLP-1 at 90 minutes. For these animals, Kel (and t _1/2 ) and AUC _inf could not be measured because there was no clear elimination phase. The average t _1/2 and AUC _inf for the other 3 animals were estimated to be 22.4 minutes and 5,734.4 minutes * pg / mL, respectively.

Formulation 5 / Group 5
During the infusion process, one animal in Group 5 delivered an additional ˜100 μl during the 5 to 10 minute time point due to mechanical failure of the pump device. This is reflected in the concentration value of> 5000 pg / mL at 10 minutes. Data for this animal was not included in the Group 5 pharmacokinetic assessment because the exact conditions of the infusion could not be determined.

The mean C _max at 10 minutes of intravenous infusion was 3.183.6 pg / mL. Three animals showed a T _max of 5 minutes and one animal showed a T _max of 10 minutes; however, the concentration of GLP-1 was 5 minutes and 10 minutes for all 4 animals. It was almost the same at that time. The final t1 _{/ 2 for} this group was estimated to be 30.9 minutes. The average AUC _last of 28,883.2 min * pg / mL was 29,149.5 min * pg / mL with AUC _inf slightly above this, so most of the exposure profile was captured.

Examination of the logarithmic profile of concentration versus time from 10 minutes (at the end of the infusion) to 20 minutes suggested excretion of two phases of GLP-1. The earlier alpha phase of the biphasic profile is generally associated with extravascular distribution and excretion, and the slower beta phase is believed to represent final excretion. Calculation of the discharge rate within this time frame indicated an initial t _{1/2 of} 2 minutes or less than 2 minutes. This initial t _1/2 would be consistent with the achievement of a clear steady state at the 10 minute trial of the infusion time.

Bioavailability GLP-1 bioavailability after nasal administration was calculated using AUC _last and AUC _inf ; these estimates are shown in Table 15. In Groups 1, 3, and 4, the concentration of inhibitor Lys (4-nitro-Z) -pyrrolizide in the formulation was 0 mM, 15 mM, or 25 mM, respectively. In the absence of inhibitor, GLP-1 bioavailability was about 2%, but by adding 15 mM Lys (4-nitro-Z) -pyrrolizide to the formulation (Group 3), GLP-1 Bioavailability increased from about 3% to 5%. GLP-1 bioavailability was further increased to about 7% to 11% by the addition of 25 mM Lys (4-nitro-Z) -pyrrolizide to the formulation (Group 1).

  Polypeptide PN159 has been demonstrated to increase peptide bioavailability compared to low molecular weight excipients. Formulation 2 (Group 2) containing PN159 and 25 mM Lys (4-nitro-Z) -pyrrolizide showed about 6% to 11% GLP-1 bioavailability when tested, which , Equivalent to the bioavailability observed for GLP-1 in the presence of 25 mM Lys (4-nitro-Z) -pyrrolizide without PN159. PN159 has an effect equivalent to 10 mM EDTA on GLP-1 IN bioavailability.

The final half-life of GLP-1 in rabbits is approximately 30 minutes, and literature (Parkes, D., et al., “Pharmaceutical Actions of” reported a final half-life of 10 minutes or less for rats and humans. Exendin-4 in the rat: Comparison with Glucagon-like Peptide-1, "Drug Development Research 53: 260-267, 2001; Deacon, C, Therapeutic Strategies Based on Glucagon-like Peptide-1, Perspectives in Diabetes 53: 2181- 2189, 2004), which was longer than expected. Rabbit t1 _{/ 2} was supported by this study as it was consistent between groups and animals.

  The final calculation depends on the portion of the logarithmic curve of concentration versus time used to determine the slope (see Kel definition in Table 13). For the above estimation, the modeling program selected the best fit for Kel estimation. Manual selection of a curve that fits from 10 minutes (at the end of the infusion) to 90 minutes showed a final half-life in the range of 10 to 12 minutes (no data disclosed).

  A second means is provided for evaluating post-dose treatment of the drug by clearance. Clearance can be considered the intrinsic ability of the body or its organs to remove drugs from blood (Basic Clinical Pharmaceuticals, 2nd ed., Applied Therapeutics, Inc. Vancouver, Washington.). In this case, however, GLP-1 can remain in the blood but can also be considered removed for purposes of assessing clearance. The basis for this is that GLP-1 exists in two different states; an active state consisting of a peptide fragment represented by amino acids 7-36, and a peptide fragment represented by amino acids 9-36. Inactive state resulting from metabolism. When converted to the inactive state, GLP-1 is considered effectively removed from the body. Since the assay used here for detection of GLP-1 was specific for the active form of GLP-1 (7-36), the presence of the inactive form of GLP-1 in the blood It does not interfere with the clearance rating of -1.

  For the intranasal group, a group average maximum clearance value (CL-F) of 15,160.0 mL / min / kg was shown for group 4. The average value for Group 3 was 6,977.0 mL / min / kg. For groups 2 and 1, the clearances were similar, 2,716.0 mL / min / kg and 3,032.8 mL / min / kg, respectively. As expected, the clearance estimate for each group is inversely proportional to systemic exposure.

  The clearance values for the intranasal group were also corrected for GLP-1 bioavailability. The corrected clearances for groups 4 and 3 were 7,580 mL / min / kg and 1,395 mL / min / kg, respectively. For Groups 1 and 2 (˜11% bioavailability, respectively), the corrected clearance values were measured to be 275 mL / min / kg and 247 mL / min / kg, respectively. The corrected clearance for Groups 1 and 3 approximated the clearance estimate of 259.8 mL / min / kg for the intravenous group (Group 5).

The total dose of Lys (4-nitro-Z) -pyrrolizide was about 0.004 mmol / kg for groups 1, 2 and 5. The blood level of Lys (4-nitro-Z) -pyrrolizide was not measured in this study and therefore bioavailability after intranasal administration is unknown. Due to the presence of Lys (4-nitro-Z) -pyrrolizide in the nasal formulation, the active GLP-1 is metabolized in the systemic circulation, assuming its metabolism in the nasal mucosa and its reasonable bioavailability. It becomes possible to protect from. Protection from metabolism by the nasal mucosa is also consistent with the higher C _max of GLP-1 when Lys (4-nitro-Z) -pyrrolizide was present in the formulation. However, since both fragments of GLP-1 will likely cross-enter into the systemic circulation, total GLP-1 (7-36) is confirmed to confirm a higher percentage of active GLP-1 Assays for amino acids and fragments of 9-36 amino acids) would be necessary.

  Protection from metabolism in the blood is indicated by the similarity in the clearance values of GLP-1 between the intravenous group (group 5) and the nasal group with the highest inhibitor concentration (groups 1 and 2).

Summary The above data are surprising and unexpected that the delivery of an intranasal pharmaceutical formulation of GLP-1 containing the DPP IV inhibitor Lys (4-nitro-Z) -pyrrolizide increased GLP-1 bioavailability. It is a proof of discovery. Furthermore, GLP-1 bioavailability was dependent on the concentration of inhibitor in the formulation. In the absence of inhibitor, GLP-1 bioavailability was approximately 2%. Inclusion of Lys (4-nitro-Z) -pyrrolizide at 15 mM increases GLP-1 bioavailability to about 5%, with 25 mM Lys (4-nitro-Z) -pyrrolizide, GLP- The bioavailability of 1 was about 11%. The data obtained suggested the possibility of local (nasal tissue) and systemic inhibition of GLP-1 metabolism. In addition to 25 mM Lys (4-nitro-Z) -pyrrolizide, a formulation containing PN159 was also investigated to evaluate the possibility of achieving higher bioavailability with this tight junction modulator. Under the conditions of this study, the effect of PN159 on the bioavailability of GLP-1 did not exceed the effect of EDTA in the formulation.

Example 6
Stability in use and as a solid of the GLP-1 formulation The “in use” stability is stored in a vial fitted with an actuator and according to an appropriate treatment plan (in this case 3 times a day (TID)). Defined by tests with formulations that are nebulized and placed at a specific storage temperature. The vial is first primed and not primed until a subsequent spray. Priming is defined as spraying until the first complete spray is visually clear and then activated one or more times before administration. Vials were always stored at 30 ° C./65% relative humidity (RH) and removed from the chamber for each dose and sprayed within 10 minutes. Vials were run three times or once a day depending on the plan chosen for each test. There was a minimum of one hour between each spray to describe the visual / physical observations.

  A “in use” test at TID / 30 ° C. was performed. The formulation used in this study contained 5 mg / mL GLP-1, 10 mg / mL EDTA, 10 mM citrate buffer (pH 3.5) and no preservative. Vials were filled, primed, operated with TID, and stored at 30 ° C./65% RH for 10 days. Tables 16 and 17 show the recovery rate and purity of GLP-1 after TID spraying for 7 days. The recovery of “in use” peptides was over 95 ± 2.2% by day 7. The total peptide purity “in use” was 98 ± 2.1% after 10 days TID / 30 ° C./65% RH.

  “Sealed” stability is stored in a sealed (ie, capped) vial and is at a high temperature condition (ie, 5 ° C., 25 ° C., 40 ° C., and / or 50 ° C.) in a specific storage location. Defined by a test using a formulation that was allowed to stand for a specific time below. “Sealed” stability studies were performed on formulations that showed good results in a series of in vitro screens. The formulation was prepared and stored at 5 ° C, 25 ° C and 35 ° C. Table 18 shows the formulations tested. Results for Formulations # 5 and # 6 (containing GLP-1 and EDTA) show that after 56 days of storage, GLP-1 recovery was 100% at 5 ° C,> 97% at 25 ° C, and 35 ° C It was> 90%.

  “Sealed” stability assays consisted of formulations made for “in use” tests (5 mg / mL GLP-1, 10 mg / mL EDTA, 10 mM citrate buffer (pH 3.5), The same batch formulation was used (no preservative). The formulation was stored at 5 ° C. The “sealed” stability results are shown in Tables 19-20 below.

Example 7
In Vitro Pharmacodynamics of Intranasal GLP-1 Formulations Three studies have shown that GLP-1 (7-36) amide and synthetic exendin-4 (exenatide) on blood glucose after intranasal (IN) administration of GLP-1 ) Was compared with or without subcutaneous injection (SQ) of DPP-IV inhibitor or exenatide. This study was conducted with the ZDF rat model of the oral glucose tolerance test (OGTT) used in human studies.

  Pharmacodynamic effects were assessed by monitoring blood glucose and blood insulin levels. The concentration of glucose in the blood was measured using a Syncron CX4 analyzer and an appropriate Glucose Reagent kit (Beckman Coulter, Blair, CA, USA). Pharmacokinetic parameters were measured using WinNonlin software (Pharsight, version 5.01, Mountain View, CA). The insulin concentration in the blood was measured by ELISA.

Acetaminophen concentration in the blood was measured using a Synchron CX4 analyzer and an Acetaminophen Reagent kit (Beckman Coulter, Blair, CA, USA). Since acetaminophen generally absorbs only to a negligible extent from the stomach, acetaminophen was used as a marker for gastric emptying. Time to peak concentration after administration (T _max ) and peak concentration (C _max ) reflect the time at which gastric emptying occurs, and the profile of gastric emptying (eg, into the systemic circulation after being released from the stomach) Absorption etc.) reflect the time to discharge. AUC reflects overall exposure. Acetaminophen was administered in tests 2 and 3 to monitor gastric emptying.

Test 1
Table 21 outlines the overall experimental design for evaluating blood glucose and insulin values in Test 1.

  The formulation for GLP-1 treated rats in Study 1 was as follows: F1 = 1O mg / mL EDTA, 10 mM citrate buffer (pH 3.5), no inhibitor; F2 = 10 mg / mL EDTA, 10 mM citrate buffer (pH 3.5) and 25 mM H-Lys (4-nitro-Z) -pyrrolizide (inhibitor is commercially available from Bachem); and F3 = 10 mg / mL EDTA, 10 mM citrate buffer (pH 3.5) and 25 mM L-proline-boroproline (Pro-boropro) are disclosed in Patent Application No. 2004/0229820 A1, Bachovin , And synthesized based on the information of William, W. et al.).

  Table 22 shows the PD results of Test 1. The AUC (0-150) of glucose in rats treated with the GLP-1 formulation (with or without DPP-IV) was ˜15% lower than rats treated with exenatide and placebo. In Test 1, glucose was administered at 2 g / kg, whereas in Test 2 and subsequent tests, glucose was administered at 1 g / kg. As a result, because of the high glucose load in Test 1, the observed glucose reduction rate was low compared to the glucose reduction rate of other tests (eg, 0-16% AUC (0-150)). In Study 1, acetaminophen was not administered to monitor gastric emptying.

  There was no difference in blood glucose between GLP-1 formulations when administered +20 minutes after oral glucose administration. When pre-administered at -10 minutes, there was a difference in blood glucose profile between GLP-1 formulations with inhibitors (F2 and F3) versus GLP-1 formulations without inhibitors (F1). Both formulations (F2 and F3) containing DPP-IV inhibitors produced a delay in Cmax (from 45 minutes to about 120 minutes). Significant levels of GLP-1 delayed gastric emptying; the rabbit PK study showed 5% higher BA% in the GLP-1 formulation with the DPP-IV inhibitor than without. In Study 2, acetaminophen was added to a formulation containing a DPP-IV inhibitor and compared to the gastric emptying of a GLP-1 formulation without the inhibitor.

  Insulin and glucose measurement data for placebo at -10 minutes, GLP-1 F1 at -10 minutes (no inhibitor) and GLP-1 F1 at +20 minutes (no inhibitor), the increase in insulin is- It is shown to be more magnified with GLP-1 F1 when administered at either 10 minutes and +20 minutes compared to placebo.

Test 2
The PD effect of GLP-1 (7-36 amide) in the presence and absence of a DPP-IV inhibitor on blood glucose after intranasal instillation (GLP-1) in a rat model of OGTT was tested. Assayed. A summary of the overall experimental design for assessing blood glucose and insulin values is shown in Table 23. The OGTT in Test 2 was performed by administering a 1 g / kg glucose solution batch administration via an oral gastric tube. Acetaminophen was co-administered with the glucose solution at a dose of 100 mg / kg. For the purpose of time measurement, OGTT was set to 0 minutes.

  Formulations in Test 2 include: F1 = 10 mg / mL EDTA, 10 mM citrate buffer (pH 3.5), no inhibitor; F3 = 10 mg / mL EDTA, 10 mM citrate Buffer (pH 3.5) and 25 mM L-proline-boroproline. Blood glucose was assessed at the following time points: before OGTT and 5, 10, 15, 30, 45, 60, 75, 90, 120, 180 and 240 minutes after OGTT. Blood insulin was assessed at the following time points: before OGTT and 15, 30, 60, 120 and 240 minutes after OGTT. APAP blood levels were assessed at the following time points: before OGTT and 5, 10, 15, 30, 45, 60, 75, 90, 120 and 180 minutes after OGTT. The results of Test 2 show that there was a decrease in glucose AUC (baseline corrected) compared to the control, especially for the formulation without inhibitor (F1). In both formulations with and without inhibitors (F1 and F3), an increase in insulin occurs after administration. IN administration of 100 ug / mL GLP-1 was shown to delay gastric release in the presence of inhibitors. GLP-1 administration at higher amounts of IN affected gastric emptying even in the absence of inhibitor. When gastric emptying was delayed due to large doses or the presence of inhibitors, glucose absorption was also delayed. Table 24 shows the difference in glucose AUC for the treatment group compared to placebo.

Test 3
In study 3, GLP-1 (7-36 amide) in the absence of an inhibitor of blood glucose after intranasal instillation (GLP-1 or saline) or exenatide subcutaneous injection in a rat model of OGTT and Exenatide's pharmacodynamic (PD) effect was assayed. Test 3 used a GLP-1 formulation without a DPP-IV inhibitor (F1 = 1O mg / mL EDTA, 10 mM citrate buffer (pH 3.5), no inhibitor). The primary purpose of Trial 3 was to compare GLP-1 F1 (IN) with exenatide (SQ). PD was assessed by monitoring blood glucose and blood insulin concentrations. Acetaminophen was administered to monitor gastric emptying (from study 2).

  In Test 3, OGTT was performed by administering a 1 g / kg glucose solution batch administration via an oral gastric tube. Where appropriate, acetaminophen was co-administered with a glucose solution at a dose of 100 mg / kg. For the purpose of time measurement, OGTT was set to 0 minutes. The time of treatment administration was relative to OGTT. A single treatment dose was administered at -10 minutes. The second administration was performed at -10 minutes for the first administration and at +35 minutes for the second administration. Therefore, saline, GLP-1 or exenatide was administered at -10 minutes, +35 minutes, or -10 minutes and +35 minutes relative to OGTT.

Blood was collected for pharmacodynamic analysis, including calculation of AUC _0-24O and Cmax. Blood glucose was assessed at the following time points: before OGTT and after OGTT (5, 15, 30, 45, 60, 75, 90, 120, 180 and 240 minutes). Blood insulin was assessed at the following time points: before OGTT and after OGTT (5, 15, 30, 45, 60, 75, 90, 120 and 180 minutes). The blood concentration of acetaminophen was assessed at the following time points: before OGTT and after OGTT (15, 30, 45, 60, 75, 90, 120 and 180 minutes). A summary of the overall design in Test 3 for evaluating blood glucose and insulin values is shown in Table 25. An overview of the overall design for assessing gastric emptying is shown in Table 26.

The results of Trial 3 _show a dramatic reduction ( _˜60 %) in corrected blood glucose AUC _0-24O after GLP-1 intranasal administration compared to saline or exenatide SQ administration. AUC _0-24O and C _max values are shown in Table 27. The change in glucose concentration corrected for endogenous glucose is shown in FIG.

  These pharmacodynamic results indicate a surprising and unexpected finding that intranasal administration of a pharmaceutical formulation of GLP-1 reduced blood glucose levels.

  Group average insulin data for the GLP-1 treatment group and placebo in trial 3 are shown in FIG. The insulin concentration in saline-treated (placebo) rats showed a slight decrease after OGTT. A single dose of 100 μg / kg GLP-1 at −10 minutes resulted in a significant increase in blood insulin concentration in response to OGTT. Administration of GLP-1 at -10 and +35 minutes resulted in insulin spikes immediately after each administration. This pattern indicates that GLP-1 is involved in insulin release in response to elevated blood glucose.

Group mean acetaminophen data (with standard deviation for placebo group) for GLP-1 treated group and placebo is shown in FIG. In trial 3, total exposure (AUC) was measured and showed that there was less than 3% difference in the total amount of acetaminophen absorbed into the systemic circulation among the three treatment groups. It was done. The AUC values are 6,804 μg * min / kg and 6,672 μg * min for the saline control (placebo), 100 μg / kg GLP-1 and 1000 μg / kg GLP-1 groups, respectively. / kg and 6,951 μg * min / kg. However, the absorption profile was different. In placebo-treated rats, the T _{max of} acetaminophen was 30 minutes after administration, the group mean C _max was 78 μg / mL, and its standard deviation was ± 21 μg / mL. A slightly lower 66 ± 21 ug / mL C _max was observed in the 100 μg / kg GLP-1 group 30 minutes after administration; however, at most time points evaluated, this group and the control group In between, the concentration of acetaminophen was close. The C _{max of} acetaminophen was 45 ± 30 ug / mL, lower in the 1000 μg / kg GLP-1 group. Furthermore, T _max was thought to occur between 30 and 90 minutes after administration, and the blood concentration of acetaminophen was significantly higher at the second half. The results for the 1000 μg / kg GLP-1 group are consistent with delayed gastric emptying and a more prolonged profile of gastric emptying after large doses of GLP-1. The results of Test 3 show that GLP-1 without inhibitor significantly reduces glucose and the dose can be administered before or after a meal. The IN GLP-1 formulation reduced glucose AUC, but did not reduce exenatide when administered SQ to ZDF rats at either 0.6 or 3 ug / kg.

Summary of PD results The pharmacodynamic results show the surprising and unexpected discovery that intranasal administration of a pharmaceutical formulation of GLP-1 reduces blood glucose levels. Furthermore, the pattern of blood insulin concentration indicates that GLP-1 is a factor involved in insulin release in response to an increase in blood glucose. The results of the acetaminophen test are consistent with delayed gastric emptying and a more prolonged profile of gastric emptying after large doses of GLP-1 in the absence of DPP-IV inhibitors. A low dose of GLP-1 effective at low blood glucose had no effect on gastric emptying. Decreasing the glucose concentration in the blood is effective for the treatment of type 2 diabetes, particularly through an increase in insulin concentration. Furthermore, a delay in gastric emptying can increase satiety and promote weight loss. These data indicate the effectiveness of the GLP-1 intranasal formulation described in these assays for use in the treatment of metabolic disorders such as obesity and diabetes.

Example 8
Transmucosal formulations containing promoters of synthetic exendin-4 (exenatide) Various excipients were tested for in vitro optimization of transmucosal exenatide formulations. A transmucosal exenatide formulation was created by combining exenatide and excipients (including permeation enhancers, solubilizers, surfactants, chelating agents, stabilizers, buffers, tonicity agents and preservatives).

  The formulation was screened multiple times and classified into two series, A and B. Series A focused on changing excipient concentrations of solubilizer (Me-β-CD), surfactant (DDPC), chelating agent (EDTA), and stabilizer (gelatin). Tests were also conducted on buffers such as citrate buffer, tartrate buffer, and glutamate (MSG). In series B, alternative excipients were screened for their ability to promote exenatide permeation. Various concentrations of cyclodextrin, glycoside, fatty acid, phosphatidylcholine, GRAS compound, PN159, gelatin and other permeation enhancer candidates were tested. In addition to screening permeation enhancer candidates, screening was also performed for different concentrations of buffer (citrate buffer, tartrate buffer) and isotonic agent / stabilizer excipients (mannitol, NaCl). Preservatives such as sodium benzoate (NaBz) and benzalkonium chloride (BAK) were also tested. The excipients tested in the in vitro screen are shown in Table 28. Of the 372 unique formulations tested, 11 were recommended for use in rabbit preclinical PK studies in vivo, see Table 29.

Example 9
The transmucosal exenatide formulation promotes the release of tight junctions in vitro The exenatide formulation described in the protocol in Example 2 above was assayed in vitro for TER, LDH, MTT and permeation. For all exenatide formulations containing accelerators, the TER decreased from about 350-700 ohms x cm ² to about 5-20 ohms x cm ² after an incubation time of 60 minutes. All exenatide formulations, except the control, contained EDTA. EDTA is known as a calcium chelating agent to release tight junctions by removing calcium. In a static environment such as the in vitro tissue culture system used here, the removal of calcium from the solution significantly releases the tight junction. No decrease in TER was observed with Exenatide + Glutamate Control (MSG) containing only Exenatide in glutamate buffer with sodium chloride as an isotonic agent. Exenatide + glutamate control indicates that opening a tight junction is not an essential property of exenatide itself. The TER of the insert after 60 minutes exposure to the glutamate control is comparable to that of the insert exposed to the media for 60 minutes. The Triton X control had the lowest potential TER, as expected, caused by killing the cell barrier.

  In order to confirm that the decrease in TER by exenatide preparation was not the result of cell death but the result of tight junction adjustment by an accelerator, LDH and MTT assays were performed using the same MatTek cell line used in TER. went. Exenatide formulation showed no significant increase in cytotoxicity as measured by% LDH. The Exenatide formulation only showed less than 20% LDH loss. Similarly, the media control also showed no cytotoxicity. In contrast, the Triton X control treatment group showed significant cytotoxicity as expected. Cell viability was assessed using the MTT assay (MTT-100, MatTek kit). Exenatide formulation is MTT% except that three formulations of JW-239-126-14, JW-239-126-19 and JW-239-126-24 showed about 50% MTT. When measured, it did not significantly increase cytotoxicity. In contrast, the exenatide formulation showed a survival rate exceeding 80% MTT. Similarly, the medium control showed no cytotoxicity. In contrast, the Triton X control treatment group showed significant toxicity as expected. The results of the transmission test are shown in Table 30.

  The permeation results of the formulation showed a 300- to 830-fold increase in permeability over the control formulation containing only 3 mg / mL exenatide and monosodium glutamate. The formulation that received half the dose showed a 1400 to 2200-fold increase in permeability over the control.

Example 10
Intra-Rabbit PK Results for Transmucosal Exenatide Formulations Exendin-4 formulations prepared for in vivo testing are shown in Table 31.

  Exendin-4 PK test was performed in rabbits and the PK results of exendin-4 administered by IV and IN were compared. The IN formulation included IN control (no accelerator), IN 1X accelerator + gelatin, IN 2X accelerator, and IN 2X accelerator + gelatin (formulation is shown in Table 31). The results of the PK test are shown in Table 32 and FIG.

  The PK results indicate that IN 2X accelerator and IN 2X accelerator + gelatin were the most performing exendin-4 formulations tested in rabbit tests. Both IN 2X accelerator and IN 2X accelerator + gelatin showed higher PK values than IV or IN controls.

Example 11
Another transmucosal exenatide formulation Another exendin-4 formulation for transmucosal administration was developed and tested for: 1) improved storage stability, 2) improved exendin-4 bioavailability, And 3) an increase in pharmacodynamic effects determined by measuring insulin and glucose concentrations.

  The modification of the exendin-4 formulation tested earlier was adjusted by removing DDPC and gelatin in all formulations except one formulation. “OEF” was used to refer to a formulation containing 80 mg / mL Me-β-CD and 5 mg / mL EDTA (without DDPC or gelatin) in 10 mM acetate buffer. The second change to the formulations tested earlier was to add arginine to some formulations. The OER formulations are listed in Table 33.

Percent Permeation In vitro permeation tests show that the OEF formulation (without DDPC and gelatin) is equivalent or better compared to the Exendin-4 formulation tested earlier (see previous tested formulations in Table 31). It shows that exendin-4 permeation was more greatly promoted while presenting cell viability. The permeation results comparing the formulations are shown in Table 34.

Pharmacokinetic Studies In vivo pharmacokinetic (PK) studies in rabbits were performed using the formulations previously tested when the “OEF + arginine” and “2X accelerator + gelatin, arginine” formulations were delivered intranasally. It was shown that exendin-4 bioavailability was improved to the same extent or beyond. The average peak exendin-4 plasma concentration was highest with the OEF + arginine formulation. Table 35 shows a comparison of the average PK parameters (T _max , C _max , AUC, t _1/2 and Kel) of the formulations tested. Table 36 shows the dispersion coefficient of each PK parameter, and Table 37 shows the absolute bioavailability (F%) of each intranasal formulation.

  The results of the PK test indicate that OEF + arginine had the highest% bioavailability (16.6%). The other two intranasal formulations, 2X accelerator and 2X accelerator plus gelatin also significantly promoted bioavailability compared to IN control (12.6% and 11.5%, respectively).

Pharmacodynamic Testing In vivo studies in rabbits showed that the formulations described above (see Table 31) when the “OEF + arginine” and “2X accelerator + gelatin, arginine” formulations were delivered intranasally. Pharmacodynamic (PD) parameters indicating that they caused insulin and glucose responses to the same extent or better). Table 38 includes average insulin concentration PD parameters (T _max , C _max and AUC), and Table 39 shows average glucose concentration PD parameters (T _min and C _min ). Surprisingly, the “2X accelerator + gelatin, arginine” formulation elicited the greatest PD response despite the least increase in exendin-4 bioavailability in the PK test.

Stability Accelerated stability studies show that a variant of the “OEF” formulation improves the stability of exendin-4 over the “2X accelerator + gelatin” formulation. The stability of the “OEF” formulation was comparable to that of commercially available BYETTA®. After 4 weeks at 40 ° C., the purity of the “OEF” formulation was 85.8-86.0%, while the purity of “2X accelerator + gelatin” was 83.7%. After 4 weeks at 50 ° C., the purity of the “OEF” formulation is comparable to BYETTA® (72.2-72.9% vs. 72.9%), while “2X accelerator + The “gelatin” formulation caused precipitation. No precipitation was observed in the simple formulation.

In summary, accelerated stability studies showed that acetate buffer improved the physical stability of exendin-4 formulations compared to tartrate buffer. Less precipitation was observed with the acetic acid buffered formulation than with the tartaric acid buffered formulation. In general, monovalent ionogenic buffers (acetic acid, arginine, lactic acid) provided better stability than poly-ionogenic buffers (tartaric acid, citric acid). In the range of pH 4.7-5.5, the chemical stability of exendin-4 was improved as compared to the case of pH ≦ 4.25. Arginine also slightly improved chemical stability. The chemical stability of exendin-4 was reduced by the addition of methionine and aspartic acid. The optimal osmolarity for exendin-4 chemical stability was 200-250 mOsm / kg H ₂ O.

  In addition to the above, accelerated stability studies indicate that a variant of the “OEF” formulation improves the stability of exendin-4 compared to the existing “2X accelerator + gelatin” formulation. The stability of the “OEF” formulation is comparable to that of commercially available BYETTA®. After 4 weeks at 40 ° C., the purity of the “OEF” formulation was 85.8-86.0%, while the purity of “2X accelerator + gelatin” was 83.7%. More dramatically, after 4 weeks at 50 ° C., the purity of the “OEF” formulation was comparable to BYETTA® (72.2-72.9% vs. 72.9%), On the other hand, the “2X accelerator + gelatin” formulation caused precipitation.

  Although not tested in this study, it is expected that a thickener could be added to the formulation. The formulation may include a preservative, which may include, but is not limited to, benzalkonium chloride, methyl and propylparaben, and / or chlorobutanol. Absent. Examples of other preservatives tested earlier that may be included in the formulation to promote antibacterial action include the following combination: 0.033% methyl paraben + 0.017% propyl paraben 0.18% methyl paraben + 0.02% propyl paraben; 0.10% to 0.50% chlorobutanol; 0.10% to 0.25% chlorobutanol + 0.033% methyl paraben + 0 0.07% propylparaben; 0.10% to 0.25% chlorobutanol + 0.18% methylparaben + 0.02% propylparaben; 0.5% benzyl alcohol; 0.5% benzyl alcohol + 0.033% methyl paraben + 0.017% propyl paraben; 0.5% benzyl alcohol + 0.18% methyl paraben + 0.02% propyl paraben 0.5% phenylethanol + 0.1 to 0.25% chlorobutanol; 0.5% phenylethanol + 0.033% methylparaben + 0.017% propylparaben; 0.5% Phenylethanol + 0.18% methylparaben + 0.02% propylparaben; 5 mg / ml EDTA; 0.01-0.1% benzalkonium chloride; 7 mg / mL ethanol; 5 mg / mL Benzyl alcohol + 2.5 mg / mL phenylethyl alcohol.

  For example, varying the concentration of exenatide, such as a concentration of 0.5 mg / ml to 6 mg / ml, can be used to achieve the desired dosage. The “OEF” and “OEF + arginine” formulations may also improve the stability and bioavailability of other GRPs, including but not limited to other GLP-1 analogs.

  An exendin-4 formulation for transmucosal administration with improved shelf life, low commodity cost, and high bioavailability has been identified by an improved OEF over exendin-4 formulation. An improved shelf life means that the product will last longer, reduce expired products and reduce the required production. High bioavailability means the potential high efficacy and therapeutic utility of a pharmaceutical product. It will also mean that costs are reduced by reducing the amount of API (Exendin-4, GLP-1) required for the certain effectiveness of the drug. Because DDPC is a novel excipient, removal of DDPC may make it easier to obtain approval for products by the FDA. Further, by removing the DDPC, the time required for manufacturing the product is shortened. Furthermore, since DDPC is an expensive excipient, the cost of goods for pharmaceuticals is significantly reduced by removing it. By removing gelatin, the time required to manufacture the product is reduced, and by removing certain excipients, the cost of manufacturing is reduced.

  The present invention also includes a GRP formulation without preservatives. Such formulations do not contain preservatives. In the absence of antimicrobial excipients, the formulation is filled into preservative-free delivery devices under aseptic conditions. Preservative-free exenatide preparations may contain preparations as shown in Table 40 and Table 41. Exenatide concentration may vary in the range of 2-12 mg / ml.

  In other embodiments, the concentration of permeation enhancing components may vary: Me-β-CD (20-80 mg / ml), DDPC (0-2 mg / ml), EDTA (2-10 mg / ml), tartrate buffer (0-30 mM), gelatin, and sodium chloride. Another embodiment may include different formulations: Me-β-CD (80 mg / ml), EDTA (5 mg / ml), arginine (2.8 or 10 mM) and / or acetate buffer. (10 mM), as well as sodium chloride (pH 4.9-5.6). The concentration of the excipient may vary. The preparation may further contain a thickener such as gelatin, hydroxymethylcellulose, carboxymethylcellulose or carbopol. By varying the concentration of exenatide, it is possible to achieve the desired dosage. For example, the concentration may be in the range of 0.5 mg / ml to 25 mg / ml.

Example 12
Single dose pharmacodynamic (PD) test by intranasal administration of glucose regulatory protein (GRP) in an oral glucose tolerance test (OGTT) rat model GLP-1 in an oral glucose tolerance test (OGTT) rat model The pharmacodynamic effects on blood glucose after intranasal injection of (7-36 amide) and exendin-4 were tested. Two exendin-4 formulations were evaluated and a single formulation of GLP-1 was used. The GLP-1 (EDTA-based) formulation (# 2) contained GLP-1 (7-36 amide) and the following ingredients: 10 mg / mL EDTA disodium; 10 mM citrate buffer (pH 3 .5). Exendin-4 (EDTA-based) formulations (# 2, # 3, # 4 and # 5) contained exendin-4 and the following ingredients: 10 mg / mL EDTA disodium; 10 mM arginine Buffer (pH 4.0). Exendin-4 (PDF-based) formulations (# 6 and # 7) contained exendin-4 and the following ingredients: 45 mg / mL Me-β-CD, 1 mg / mL DDPC, 1 mg / mL disodium EDTA, 100 mM sorbitol, 25 mM lactose, 5 mg / mL CB, and 10 mM arginine buffer (pH 4.0). A summary of the formulations tested is shown in Table 42. Table 43 shows the experimental design and group names.

  Pharmacodynamics were assessed by monitoring blood glucose and blood insulin concentrations; gastric emptying was monitored by co-administration of acetaminophen with glucose. This study included a single dose treatment of ZDF rats (5 rats in each treatment group) about 11 weeks of age. For OGTT, 1 g / kg glucose solution was administered by oral gavage. Acetaminophen was administered at a dose of 100 mg / kg. For the purpose of time measurement, OGTT was set to 0 minutes. Administration of saline, GLP-1 or exendin-4 was performed at -10 minutes relative to OGTT. Blood glucose was assessed at the following time points: before OGTT (twice) and 5, 15, 30, 45, 60, 75, 90, 120, 180 and 240 minutes after OGTT. Blood insulin was assessed at the following time points: before OGTT and 5, 15, 30, 45, 60, 75, 90, 120 and 180 minutes after OGTT. APAP blood levels were assessed at the following time points: before OGTT and 15, 30, 45, 60, 75, 90, 120 and 180 minutes after OGTT.

Results of Pharmacodynamic Test Table 44 shows the results of the pharmacodynamic test for glucose concentration. The peak glucose concentration (C _max ) was 70% of the control in animals receiving GLP-1; similarly, the AUC of glucose in the time frame from 0 to 60 minutes after OGTT (AUC _0-60 ) Was 70% of the control. Exendin-4 administration had only minimal effect on C _max (range 86% to 99% of control) and AUC _0-60 (range 91% to 103% of control). There was no clear dose response for exendin-4.

Table 45 shows the results of a pharmacodynamic test of glucose concentration after correction for endogenous glucose. The peak glucose concentration (C _max ) was 51% of the control in animals receiving GLP-1; similarly, the AUC of glucose in the _0-60 minute time frame after OGTT (AUC _0-60 ) Was 46% of the control, and AUC _0-240 (0 to 240 minutes after OGTT) was 88% of the control. Exendin-4 administration in EDTA or PDF formulations was moderate relative to C _max (range 73% to 87% of control) and AUC _0-60 (range 91% to 103% of control). It showed a degree of action. The effect on AUC _0-240 was minimal to none, 77% to 125% of the control.

  Insulin responsiveness (corrected for endogenous insulin) results showed that the post-OGTT insulin concentrations in control animals were comparable or slightly lower compared to pre-dose values. It was. Nasal administration of GLP-1 at 100 μg / kg was accompanied by a maximum 2-fold increase in insulin 5 to 45 minutes after OGTT. The insulin concentration after administration of exendin-4 at 2, 10 and 20 μg / kg (EDTA-based preparation) showed a dose-dependent increase in insulin, and the peak concentration was the value before the above administration for each group. Were about 0.7 times, 1.5 times and 4 times, respectively. The reaction was limited to about 45 minutes after OGTT. After nasal administration of exendin-4 at a dose of 2 or 10 μg / kg in the PDF formulation, the peak insulin concentration was 1.5 times or 2.7 times that before the administration, respectively. The reaction was limited to about 45 minutes after OGTT.

Gastric emptying results Table 46 shows the results for acetaminophen (gastric emptying). In control, the peak amphetamine concentration of 73 ng / mL _{(C max)} occurred at 30 minutes post-dose _{(T max).} Slightly lower C _max (66 ng / mL) was observed in animals administered GLP-1; however, T _max was longer, suggesting delayed gastric emptying at this dose level. Exendin-4 administration at 2 or 10 μg / kg in EDTA formulations or at 2 μg / kg in PDF formulations gives T _max (30 to 45 min) and C _max (about 70 to 73 ng). / mL) was similar to the control. Exendin-4 administration at 20 μg / kg in the EDTA-based formulation or 10 μg / kg in the PDF-based formulation showed a decrease in C _max (about 51-54 ng / mL), and For at least 20 μg / kg (EDTA-based formulation), a slightly prolonged exposure profile was shown. The results suggested that gastric emptying was affected by these dose levels.

Summary Based on the pharmacological effects of GLP-1 and exendin-4 on stimulation of glucose-dependent insulin release, the described EDTA and PDF intranasal formulations effectively deliver active drugs to systemic targets did. The stimulation of insulin release was dose dependent.

Animals administered GLP-1 or exendin-4 showed changes in peak blood glucose concentration (C _max ) and exposure (AUC) compared to controls. Stimulation of insulin release and regulation of gastric emptying by GLP-1 and exendin-4 is probably the pharmacological basis for changes in blood glucose profile after oral glucose infusion.

Inhibition of gastric emptying is a known pharmacological action of GLP-1 and exendin-4. The change in time to peak concentration (T _max ) or peak concentration (C _max ) for acetaminophen was consistent with GLP-1 and exendin-4 induced pharmacological properties. These data support the effectiveness of using the GLP-1 and exendin-4 formulations described in the above assay in the treatment of metabolic disorders such as obesity and diabetes.

Although the foregoing invention has been described in detail by way of example for purposes of clarity of understanding, those skilled in the art will recognize certain changes and modifications from the present disclosure and are intended to be illustrative and not restrictive. It will be apparent that these changes and modifications can be practiced without undue experimentation within the scope of the appended claims.

Claims (42)

  A glucose regulating peptide (GRP) epithelium comprising a glucose regulating peptide (GRP) and a penetration enhancer, wherein the enhancer comprises a solubilizer, a surfactant, a monovalent ionogenic buffer and a thickener. Pharmaceutical formulation for delivery.   The formulation of claim 1, wherein the enhancer increases GRP permeability by at least 10%.   The formulation of claim 1, wherein the enhancer increases GRP permeability by at least 15-fold.   2. The formulation of claim 1, wherein the enhancer increases GRP bioavailability by at least about 25-fold.   The formulation of claim 1, wherein the enhancer increases GRP bioavailability by at least about 50-fold.   The formulation of claim 1, wherein the enhancer has a bioavailability of GRP of at least about 1% for delivery by subcutaneous injection.   The formulation of claim 1, wherein the enhancer has a GRP bioavailability of at least about 5% for delivery by subcutaneous injection.   The formulation of claim 1, wherein the enhancer has a GRP bioavailability of at least about 10% for delivery by subcutaneous injection. The formulation of claim 1, wherein the accelerator has a time T _max for reaching a maximum concentration in the circulation in the animal of less than about 45 minutes. The formulation of claim 1, wherein the time period T _max for the promoter to reach its maximum concentration in the circulation in the animal is greater than about 60 minutes. The formulation of claim 1, wherein the accelerator has a time T _max to reach a maximum concentration in the circulation in the animal of less than about 30 minutes.   The formulation of claim 1 further comprising a chelating agent.   13. The formulation of claim 12, wherein the chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid and ethylene glycol tetraacetic acid.   The formulation of claim 1, wherein the solubilizer is selected from the group consisting of cyclodextrin, hydroxypropyl-β-cyclodextrin, sulfobutyl ether-β-cyclodextrin and methyl-β-cyclodextrin.   15. The formulation of claim 14, wherein the solubilizer is methyl-β-cyclodextrin.   The surfactant is a nonionic polyoxyethylene ether, fusidic acid and its derivatives, sodium taurodihydrofusidate, L-α-phosphatidylcholine didecanoyl, polysorbate 80, polysorbate 20, polyethylene glycol, cetyl alcohol, polyvinylpyrrolidone, The formulation of claim 1 selected from the group consisting of polyvinyl alcohol, lanolin alcohol and sorbitan monooleate.   The formulation of claim 16, wherein the surfactant is L-α-phosphatidylcholine didecanoyl.   The formulation according to claim 1, comprising GRP, methyl-β-cyclodextrin and L-α-phosphatidylcholine didecanoyl.   The formulation of claim 1, wherein the GRP is present at a concentration greater than about 50 mg / mL.   The formulation of claim 1, wherein the GRP is present at a concentration of about 0.1 to about 50 mg / mL.   The formulation of claim 1, wherein the GRP is present at a concentration of about 0.25 to about 10 mg / mL.   The formulation of claim 1, wherein the GRP consists of GLP-1.   The formulation of claim 1, wherein the GRP consists of exendin-4.   24. The formulation of claim 22 or 23, wherein the GRP derivative comprises GRP covalently bound to a hydrophobic moiety.   25. The formulation of claim 24, wherein the hydrophobic moiety comprises an alkyl chain.   25. A formulation according to claim 24, wherein the hydrophobic moiety comprises a fatty acid chain.   The preparation according to claim 1, wherein the preparation is a non-sterile preparation.   Further comprising a preservative selected from the group consisting of chlorobutanol, methylparaben, propylparaben, butylparaben, benzalkonium chloride, benzethonium chloride, sodium benzoate, sorbic acid, phenol, and ortho-, meta- or para-cresol 28. A formulation according to claim 27.   The preparation according to claim 1, wherein the preparation is a sterile preparation.   24. The formulation of claim 22, further comprising a dipeptidyl aminopeptidase (DPP) IV inhibitor.   31. The formulation of claim 30, wherein the DPP IV inhibitor is lys (4-nitro-Z) -pyrrolizide.   32. The formulation of claim 31, wherein the lys (4-nitro-Z) -pyrrolizide is at a concentration of at least about 5 mM.   32. The formulation of claim 31, wherein the lys (4-nitro-Z) -pyrrolizide is at a concentration of at least about 30 mM.   32. The formulation of claim 31, wherein the lys (4-nitro-Z) -pyrrolizide is at a concentration of at least about 50 mM.   The formulation of claim 1, wherein the formulation is suitable for intranasal delivery of GLP-1.   The formulation of claim 1, wherein the formulation has a pH of about 3 to about 6.   The formulation of claim 1, wherein the formulation has a pH of 3.5 ± 0.50.   The formulation of claim 1, comprising GLP-1, 45 mg / ml methyl-β-cyclodextrin, 1 mg / ml L-α-phosphatidylcholine didecanoyl, and 1 mg / ml EDTA.   A drug for the treatment of metabolic syndrome in a mammal, wherein said drug comprises a formulation according to any one of claims 1 to 38, and such drug is pharmaceutically effective Use of GRP in the manufacture of a medicament comprising an amount of GRP.   40. Use according to claim 39, wherein epithelial administration of the drug to the mammal increases plasma insulin concentration.   40. Use according to claim 39, wherein epithelial administration of the drug to the mammal reduces blood glucose concentration.   40. Use according to claim 39, wherein epithelial administration of the drug to the mammal delays gastric emptying.

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