JP5415938B2 - Glucagon-like peptide 1 (GLP-1) pharmaceutical preparation - Google Patents

Description

Detailed Description of the Invention

This application is a continuation-in-part of US patent application Ser. No. 10 / 632,878 filed on Jul. 22, 2003 and is subject to section 119 (e) of the US Patent Act. No. 60 / 744,882, filed Apr. 14, 2006, for the benefit of US Provisional Patent Application No. 60 / 744,882. Each priority application mentioned above is incorporated herein by reference in its entirety.

TECHNICAL FIELD The present invention relates to the field of pharmaceutical formulations. The present invention discloses a dry powder formulation comprising diketopiperazine (DKP) particles in combination with glucagon-like peptide 1 (GLP-1). The present invention is useful as a pharmaceutical preparation for treating diseases such as diabetes, cancer and obesity, but is not limited to such diseases. More particularly, the present invention is useful as a pharmaceutical formulation for intrapulmonary delivery.

BACKGROUND ART As disclosed in the literature, glucagon-like peptide 1 (GLP-1) is an incretin consisting of 30 or 31 amino acids, in response to dietary fat, carbohydrate intake and protein. It is released from enteroendocrine L cells. It has been found that secretion of this peptide hormone is reduced in individuals with type 2 diabetes, and in type 2 diabetes this peptide hormone can be a candidate for the treatment of the disease and other related diseases.

  In the disease-free state, GLP-1 is secreted from the intestinal L cells in response to orally ingested nutrients (especially sugars), thereby causing a diet-induced release of insulin from the pancreas. Stimulated, glucagon release from the liver and other effects on the gastrointestinal tract and brain are suppressed. The action of GLP-1 in the pancreas is glucose dependent, thereby minimizing the risk of hypoglycemia during exogenous peptide administration. GLP-1 also promotes all steps in insulin biosynthesis and directly stimulates beta cell growth, survival and differentiation. By combining these actions, the β cell population grows. Furthermore, GLP-1 receptor signaling reduces β-cell apoptosis, which further contributes to the growth of the β-cell population.

  In the gastrointestinal tract, GLP-1 suppresses gastrointestinal motility, increases insulin secretion in response to glucose, and decreases glucagon secretion, thereby contributing to a decrease in glucose excursion. is there. Central administration of GLP-1 has been shown to inhibit rodent food intake, suggesting that GLP-1 released in the periphery can directly affect the brain. This is plausible as circulating GLP-1 has been shown to be able to access GLP-1 receptors in specific brain regions (ie, the subfornical organ and the last cortex). These areas of the brain are known to be involved in appetite regulation and energy homeostasis. Interestingly, gastric distension activates neurons containing GLP-1 in the caudate nucleus of the solitary tract, which predicts a role for GLP-1 expressed centrally as an appetite suppressant . These hypotheses are supported by studies using GLP-1 receptor antagonists (exendin (9-39) with adverse effects). In humans, administered GLP-1 has a satiety effect (Verdich et al., 2001), and diabetic patients show decreased appetite when administered by continuous subcutaneous infusion over a 6 week dosing regimen, thereby reducing body weight Decreased significantly (Zander et al., 2002).

  GLP-1 has also been shown to be effective in patients with type 2 diabetes, increasing insulin secretion and normalizing both fasting and postprandial blood glucose when administered as a continuous intravenous infusion (Nauck et al., 1993). Furthermore, infusion of GLP-1 reduced glucose levels in patients who had already been administered non-insulin oral drugs or in patients who needed insulin treatment after ineffective treatment with sulfonylurea (Nauck et al., 1993). However, the results provided by the single subcutaneous injection effect of GLP-1 were disappointing, as shown in the art and discussed herein below. Despite the achievement of high plasma immunoreactive GLP-1 levels, insulin secretion quickly returned to pre-dose values and blood glucose levels were not normalized (Nauck et al., 1996). The effect on fasting blood glucose was equivalent to intravenous administration only when repeated subcutaneous administration was performed (Nauck et al., 1996). It was revealed that fasting glucose concentration and postprandial glucose concentration decreased and HbA1c concentration decreased after 6 weeks of continuous subcutaneous administration (Zander et al., 2002). The short effectiveness of a single subcutaneous injection of GLP-1 was related to its circulatory instability. GLP-1 was metabolized by plasma in vitro and it was found that the enzyme dipeptidyl peptidase IV (DPP-IV) was responsible for this degradation (Mentlein et al., 1993).

  Along with the physiological importance of GLP-1 in diabetes and the fact that exogenous GLP-1 has been shown to rapidly degrade at the amino terminus in both healthy and type 2 diabetic subjects In their studies, a new approach to anti-diabetic drugs for the treatment of diabetes has been focused on the possibility of manipulating the stability of GLP-1 in vivo (Deacon et al., 2004). As two separate approaches, 1) the development of analogs of GLP-1 that are not sensitive to enzymatic degradation, and 2) the concentration of biologically active peptides that prevent degradation and are intact in vivo. The use of selective enzyme inhibitors to enhance has been promoted. Long-acting GLP-1 analogues (eg Liraglutide (Novo Nordisk, Copenhagen, Denmark); exenatide (exendin-4; Byetta®), which is resistant to degradation and is called an “incretin mimetic” ( Amylin Inc., San Diego, California, USA) and (Exenatide-LAR, Eli Lilly, Indianapolis, IN) have been studied in clinical trials, dipeptidyl peptidase IV that inhibits the enzyme responsible for the degradation of incretin. Inhibitors such as Vildagliptin (Galvus) developed by Novartis (Basel, Switzerland) and Januvia (Sitagliptin) developed by Merck (White House Station, New Jersey, USA) are also under investigation (Deacon et al., 2004). Thus, multiple mechanisms of action of GLP-1 (eg, Surin increased release, or delayed gastric emptying, satiety increasing), together with its low propensity for hypoglycemia appear to what gives better features than currently available treatments.

  However, despite these efforts / advancements in treatment with GLP-1, currently available drugs for diabetes that can reach therapeutic targets in all patients (HbA1c, fasting blood glucose, glucose excursion) It does not exist, and there are no drugs that have no side effects such as toxicity, stress due to hypoglycemia, weight gain, nausea and vomiting. Therefore, there is a need in the art for stable GLP-1 formulations that have long-term efficacy and optimal absorption when administered as pharmaceuticals.

Summary of the Invention There is a deficiency in the art for stable, inhalable glucagon-like peptide 1 (GLP-1) formulations for use as pharmaceuticals. To solve the shortage in the art, the present invention provides a formulation of GLP-1 in combination with diketopiperazine (DKP) particles as a pharmaceutical.

  Therefore, in a particular aspect of the invention, a dry powder composition is provided comprising a GLP-1 molecule and diketopiperazine or a pharmaceutically acceptable salt thereof. In a further aspect, the dry powder composition of the invention comprises a natural GLP-1, GLP-1 metabolite, GLP-1 analog, GLP-1 derivative, dipeptidyl peptidase IV (DPP-IV) protected GLP-1 A GLP-1 molecule selected from the group consisting of a GLP-1 mimetic, exendin, a GLP-1 peptide analog or a biosynthetic GLP-1 analog. In a further aspect of the invention, the dry powder composition comprises a diketopiperazine having the chemical formula: 2,5-diketo-3,6-di (4-X-aminobutyl) piperazine, wherein X is Selected from the group consisting of succinyl, glutaryl, maleyl and fumaryl. In another aspect, the dry powder composition comprises a diketopiperazine salt. In yet another aspect of the present invention, a dry powder composition is provided wherein the diketopiperazine is 2,5-diketo-3,6-di (4-fumaryl-aminobutyl) piperazine.

  The present invention relates to a method wherein a GLP-1 molecule is a natural GLP-1 or an amidated GLP-1 molecule, wherein the amidated GLP-1 molecule is GLP-1 (7-36) amide. Further contemplated are powder compositions.

  In yet another particular aspect of the invention, providing a GLP-1 solution comprising GLP-1 molecules; providing a solution of particle-forming diketopiperazine or a suspension of particles of diketopiperazine; And a step of mixing the GLP-1 solution with the diketopiperazine solution or suspension, a method of preparing a particle comprising a GLP-1 molecule and a diketopiperazine is provided. In another particular aspect of the invention, a method of preparing particles comprising GLP-1 molecules and diketopiperazine, further comprising removing the solvent from the solution or suspension by lyophilization, filtration or spray drying. Is provided. In a further embodiment, the particles of the invention are formed by removing the solvent or formed prior to removing the solvent.

  In one aspect of the invention, a method for preparing particles having a GLP-1 molecule and a diketopiperazine in a natural GLP-1, GLP-1 analog, GLP-1 derivative, dipeptidyl peptidase IV (DPP-IV). ) GLP-1 molecules selected from the group consisting of protected GLP-1, GLP-1 mimics, exendins, GLP-1 peptide analogs or biosynthetic GLP-1 analogs are provided. In another aspect, a method of preparing particles having a GLP-1 molecule and a diketopiperazine comprises diketopiperazine provided as a suspension of particles. In a further aspect, the diketopiperazine is provided in the form of a solution, and the method includes adjusting the pH of the solution to precipitate the diketopiperazine to form particles.

  In another specific embodiment of the invention, the GLP-1 solution has a concentration of about 1 μg / mL to 50 mg / mL, more preferably about 0.1 mg / mL to 10 mg / mL. In yet another specific aspect of the invention, the GLP-1 solution has a concentration of about 0.25 mg / mL.

In another method of preparing particles comprising a GLP-1 molecule and a diketopiperazine, the method further comprises adding an agent to the solution, wherein the agent comprises a salt, a surfactant, an ion , Osmolite, chaotrope and lyotrope, acids, bases and organic solvents. The agent promotes the association between GLP-1 and diketopiperazine particles and also improves the stability and / or efficacy of the GLP-1 molecule. In some embodiments of the invention, the agent is a salt such as but not limited to sodium chloride. The agent may be a surfactant such as, but not limited to, Tween, Triton, pluronic acid, CHAPS, cetrimide, Brij, H (CH ₂ ) ₇ SO ₄ Na. it is conceivable that. The agent can be an ion (eg, cation or anion). The agent is not limited, but osmolite (stable) such as hexylene glycol (Hex-Gly), trehalose, glycine, polyethylene glycol (PEG), trimethylamine N-oxide (TMAO), mannitol, proline and the like. Agent). The agent may be, but is not limited to, cesium chloride, chaotrope such as sodium citrate, sodium sulfate, or liotrope. The agent may be an organic solvent such as an alcohol selected from methanol (MeOH), ethanol (EtOH), trifluoroethanol (TFE) and hexafluoroisopropanol (HFIP).

  In another particular aspect of the present invention, a method of preparing particles comprising GLP-1 molecules and diketopiperazine is contemplated, wherein the method adjusts the pH of the particle suspension to about 4 or higher. Including that. In a further aspect of the invention, the method of preparing the particle comprises a GLP-1 molecule and a diketopiperazine, wherein the GLP-1 molecule within the particle has greater stability.

  Further contemplated by the present invention is a method of administering an effective amount of a GLP-1 molecule to a subject in need thereof, comprising providing the subject with GLP-1 / diketopiperazine particles. . The method of administration may be, but is not limited to, intravenous, subcutaneous, oral, nasal, buccal, rectal, or pulmonary delivery. In one aspect, the method of administration is by pulmonary delivery. In yet another aspect of the present invention, the administration method comprises diabetes, ischemia, reperfusion tissue damage, dyslipidemia, diabetic cardiomyopathy, myocardial infarction, acute coronary syndrome, obesity, postoperative catabolic Treatment of symptoms or diseases selected from the group consisting of alterations, hyperglycemia, irritable bowel syndrome, stroke attacks, neurodegenerative disorders, memory disorders and learning disorders, islet cell transplantation and regeneration therapy.

  In another aspect of the present invention, a method of administering a GLP-1 / diketopiperazine particle composition results in improved pharmacokinetic half-life and bioavailability of GLP-1.

  In a further particular aspect of the invention, providing a solution of GLP-1 molecules; providing a particle-forming diketopiperazine; forming a particle; and mixing GLP-1 and diketopiperazine A method of preparing a dry powder composition having an improved pharmacokinetic profile, comprising: removing a solvent by drying to obtain a dry powder, wherein the dry powder is an improved drug Has a kinetic profile. The improved pharmacokinetic profile includes a higher half-life of GLP-1 and / or improved bioavailability of GLP-1. The higher half-life of GLP-1 is 7.5 minutes or more.

  In one aspect of the invention, a dry powder composition is provided comprising a GLP-1 molecule and diketopiperazine or a pharmaceutically acceptable salt thereof. In another aspect, the GLP-1 molecule is a natural GLP-1, GLP-1 metabolite, GLP-1 analog, GLP-1 derivative, dipeptidyl peptidase IV (DPP-IV) protected GLP-1, It is selected from the group consisting of GLP-1 mimetics, GLP-1 peptide analogs or biosynthetic GLP-1 analogs.

  In one aspect of the invention, the diketopiperazine is a diketopiperazine having the formula: 2,5-diketo-3,6-di (4-X-aminobutyl) piperazine, wherein X is Selected from the group consisting of succinyl, glutaryl, maleyl and fumaryl. In another embodiment, the diketopiperazine is a diketopiperazine salt. In another aspect, the diketopiperazine is 2,5-diketo-3,6-di (4-fumarylaminobutyl) piperazine.

  In one aspect of the invention, the GLP-1 molecule is native GLP-1. In another aspect, the GLP-1 molecule is an amidated GLP-1 molecule. In another aspect, the amidated GLP-1 molecule is GLP-1 (7-36) amide.

  In one aspect of the invention, providing a GLP-1 molecule; providing a form of diketopiperazine selected from particle-forming diketopiperazine, diketopiperazine particles and combinations thereof; and a common solution of Mixing the GLP-1 molecule and the diketopiperazine in a form, wherein a particle comprising the GLP-1 molecule and the diketopiperazine is formed, comprising a particle comprising the GLP-1 molecule and the diketopiperazine A method of forming is provided.

  In one aspect of the invention, the method further comprises removing the solvent from the common solution by lyophilization, filtration or spray drying. In another aspect, the particles comprising GLP-1 molecules and diketopiperazine are formed by removing the solvent. In another aspect, the particles comprising the GLP-1 molecule and the diketopiperazine are formed prior to removing the solvent.

  In another aspect, the GLP-1 molecule is a natural GLP-1, GLP-1 analog, GLP-1 derivative, dipeptidyl peptidase IV (DPP-IV) protected GLP-1, GLP-1 mimic , A GLP-1 peptide analog or a biosynthetic GLP-1 analog. In another aspect, the GLP-1 molecule is provided in the form of a solution comprising GLP-1 at a concentration of about 1 μg / mL to 50 mg / mL. In another aspect, the GLP-1 molecule is provided in the form of a solution comprising GLP-1 at a concentration of about 0.1 mg / mL to 10 mg / mL. In another aspect, the GLP-1 molecule is provided in the form of a solution comprising GLP-1 at a concentration of about 0.25 mg / mL.

  In another aspect of the invention, the diketopiperazine is provided in the form of a suspension of diketopiperazine particles. In another aspect, the diketopiperazine is provided in the form of a solution comprising particle-forming diketopiperazine, the method further comprising adjusting the pH of the solution to form diketopiperazine particles. Including. In another aspect, the method further comprises adding an agent to the solution or suspension, wherein the agent is a salt, surfactant, ion, osmolyte, chaotrope and lyotrope, Selected from the group consisting of acids, bases and organic solvents. In another aspect, the agent promotes association between the GLP-1 molecule and the diketopiperazine particles or the particle-forming diketopiperazines. In another aspect, the agent improves the stability or efficacy of the GLP-1 molecule. In another aspect, the agent is sodium chloride.

  In another aspect of the invention, the method further comprises adjusting the pH of the suspension or solution. In another aspect, the pH is adjusted to about 4.0 or higher. In yet another aspect, the GLP-1 molecule within the particle has greater stability than native GLP-1.

  In another aspect, the common solution comprises GLP-1 at a concentration of about 1 μg / mL to 50 mg / mL. In another aspect, the common solution comprises GLP-1 at a concentration of about 0.1 mg / mL to 10 mg / mL. In another aspect, the common solution comprises GLP-1 at a concentration of about 0.25 mg / mL.

  In yet another aspect of the invention, the method further comprises adding an agent to the common solution, wherein the agent is a salt, surfactant, ion, osmolyte, chaotrope and It is selected from the group consisting of lyotrope, acid, base and organic solvent. In another aspect, the agent promotes association between the GLP-1 molecule and the diketopiperazine particles or the particle-forming diketopiperazines. In another aspect, the agent improves the stability or efficacy of the GLP-1 molecule. In another aspect, the agent is sodium chloride.

  In another aspect, the method further comprises adjusting the pH of the common solution. In another aspect, the pH is adjusted to about 4.0 or higher.

  In another aspect of the invention, a method of administering an effective amount of a GLP-1 molecule to a subject in need thereof, comprising providing the subject with particles comprising GLP-1 and a diketopiperazine. A method is provided that includes: In another aspect, the providing is performed by intravenous, subcutaneous, oral, nasal, buccal, rectal or pulmonary delivery. In another aspect, the providing is by pulmonary delivery.

  In another aspect, the subject in need is diabetes, ischemia, reperfused tissue damage, dyslipidemia, diabetic cardiomyopathy, myocardial infarction, acute coronary syndrome, obesity, postoperative catabolic changes, high Treatment of symptoms or diseases selected from the group consisting of blood glucose, irritable bowel syndrome, stroke attacks, neurodegenerative disorders, memory disorders and learning disorders, islet cell transplantation and regeneration therapy.

  In another aspect, the provision of the particles results in improved pharmacokinetic half-life and bioavailability of GLP-1 over native GLP-1.

  In one aspect of the invention, providing a GLP-1 molecule; providing a particle-forming diketopiperazine in the form of a solution; forming a diketopiperazine particle; forming the GLP to form a common solution Mixing the molecule with the solution; and removing the solvent from the common solution by spray drying to form a powder with an improved GLP-1 pharmacokinetic profile. A method is provided for forming a powder composition having an improved kinetic profile.

  In another aspect, the improved GLP-1 pharmacokinetic profile comprises a higher GLP-1 half-life. In another aspect, the higher GLP-1 half-life is 7.5 minutes or greater. In another aspect, the improved GLP-1 pharmacokinetic profile comprises improved bioavailability of GLP-1 over native GLP-1.

BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood with reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

1A-1D. Structural analysis of GLP-1 at various concentrations (pH 4, 20 ° C.). FIG. 1A: Far-UV circular dichroism (CD) of GLP-1 indicates that as the concentration increases, the secondary structure of the peptide changes from a largely unstructured conformation to a helical structure. Indicates. FIG. 1B: Near-UV CD shows that tertiary structure increases with increasing peptide concentration, suggesting that GLP-1 self-associates. FIG. 1C: Fluorescence emission of GLP-1 at various concentrations (pH 4, 20 ° C.) generated by tryptophan excitation at 280 nm. FIG. 1D: Transmission type FTIR of GLP-1 at various concentrations (pH 4, 20 ° C.). The amide I band at 1656 cm ⁻¹ indicates that GLP-1 has an α-helix structure at concentrations of 2 mg / mL or higher.

  2A-2D. Structural analysis of low concentration GLP-1 at various ionic strengths (pH 4, 20 ° C.). FIG. 2A: Far UV CD of 1.0 mg / mL GLP-1 shows that as the salt concentration increases, the disordered structure of GLP-1 changes to a more regular α-helix structure. FIG. 2B: Near UV CD of 1.0 mg / mL peptide shows that the tertiary structure of GLP-1 also increases with increasing NaCl concentration. FIG. 2C: Internal fluorescence emission of 1.0 mg / mL GLP-1 at various NaCl concentrations (pH 4, 20 ° C.) after tryptophan excitation at 280 nm. At high peptide concentrations, the intensity maxima decrease and shift to shorter wavelengths, indicating a clear tertiary structure. FIG. 2D: Tertiary structure analysis of 10 mg / mL GLP-1 at various ionic strengths (pH 4, 20 ° C.). The near ultraviolet CD spectrum shows that the tertiary structure of self-associated GLP-1 increases with increasing ionic strength.

  3A-3B. Structural analysis of 10 mg / mL GLP-1 at various temperatures (pH 4). FIG. 3A: Near-UV CD shows that GLP-1 oligomers separate with increasing temperature. FIG. 3B: Structural analysis of 10 mg / mL GLP-1 at various temperatures (pH 4). FIG. 3C: Structural analysis of 0.05 mg / mL GLP-1 at various temperatures (pH 4). Deep UV CD indicates that the peptide is insensitive to temperature.

  4A-4B. Structural analysis of GLP-1 at various pH (20 ° C.). FIG. 4A: Far UV CD of 10 mg / mL GLP-1 at various pH (20 ° C.). As pH increases, self-associated GLP-1 precipitates between pH 6.3 and 7.6, but at pH 1.5 and 11.7, the helix structure is maintained. FIG. 4B: The increase in the spectrum at pH 7.6 reveals that the secondary structure of GLP-1 becomes irregular as a result of the decrease in concentration.

FIG. Resistance of 1 mg / mL GLP-1 to amidolysis and oxidation as shown by HPLC. The conditions for deamidation were achieved by incubating GLP-1 at pH 10.5 for 5 days at 40 ° C. Oxidation conditions were achieved by incubating GLP-1 in 0.1% H ₂ O ₂ for 2 hours at room temperature.

  6A-6B. Effect of stirring on the tertiary structure of 1.5 and 9.4 mg / mL GLP-1 (pH 4). Near-UV CD (FIG. 6A) and GLP-1 fluorescence emission (FIG. 6B) both indicate that the tertiary structure of the GLP-1 peptide does not change significantly due to agitation. Samples were stirred for 30 and 90 minutes at room temperature and fluorescence emission spectra were collected after tryptophan excitation at 280 nm.

  7A-7C. Effect of 10 freeze-thaw cycles on the tertiary structure of GLP-1 (pH 4) at 1.6, 5.1 and 8.4 mg / mL. Near-UV CD (FIG. 7A) and GLP-1 fluorescence emission (FIG. 7B) both indicate that the tertiary structure of the peptide does not change significantly with multiple freeze-thaw cycles. Samples were frozen at −20 ° C. and thawed at room temperature. Fluorescence emission spectra were collected after excitation of tryptophan at 280 nm. A similar experiment was performed showing the effect of 11 freeze-thaw cycles on the secondary structure of 10 mg / mL GLP-1 (pH 4) by far UV CD (FIG. 7C).

  8A-8B. Salt test. Loading curve for GLP-1 / FDKP as a function of pH and NaCl concentration (FIG. 8A). Loading was performed with 5 mg / mL FDKP and 0.25 mg / mL GLP-1. The NaCl concentration was expressed in mM. FIG. 8B: Represents the amount of GLP-1 detected in a reconstituted FDKP-free control sample as a function of pH and NaCl concentration.

  9A-9B. Surfactant test. Loading curve for GLP-1 / FDKP as a function of pH and surfactant (FIG. 9A). Loading was performed with 5 mg / mL FDKP and 0.25 mg / mL GLP-1. FIG. 9B: Represents the amount of GLP-1 detected in a control sample without reconstituted FDKP as a function of pH and added surfactant.

  10A-10D. Ion test. Loading curve for GLP-1 / FDKP as a function of pH and ion. Loading was performed with 5 mg / mL FDKP and 0.25 mg / mL GLP-1 (FIGS. 10A and 11C). The ion concentration is indicated in the legend (mM). The right curve represents the results for 1M NaCl. FIGS. 10B and 10D: Represents the amount of GLP-1 detected in a reconstituted, FDKP-free control sample as a function of pH, ion and 1M NaCl.

  11-11B. Osmolite test. Loading curve for GLP-1 / FDKP as a function of pH in the presence of common stabilizers (osmolite; FIG. 11A). Loading was performed with 5 mg / mL FDKP and 0.25 mg / mL GLP-1. FIG. 11B: Represents the amount of GLP-1 detected in a reconstituted FDKP-free control sample as a function of pH and osmolyte. “N / A” indicates that no osmolyte was present in the sample.

  12A-12B. Chaotrope / liotrope test. Load curve for GLP-1 / FDKP as a function of chaotrope or lyotrope concentration at pH 3.0 (FIG. 12A) and pH 4.0 (FIG. 12C). Loading was performed with 5 mg / mL FDKP and 0.25 mg / mL GLP-1. 12B and 12D: Represents the amount of GLP-1 detected in a reconstituted FDKP-free control sample as a function of pH in the presence of various chaotropes or liotropes. “N / A” indicates that no chaotrope or liotrope was present in the sample.

  13A-13B. Alcohol test. Loading curve for GLP-1 / FDKP as a function of pH and alcohol. Loading was performed with 5 mg / mL FDKP and 0.25 mg / mL GLP-1. Four alcohol concentrations (5% v / v, 10% v / v, 15% v / v and 20% v / v alcohol each) were evaluated (FIG. 13A). TFE = trifluoroethanol; HFIP = hexafluoroisopropanol. FIG. 13B: Represents the amount of GLP-1 detected for a reconstituted FDKP free control sample as a function of pH and alcohol (20%).

  14A-14B. Load from GLP-1 / FDKP concentration test (FIG. 14A). Loading was done with 5 mg / mL FDKP and the analyzed GLP-1 concentration is shown on the X-axis. FIG. 14B: Scanning electron microscope (SEM) images (10000 × magnification) of multiple GLP-1 / FDKP formulations represent clusters of spherical and rod-like GLP-1 / FDKP particle formulations. (Panel A) 0.5 mg / mL GLP-1 and 2.5 mg / mL FDKP; (Panel B) 0.5 mg / mL GLP-1 and 10 mg / mL FDKP; (Panel C), pH 4.0 0.5 mg / mL GLP-1 and 10 mg / mL FDKP in 20 mM sodium chloride, 20 mM potassium acetate and 20 mM potassium phosphate; and (panel D) 20 mM sodium chloride, 20 mM potassium acetate and 20 mM phosphorus at pH 4.0 10 mg / mL GLP-1 and 50 mg / mL FDKP in potassium acid.

  FIG. Figure 6 represents the effect of stress on multiple GLP-1 / FDKP formulations. The legend shows the mass to mass percentage of GLP-1 relative to FDKP particles and other components present in the solution prior to lyophilization. Samples were incubated at 40 ° C. for 10 days.

  16A-16C. The structure of GLP-1. FIG. 16A represents the glycylated form (SEQ ID NO: 1) and amidated form (SEQ ID NO: 2) of GLP-1. FIG. 16B: Inhibition of DPPIV activity by aprotinin. FIG. 16C: Inhibition of DPPIV activity by DPPIV inhibitors.

  FIG. Detection of GLP-1 after incubation in lung lavage fluid.

  18A-18B. Represents quantification of GLP-1 in plasma. FIG. 18A shows quantification at a 1: 2 dilution of plasma. FIG. 18B shows quantification at a 1:10 dilution of plasma.

  19A-19B. Effect of GLP-1 and GLP-1 analogs on cell survival. Effect of GLP-1 on rat pancreatic epithelial (ARIP) cell death (FIG. 19A). Annexin V staining representing inhibition of apoptosis in the presence of GLP-1 and staurosporine (Stau) as single agent and in combination (FIG. 19B). The concentration of GLP-1 is 15 nM and the concentration of staurosporine is 1 μM.

  FIG. Effect of exendin-4, a GLP-1 analog, on cell viability. ARIP cells received 0, 10, 20 and 40 nM exendin 4 for 16, 24 and 48 hours.

  FIG. Effect of multiple GLP-1 / FDKP formulations on staurosporine-induced cell death. ARIP cells pre-administered with GLP-1 samples were exposed to 5 μM staurosporine for 4 hours and analyzed by Cell Titer-Glo® to determine cell viability. Samples were stressed at 4 ° C. and 40 ° C. for 4 weeks. Control samples shown on the right side (medium, GLP-1, STAU, GLP + STAU) are GLP-1, staurosporine, and GLP-1 and stauros in medium (no GLP-1 or staurosporine) Shows cell viability in porin (Note: Graph legend does not apply to control samples). All of the results shown are the average of 3 replicates.

  22A-22B. Pharmacokinetic study showing a single intravenous injection (IV; FIG. 22A) and pulmonary inhalation (IS; FIG. 22B) in rats using various concentrations of GLP-1 / FDKP formulation. The legend shows the GLP-1 mass to mass percentage for FDKP particles for the formulation analyzed.

  23A-23B. Reduction in cumulative food intake of rats administered GLP-1 / FDKP formulation at 2 hours (FIG. 23A) and 6 hours (FIG. 23B) after administration.

  FIG. Pharmacodynamic study of GLP-1 / FDKP administered by intrapulmonary inhalation in male obese Zucker rats. Data represent glucose measurements at 0, 15, 30, 45, 60 and 90 minutes for control (air; group 1) and GLP-1 / FDKP administration (group 2).

  FIG. Pharmacodynamic study of GLP-1 / FDKP administered by intrapulmonary inhalation in male obese Zucker rats. Data represent GLP-1 measurements at 0, 15, 30, 45, 60 and 90 minutes for control (air; group 1) and GLP-1 / FDKP administration (group 2).

  FIG. Pharmacodynamic study of GLP-1 / FDKP administered by intrapulmonary inhalation in male obese Zucker rats. Data represent insulin measurements at 0, 15, 30, 45, 60 and 90 minutes for control (air; group 1) and GLP-1 / FDKP administration (group 2).

  FIG. Pharmacokinetic study of GLP-1 / FDKP at various GLP-1 concentrations administered by pulmonary inhalation in female rats. Data are 0, 2, 5, 10, for control (air; group 1) and GLP-1 / FDKP administration groups 2, 3 and 4 to which 5%, 10% and 15% GLP-1 were administered, respectively. Represents GLP-1 measurements at 20, 30, 40 and 60 minutes.

  FIG. Pharmacokinetic study of GLP-1 / FDKP at various GLP-1 concentrations administered by pulmonary inhalation in female rats. Data are 0, 2, 5, 10, for control (air; group 1) and GLP-1 / FDKP administration groups 2, 3 and 4 to which 5%, 10% and 15% GLP-1 were administered, respectively. Represents FDKP measurements at 20, 30, 40 and 60 minutes.

  Figure 29 in female rats (n = 10) administered GLP-1 / FDKP containing 15% GLP-1 (0.3 mg GLP-1) by pulmonary inhalation once a day for 4 consecutive days Pharmacodynamic test of GLP-1 / FDKP. The data represents the average food intake measured for 4 consecutive days before administration, 1 hour, 2 hours, 4 hours and 6 hours after administration.

  Figure 30 in female rats (n = 10) administered GLP-1 / FDKP containing 15% GLP-1 (0.3 mg GLP-1) by pulmonary inhalation once daily for 4 consecutive days Pharmacodynamic test of GLP-1 / FDKP. Data represent mean body weight measured for 4 consecutive days before administration, 1 hour, 2 hours, 4 hours and 6 hours after administration.

Figure 31: GLP-1 / FDKP toxicokinetic study in monkeys administered GLP-1 / FDKP by nasal or oral administration once daily for 30 consecutive days (30 minutes per day). The data represents the peak value (C _max ) of GLP-1 concentration in male and female plasma. Animals are control (air; group 1), 2 mg / kg FDKP (group 2) or 0.3, 1.0 or 2.0 mg / kg GLP-1 / FDKP (groups 3, 4 and 5 respectively) Of administration.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS There is a shortage in the art of stable inhalable glucagon-like peptide 1 (GLP-1) formulations for use in pharmaceuticals. This is due to instability of the GLP-1 peptide in vivo. GLP-1 compounds tend to remain in solution under many conditions and have a relatively short in vivo half-life when administered as a solution formulation. Furthermore, dipeptidyl peptidase IV (DPP-IV), which is known to be present in various body fluids (eg lung fluid and blood), greatly reduces the biological half-life of GLP-1 molecules. For example, the biological half-life of GLP-1 (7-37) has been shown to be 3-5 minutes (see US Pat. No. 5,118,666). GLP-1 has also been shown to be rapidly absorbed in vivo after parenteral administration. Similarly, amide GLP-1 (7-36) has a half-life of about 50 minutes when administered subcutaneously (see also US Pat. No. 5,118,666).

  The shortcomings of GLP-1 compositions in the art, such as rapid elimination from the body and short half-life, are solved by the present invention. The present invention solves the shortcomings in the art by providing an optimized natural GLP-1 / FDKP (Fumaryl diketopiperazine) formulation that is particularly suitable for intrapulmonary delivery. In another specific aspect, the present invention provides a formulation of natural GLP-1 molecules that can elicit a GLP-1 response in vivo. The use of natural GLP-1 variants in such formulations is also contemplated.

  To solve the deficiencies in the art, the present invention provides a formulation of GLP-1 in combination with diketopiperazine (DKP) particles. In one particular aspect of the invention, a GLP-1 / DKP formulation is provided for administration to a subject. In a further specific aspect, the GLP-1 / DKP formulation includes, but is not limited to, fumaryl diketopiperazine (FDKP), other DKP (asymmetric DKP, xDKP) (eg, 2,5-diketo- Asymmetric diketopiperazines such as 3,6-di (4-succinyl-aminobutyl) piperazine (SDKP), substituted at only one position on the DKP ring (eg, “one-arm” analogs of FDKP) and DKP In other specific embodiments of the invention, administration of the GLP-1 / FDKP formulation is by pulmonary delivery.

  During the development of therapeutic formulations for GLP-1 molecules, the structural properties of GLP-1 in solution include deep ultraviolet circular dichroism (far ultraviolet CD) and near ultraviolet circular dichroism (near ultraviolet CD). ), Internal fluorescence, Fourier transform infrared spectroscopy (FTIR), high performance liquid chromatography (HPLC), mass spectrometry (MS) and various other biophysical and analytical techniques. The circular dichroism (CD) technique is a powerful tool used to analyze structural changes in proteins under various experimental conditions and is well known in the art. Experimental conditions under which these analyzes were performed included concentration, ionic strength, temperature, pH, oxidative stress, agitation, and the effects of multiple freeze-thaw cycles on the GLP-1 peptide. These analyzes will also reveal the major pathways of degradation to achieve preferred GLP-1 / DKP formulations with desirable pharmacokinetic (PK) and pharmacodynamic (PD) properties, and Designed to establish conditions that manipulate the structure of the GLP-1 peptide.

  It was observed that as the concentration of GLP-1 increased, the secondary structure of the peptide changed from a largely unstructured conformation to a more helical conformation. With increasing ionic strength in solution, the structure of GLP-1 increased until it precipitated reversibly. The presence of NaCl increases the tertiary structure of GLP-1 as evidenced by the increased intensity of the near CD band as shown in FIG. 2D. This also occurs for low concentrations of peptides with no evidence of self-association. Due to the increase in ionic strength, unstructured GLP-1 is in the form of an α-helix as represented by the migration of the far CD minimum to 208 nm and 222 nm (FIG. 2A), and in FIGS. 2B and 2D. It easily changes to a self-assembled conformation as represented by a shift in tryptophan emission to lower wavelengths with increasing salt and near CD pattern. The effect of temperature and pH on the conformation of GLP-1 was different in that the disordered structure of GLP-1 was not altered by either of these parameters. On the other hand, the self-associated conformation of GLP-1 was found to be sensitive to heat denaturation and its solubility was between GLP- and pH 6.3-7.6 at a peptide concentration of 10 mg / mL. It was found to be sensitive to pH, as shown in FIGS. 4A and 4B showing that one peptide precipitates reversibly. Various conformations of GLP-1 were found to be generally stable to agitation and multiple freeze-thaw cycles. Neither deamidation nor oxidation was observed for GLP-1.

  Adsorption of GLP-1 on FDKP particles was also observed under various conditions, including changes in pH, changes in GLP-1 concentration, and various surfactants, salts, ions, chaotropes. And changes in the concentrations of lyotrope, stabilizer and alcohol. It has been found that the absorption of GLP-1 into FDKP particles is strongly influenced by pH, and specifically, it is found to be strongly influenced by the binding that occurs at pH 4.0 and above. The effect of other excipients on GLP-1 absorption into FDKP particles was found to be limited.

  In developing the GLP-1 / DKP formulation of the present invention, a number of parameters that were thought to affect and affect their delivery and absorption in vivo were evaluated. Such parameters include, for example, the structure of the GLP-1 peptide, the surface charge of the molecule under certain formulation conditions, solubility and stability as a formulation, and sensitivity to serine protease degradation, in vivo stability. However, all of them play an important role in the preparation of a readily absorbable formulation, which exhibits an increased biological half-life.

  The stability of the resulting GLP-1 / FDKP formulation was tested under various conditions both in vitro and in vivo. The stability of GLP-1 was analyzed by HPLC analysis and cell-based assays. In addition, the stability of GLP-1 was tested with lung lavage fluid (including DPP-IV). It was also found that the stability of natural GLP-1 depends on the concentration in solution.

  Also, in vitro testing of GLP-1 biological activity was used to test GLP-1 / FDKP loading and was also used to measure in vivo effects. This strategy contributed to further identification of the main GLP-1 / FDKP formulation method. Furthermore, based on the fact that GLP-1 has been shown to play a role in the proliferation of β cell populations by inhibiting apoptosis (stimulation of β cell proliferation and islet neogenesis), the GLP-1 / FDKP formulation of the present invention Proliferation potential and anti-apoptotic potential were investigated by cell-based assays.

  Thus, the present invention provides an optimized formulation comprising natural human GLP-1 in combination with fumaryl diketopiperazine (FDKP) that is stable and resistant to degradation.

II. GLP-1 Molecules In certain aspects of the invention, there are provided optimized formulations comprising natural human glucagon-like peptide 1 (GLP-1) in combination with a diketopiperazine such as fumaryl diketopiperazine (FDKP). Is done. Such GLP-1 / FDKP formulations of the present invention are stable and resistant to degradation.

  Human GLP-1 is well known in the art and originates from a preproglucagon polypeptide synthesized in L cells in the terminal ileum, pancreas and brain. GLP-1 is a 30-31 amino acid peptide that exists in two molecular forms, 7-36 and 7-37 (mainly the 7-36 form). Processing of preproglucagon into GLP-1 (7-36) amide and GLP-1 (7-37) extended forms occurs primarily in L cells. It has been shown in the art that fasting plasma GLP-1 concentration is about 40 pg / mL. After a meal, plasma GLP-1 concentration increases rapidly to about 50-165 pg / mL.

As used herein, the term “GLP-1 molecule” means a GLP-1 protein, peptide, polypeptide, analog, mimetic, derivative, isoform, fragment, and the like. Such GLP-1 molecules include naturally occurring GLP-1 polypeptides (GLP-1 (7-37) OH, GLP-1 (7-36) NH ₂ ) and GLP-1 metabolites (eg, GLP- 1 (9-37)). Accordingly, in a particular embodiment of the invention, GLP-1 molecules include: natural GLP-1, GLP-1 analogs, GLP-1 derivatives, dipeptidyl peptidase IV (DPP-IV) protected GLP-1, GLP -1 mimetics, GLP-1 peptide analogs or biosynthetic GLP-1 analogs.

  As used herein, an “analog” includes a compound that has structural similarity to another compound. For example, the antiviral compound acyclovir is a nucleoside analog and is structurally similar to guanosine, a nucleoside derived from the base guanine. Thus, acyclovir is similar (biologically similar) to guanosine, interfering with DNA synthesis by substituting (or competing with) guanosine residues in the viral nucleic acid and preventing translation / transcription. Thus, a compound that has structural similarity to another (parent compound) that is similar to the biological or chemical activity of the parent compound is an analog. Necessary to consider a compound as an analog if the analog can be similar, either identical, complementary or competitive, to the biological or chemical properties of the parent compound in any relevant manner There is no minimum or maximum number of element or functional group substitutions taken. Analogs can often be derivatives of the parent compound (see “Derivatives” below). The activity of analogs of the compounds disclosed herein can be equal to, less than, or greater than their parent compounds.

  As used herein, a “derivative” is a compound made (or derived) either naturally or synthetically from a parent compound. Derivatives can be analogs (see “analogs” above) and thus can have chemically or biologically similar activities. However, unlike analogs, derivatives do not necessarily have to resemble the biological or chemical activity of the parent compound. There is no minimum and maximum number of substitutions of elements or functional groups required to consider a compound as a derivative. For example, the antiviral compound ganciclovir is a derivative of acyclovir, but ganciclovir has a different range of antiviral activity and different toxicological properties than acyclovir. The activity of the derivatives of the compounds disclosed herein may or may not be equal, smaller, larger or similar when compared to their parent compounds.

  As used herein, “metabolite” is any intermediate or product of metabolism and includes both large and small molecules. As used herein and where appropriate, the definition applies to both primary and secondary metabolites. Primary metabolites are directly involved in the normal growth, development and regeneration of biological systems. Secondary metabolites are not directly involved in these processes, but usually have important ecological functions (eg, antibiotics).

  As used herein, the term “biosynthetic” means any production of a chemical compound by a biological system.

  As used herein, “particle-forming” refers to a chemical, biosynthetic or biological component or compound capable of forming solid particles, usually in a liquid medium. Point to. Particle formation usually occurs when the particle-forming component is exposed to certain conditions (eg, changes in pH, temperature, moisture and / or osmolality / osmolality). For example, binding, fusion, coagulation and / or dehydration can occur such that exposure to the conditions results in the formation of particles. A precipitation reaction is an example of a particle formation event.

  As used herein, a “co-solution” is any medium that contains at least two chemical, biological and / or biosynthetic components. For example, a common solution may be a liquid containing at least one chemical, biological and / or biosynthetic component and a solid containing a chemical, biological and / or biosynthetic component. It can be generated by combining. In another example, the common solution may be a liquid containing at least one chemical, biological and / or biosynthetic component, a chemical, biological and / or biosynthetic component. It can be formed by combining with another liquid containing. In a further example, the common solution is generated by adding at least two solids, each containing at least one chemical, biological and / or biosynthetic component, to a single solution. Can do.

  Natural GLP-1 contemplated in the present invention is a polypeptide having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. Natural GLP-1 peptides are rapidly cleaved and inactivated within a few minutes in vivo.

  Exendin may be mentioned as a GLP-1 analog of the present invention. Exendin is a peptide that has been found to be a GLP-1 receptor agonist, and such analogs can further include exendins 1-4. Exendin is found in the venom of the American dragon lizard and has about 53% amino acid homology with mammalian GLP-1. Exendin also has similar binding affinity for the GLP-1 receptor. Exendin-3 and exendin-4 have been reported to stimulate cAMP production and release of amylase from pancreatic acinar cells (Malhotra et al., 1992; Raufman et al., 1992; Singh et al., 1994). ). It has been proposed to use exendin-3 and exendin-4 as insulin secretagogues for diabetes treatment and prevention of hyperglycemia (US Pat. No. 5,424,286).

  It has been reported that the carboxy-terminal fragment of exendin (eg exendin [9-39]), carboxyamidated molecule, and fragments 3-39-9-39 are potent selective GLP-1 antagonists. (Goke et al., 1993; Raufman et al., 1991; Schepp et al., 1994; Montrose-Rafizadeh et al., 1996). The literature also shows that exendin [9-39] blocks endogenous GLP-1 in vivo, thereby reducing insulin secretion (Wang et al., 1995; D'Alessio et al., 1996). Exendin-4 binds strongly to the GLP-1 receptor on insulin-secreting β-TC1 cells, to dispersed acinar cells from the pancreas, and to mural cells from the stomach. Exendin-4 peptides also play a role in stimulating somatostatin release in the isolated stomach and suppressing gastrin release (Goke et al., 1993; Schepp et al., 1994; Eissele et al., 1994). In cells transfected with the cloned GLP-1 receptor, exendin-4 is reportedly an agonist, ie it increases cAMP, while exendin [9-39] Identified as an antagonist, ie, it blocks the stimulatory action of exendin-4 and GLP-1. Exendin has also been found to be resistant to degradation.

  In another aspect, the present invention contemplates the use of peptidomimetics. Peptidomimetics are peptides that biologically mimic activity determinants in hormones, cytokines, enzyme substrates, viruses or other biomolecules, as known to those skilled in the art, and antagonize the biological activity of natural ligands Can be stimulated or otherwise adjusted. Peptidomimetics are particularly useful in drug development. See, for example, Johnson et al., “Peptide Turn Mimetics” in BIOTECHNOLOGY AND PHARMACY, Pezzuto et al., Eds., Chapman and Hall, New York (1993). The rationale underlying the use of peptidomimetics is that the peptide backbone of the protein exists with amino acid side chains positioned primarily to facilitate molecular interactions. Peptidomimetics are thought to allow molecular interactions similar to natural molecules.

  In a further aspect, the GLP-1 molecules of the invention have at least one biological activity of native GLP-1 (eg, the ability to bind to the GLP-1 receptor) and produce insulin secretagogue activity It is thought to initiate the signal transduction pathway. In another aspect of the invention, the GLP-1 molecule may be a peptide, polypeptide, protein, analog, mimetic, derivative, isoform, fragment, etc., and at least one of naturally occurring GLP-1 Retains two biological activities. GLP-1 molecules also include pharmaceutically acceptable salts, prodrugs, prodrug salts, polymorphs, hydrates, solvates, biologically active fragments of naturally occurring human GLP-1. Includes biologically active variants and stereoisomers, as well as naturally occurring agonists, mimetics and antagonist variants of human GLP-1, exendin families such as exendins 1-4, and polypeptide fusions thereof be able to. The GLP-1 molecules of the invention can also include a dipeptidyl peptidase IV (DPP-IV) protected GLP-1 that prevents or inhibits degradation of GLP-1.

  The GLP-1 molecule of the present invention includes peptides, polypeptides, proteins that contain amino acid substitutions, improve solubility, impart antioxidant properties, increase biological efficacy, or increase half-life in circulating blood And derivatives thereof. Thus, the GLP-1 molecules contemplated in the present invention contain amino acid substitutions, deletions or additions, where the amino acids are selected from those well known in the art. The N-terminus or C-terminus of the molecule can be modified by, for example, acylation, acetylation, or amidation, but is not limited thereto. Thus, in the present invention, the term “amino acid” refers to natural and non-natural amino acids as well as amino acid analogs and amino acid mimetics that perform similar functions as natural amino acids. Naturally encoded amino acids include 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, Tryptophan, tyrosine and valine), pyrrolysine and selenocysteine. Amino acid analogs are compounds having the same basic chemical structure as natural amino acids (ie, α-carbon bonded to hydrogen, carboxyl group, amino group and R group) (for example, homoserine, norleucine, norvaline, methionine sulfoxide, Methionine / methylsulfonium, citrulline, hydroxyglutamic acid, hydroxyproline, praline). Such analogs have modified R groups (eg, norleucine) but retain the same basic chemical structure as a naturally occurring amino acid. The amino acids contemplated in the present invention include β-amino acids that are similar to α-amino acids in that they include an amino terminus and a carboxyl terminus. However, within the β-amino acid, these functional ends are separated by two carbon atoms. Β-amino acids with specific side chains can exist as R isomers or S isomers at either the α (C2) or β (C3) carbons. This allows for a total of four diastereoisomers for any given side chain.

  The GLP-1 molecules of the present invention can also include hybrid GLP-1 proteins, fusion proteins, oligomers and multimers, homologues, glycosylation pattern variants, and mutant proteins thereof, where GLP-1 The molecule retains at least one biological activity of the natural molecule and, furthermore, a method of synthesizing or producing it (made from recombinant methods (cDNA, genomic DNA, synthetic DNA or other forms of nucleic acids). (Including, but not limited to) synthetic methods and gene activation methods). Recombinant DNA technology is well known to those skilled in the art (see Russell, D.W. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 2001).

III. Diketopiperazine Diketopiperazine is well known for its ability to form microparticles useful for drug delivery and stabilization. In the present invention, diketopiperazine is used to facilitate the absorption of GLP-1 molecules, thereby providing a stable formulation that is resistant to degradation.

  Although various methods can be used, diketopiperazine can be formed into particles that incorporate GLP-1 molecules or particles onto which GLP-1 molecules can be adsorbed. This involves mixing the diketopiperazine solution with a solution or suspension of GLP-1 molecules, then precipitating and then forming particles containing diketopiperazine and GLP-1. Alternatively, diketopiperazine can be deposited to form particles and then mixed with a solution of GLP-1 molecules. The association between the diketopiperazine particles and the GLP-1 molecule is facilitated by solvent removal, or certain steps (eg pH adjustment) can be included prior to drying to facilitate the association.

  In a preferred embodiment, the diketopiperazine of the present invention includes 3,6-di (fumaryl-4-aminobutyl) -2,5-diketopiperazine, also known as (E) -3,6-bis [4- (N-carboxyl-2-propenyl) amidobutyl] -2,5-diketopiperazine (also referred to as fumaryl diketopiperazine or FDKP), but is not limited to this.

  Other diketopiperazines contemplated in the present invention include derivatives of 3,6-di (4-aminobutyl) -2,5-diketopiperazine (eg, 3,6-di (succinyl-4-aminobutyl) ) -2,5-diketopiperazine (referred to herein as 3,6-bis (4-carboxypropyl) amidobutyl-2,5-diketopiperazine; referred to as succinyl diketopiperazine or SDKP); (Maleyl-4-aminobutyl) -2,5-diketopiperazine; 3,6-di (citraconyl-4-aminobutyl) -2,5-diketopiperazine; 3,6-di (glutaryl-4-amino) Butyl) -2,5-diketopiperazine; 3,6-di (malonyl-4-aminobutyl) -2,5-diketopiperazine; 3,6-di (oxalyl-4-aminobutyl) 2,5 diketopiperazine and derivatives thereof), and the like, but not limited thereto.

  In other embodiments, the present invention contemplates the use of diketopiperazine salts. Such salts include, for example, any pharmaceutically acceptable salt (eg, diketopiperazine Na, K, Li, Mg, Ca, ammonium, or monoalkylammonium, dialkylammonium salts). , Salts of trialkylammonium (derived from triethylamine, butylamine, diethanolamine, triethanolamine or pyridines). The salt may be a mono salt, a di salt or a mixed salt. Higher salts are also contemplated for diketopiperazines where the R group contains two or more acidic groups. In another aspect of the invention, the base form of the agent can be mixed with diketopiperazine to form a diketopiperazine drug salt, so that the drug is a counter-cation of diketopiperazine. . An example of a salt contemplated herein includes, but is not limited to, FDKP diNa. Drug delivery using DKP salts is taught in US patent application Ser. No. 11 / 210,710, the disclosure of which is incorporated herein by reference for all that it contains with respect to DKP salts.

  As disclosed elsewhere herein, the present invention also provides a novel asymmetric analog of FDKP ((E) -3- (4- (3,6-dioxopiperazin-2-yl) butyrate). Rucarbamoyl) -acrylic acid; (E) -3- (3- (3,6-dioxopiperazin-2-yl) propyl-carbamoyl) acrylic acid; and (E) -3- (4- (5-isopropyl- 3,6-dioxopiperazin-2-yl) -butylcarbamoyl) xDKP) such as acrylic acid, which was filed on the same date as the present application "Asymmetrical FDKP Analogs for Use as Drug Delivery Agents" The disclosure of which is incorporated herein in its entirety (Atty Docket No. 51300-00041).

  Diketopiperazines can be prepared by cyclic dimerization of amino acid ester derivatives, cyclization of dipeptide ester derivatives, or by Kopple et al. (J. Amer. Chem. Soc. 68: 879-80; 1946). Org. Chem. 33: 862-64; 1968) can be formed by thermal dehydration of amino acid derivatives in high boiling solvents, the teachings of which are incorporated herein.

  Methods of synthesis and preparation of diketopiperazine are well known to those skilled in the art and are described in US Pat. Nos. 5,352,461; 5,503,852; 6,071,497; 331, 318; 6,428,771 and U.S. Patent Application 20060040953. US Pat. Nos. 6,444,226 and 6,652,885 describe diketopiperazine in the form of an aqueous suspension to which a solution of the active agent is added to bind the active agent to the particles. The preparation and provision of microparticles is described. These patents further describe a method of removing the liquid medium by lyophilization to produce microparticles containing the active agent, and such suspensions to promote binding of the active agent to the particles. Changing the solvent conditions of US Patent Application Nos. 60 / 717,524 and 11 / 532,063, both entitled “Method of Drug Formulation Based on Increasing the Affinity of Active Agents for Crystalline Microparticle Surfaces”. No. 11 / 532,065 entitled “Method of Drug Formulation Based on Increasing the Affinity of Active Agents for Crystalline Microparticle Surfaces”. See also U.S. Patent No. 6,440,463 and U.S. Patent Application No. 11 / 210,709 filed on August 23, 2005 and U.S. Patent Application No. 11 / 208,087. In some cases, the loaded diketopiperazine particles of the present invention may be obtained, for example, from a US patent application entitled “A Method For Improving the Pharmaceutic Properties of Microparticles Comprising Diketopiperazine and an Active Agent” filed on Feb. 22, 2006. It is intended to be dried by the spray drying method disclosed in 11 / 678,046. Each of these patents and patent applications is hereby incorporated by reference for all that they contain with respect to diketopiperazine.

IV. GLP-1 / DKP Particle Therapeutic Formulation The present invention further provides a GLP-1 / FDKP formulation for administration to a subject in need of treatment. The subject considered in the present invention may be a domestic pet or a human. In certain embodiments, the treatment is for Type II diabetes, obesity, cancer or any related disease resulting therefrom and / or for related symptoms. Human is a particularly preferred subject.

  Other diseases or conditions contemplated in the present invention include irritable bowel syndrome, myocardial infarction, ischemia, reperfusion tissue damage, dyslipidemia, diabetic cardiomyopathy, acute coronary syndrome, metabolic syndrome, postoperative These include, but are not limited to, catabolic changes, neurodegenerative disorders, memory disorders and learning disorders, islet cell transplantation and regenerative treatment or stroke attacks. Other diseases and / or symptoms contemplated in the present invention are any diseases related to the above that can be treated by administering a GLP-1 / FDKP dry powder formulation to a subject in need thereof And / or symptoms. In addition, the GLP-1 / FDKP dry powder formulation of the present invention can be used in the treatment for inducing differentiation of β cells in human cells with type II diabetes and hyperglycemia.

  In a further aspect of the invention, the subject comprises a rat, rabbit, hamster, guinea pig, gerbil, woodchuck, cat, dog, sheep, goat, pig, cow, horse, monkey and ape (chimpanzee, gibbon and baboon). It is contemplated that it may be a domestic pet or animal that includes.

  Furthermore, it is believed that the GLP-1 / FDKP particle formulations of the present invention can be administered by various routes of clinical or non-clinical purposes known to those skilled in the art. The GLP-1 / FDKP composition of the present invention can also be administered to any targeted biological membrane (preferably the mucosa of a subject). Administration can be by any route, including oral, nasal, buccal, systemic intravenous injection, subcutaneous, blood supply, directly to the affected area or by local means Alternatively, local administration by supplying lymph fluid may be mentioned, but it is not limited thereto. In a preferred embodiment of the invention, administration of the GLP-1 / FDKP composition is by pulmonary delivery.

  Other alternative routes of administration that can be used in the present invention include: aerosols by catheters, lavage fluids, creams, lipid compositions (eg, liposomes), or other methods known to those skilled in the art or any combination of the above. Intradermal, intraarterial, intraperitoneal, intralesional, intracranial, joint, intraprostatic, intrathoracic, intratracheal, intravitreal, vagina using injection, infusion, continuous infusion, local perfusion directly immersing target cells Internal, rectal, intratumoral, intramuscular, intravesical, mucosal, intrapericardial, topical bronchial administration may be mentioned (see, eg, Remington's Pharmaceutical Sciences, 1990, all of what it includes with respect to the method of administration is herein incorporated by reference. Incorporated into).

  As a dry powder formulation, the GLP-1 / DKP particles of the present invention can be delivered by inhalation to specific areas of the respiratory system, depending on particle size. Furthermore, GLP-1 / DKP particles can be made small enough for incorporation into an intravenous suspension dosage form. For oral delivery, the particles can be incorporated into suspensions, tablets or capsules. The GLP-1 / DKP composition can be delivered from inhalation devices such as nebulizers, metered dose inhalers, dry powder inhalers, nebulizers and the like.

  In a further aspect, it is contemplated that an “effective amount” of a GLP-1 / DKP formulation is administered to a patient in need thereof. An “effective amount” of a GLP-1 / DKP dry powder formulation contemplated by the present invention is a GLP-1 compound, analog or peptide that alleviates to some extent one or more of the diseases, symptoms or symptoms of the disorder being treated. It means the amount of mimics. In one aspect, the “effective amount” of the GLP-1 / DKP dry powder formulation is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, With the amount of GLP-1 molecule to treat diabetes by increasing plasma insulin levels to 35, 40, 45, 50% or more, decreasing or decreasing fasting blood glucose levels, and expanding the pancreatic beta cell population There is, but is not limited to such. In another preferred embodiment, the present invention contemplates treating obesity by administering a pharmaceutically effective amount of a GLP-1 molecule to a subject in need of such treatment. is there. In such examples, an “effective amount” of the GLP-1 / DKP dry powder formulation reduces or reduces body weight by at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50% or more. Is the amount of GLP-1 molecule for treating obesity, but is not limited to such. The invention also provides an “effective amount” of GLP-1 / DKP drying for controlling satiety by administering a pharmaceutically effective amount of a GLP-1 molecule to a subject in need of such treatment. It is intended to administer a powder formulation. Although not limited thereto, the GLP-1 molecule can be an exendin molecule such as exendin-1 or exendin-4. In such examples, the “effective amount” of the GLP-1 / DKP dry powder formulation is at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, up to 50% or more hunger and food intake (eg, by mass or calorie content) Is the amount of GLP-1 molecule that decreases (measured), but is not limited to such. An “effective amount” of a GLP-1 / DKP dry powder formulation is further defined as an amount sufficient to detectably and repeatedly reduce, reduce, minimize or limit the degree of the disease or condition or symptom. Is possible. Also, removal, elimination or healing of a disease or condition may be possible by utilizing an “effective amount” of a formulation of the invention.

  When administering the GLP-1 / FDKP composition of the present invention to a subject in need thereof, the actual dosage of the composition is: body weight, severity of symptoms, type of disease being treated, previous or It can be determined based on physical or physiological factors such as current therapeutic intervention, patient onset, route of administration and the like. One of ordinary skill in the art can determine the actual dosage based on one or more of these factors.

  The GLP-1 / DKP formulation of the present invention can be administered once or more than once depending on the disease or condition being treated. Administration of the GLP-1 / DKP formulation can be provided to the subject at intervals of minutes, hours, days, weeks or months. In some cases, the timing of the treatment can be related to the half-life of the GLP-1 molecule at the time of administration. In a further aspect, it may be desirable to administer a GLP-1 / DKP formulation of the invention having a pharmaceutical excipient or agent when treating a specific or complex disease or condition (eg, cancer, etc.). is there. In such cases, the mode of administration can be determined by the pharmaceutical excipient or agent.

V. Examples The following examples are intended to illustrate certain embodiments of the present invention. It should be appreciated by those skilled in the art that the techniques disclosed in the examples describe representative techniques that perform well in practicing the present invention. However, one of ordinary skill in the art, in light of the present disclosure, may make many changes in the specific embodiments disclosed, and similar or similar results without departing from the spirit and scope of the present invention. Admit that it is possible to obtain further.

Example 1
Biophysical and analytical analysis of the structure of GLP-1 A number of biophysical and analytical techniques were used to analyze the structure and action of GLP-1. These methods include deep ultraviolet circular dichroism (far ultraviolet CD), near ultraviolet circular dichroism (near ultraviolet CD), internal fluorescence, Fourier transform infrared spectroscopy (FTIR), high performance liquid chromatography (HPLC ) And mass spectrometry (MS), all of which are well known to those skilled in the art. A wide range of conditions are used to investigate the effects of concentration, ionic strength, temperature, pH, oxidative stress, agitation and multiple freeze-thaw cycles on GLP-1 peptides, all of which are further detailed below. . In addition, in order to realize a specific GLP-1 / DKP formulation, these analyzes were used to clarify the main pathway of degradation and establish conditions for manipulating the peptide structure of GLP-1.

Experimental Procedures GLP-1 was purchased from either American Peptide (Sunnyvale, Calif., USA) or AnaSpec (San Jose, CA, USA) or prepared in-house (MannKind Corporation, Valencia, CA, USA). Various concentrations of aqueous GLP-1 samples were analyzed at pH 4.0 and 20 ° C. unless otherwise specified. Samples were generally freshly prepared and mixed with appropriate additives (eg, salt, pH buffer, H ₂ O ₂ etc., if any) before each experiment. In addition, secondary structure measurements of GLP-1 under various conditions were collected by far ultraviolet CD and transmission Fourier transform infrared spectroscopy (FTIR). In addition, using near-UV CD and internal fluorescence, the tertiary structure of GLP-1 was analyzed by monitoring the environment surrounding its aromatic residue (ie tryptophan).

GLP-1 concentration-dependent structure Using circular dichroism (CD) spectrum, α-helix, random coil, β-pleated sheet, β-turn and random coil represented by molecules such as proteins and peptides Analyzed. In particular, deep ultraviolet CD was used to determine the types of secondary structures of proteins and peptides, such as pure α-helix and β-sheet. On the other hand, near ultraviolet CD was used for analyzing the tertiary structure of the molecule. Therefore, far ultraviolet CD and near ultraviolet CD techniques were used to examine the effect of concentration on the GLP-1 structure.

  By far-UV CD in FIG. 1A, GLP-1 is expressed in α-helix and over a wide range of concentrations (eg: 1.8, 4.2, 5.1, 6.1, 7.2 and 8.6 mg / mL). It has been found that it forms two different structures including random coils. At low concentrations (less than 2 mg / mL), GLP-1 is primarily unstructured as determined by a large single minimum at 205 nm. As the concentration increases, the peptide adopts an α-helical structure as determined by the two local minima at 208 nm and 224 nm (FIG. 1A).

  Tertiary structure analysis suggests that the high concentration structure of GLP-1 is a self-associated conformation (ie oligomer). Both near-UV CD and fluorescence emission data support this hypothesis. The 250-300 nm positive band in the near ultraviolet CD (FIG. 1B) reveals that GLP-1 has a well-defined tertiary structure that increases with increasing concentration. More specifically, these bands indicate that the aromatic residues of the peptide are almost fixed and exist in a well-defined environment.

  Similarly, the fluorescence emission of GLP-1 at various concentrations (pH 4.0, 20 ° C.) revealed that the aromatic residue tryptophan (which also showed a strong band in the near-ultraviolet CD spectrum) was a clear third order. It was revealed that it was present in the structure and the data shown was obtained from tryptophan excitation at 280 nm (FIG. 1C). The maximum fluorescence at 355 nm for low concentrations of GLP-1 showed that tryptophan was exposed to the solvent and there was no significant tertiary structure. At high peptide concentrations, the maximum intensity decreases and shifts to lower wavelengths, indicating a clearer tertiary structure.

In order to further determine the secondary structure underlying the self-associated conformation of GLP-1, FTIR analysis was performed at various concentrations (pH 4.0, 20 ° C.). The amide I band at 1656 cm ⁻¹ clearly shows that GLP-1 has an α-helical structure at concentrations of 2 mg / mL and higher (FIG. 1D). Therefore, GLP-1 does not form a β-sheet structure, but instead the peptide appears to produce a helix bundle at high concentrations.

  Furthermore, it has been experimentally revealed that these various structures of GLP-1 are not formed by sample manipulation. Similar far ultraviolet CD, near ultraviolet CD and fluorescence emission spectra were obtained by dilution from a concentrated stock solution compared to GLP-1 prepared by directly dissolving the peptide in buffer.

Effect of ionic strength on GLP-1 A test was also conducted to determine the effect of ionic strength on GLP-1 peptide. FIG. 2A (far UV CD) shows that the irregular structure of GLP-1 is changed to an α-helical structure by increasing the salt concentration (100 mM to 1000 mM) as evidenced by the minimum values at 208 nm and 224 nm. Is shown to do. When the NaCl concentration is increased to 1M, much of the peptide (1.0 mg / mL) precipitates out of solution (FIG. 2A). However, it has become clear that this type of precipitate dissolves when diluted with water, so it was proved that GLP-1 may be reversibly precipitated at high ionic strength.

  The salt was also found to yield and improve the tertiary structure of GLP-1. This is illustrated in FIG. 2B (near-UV CD), where in the absence of salt, 1.0 mg / mL GLP-1 shows no signal but increases with increasing ionic strength. The following structure is shown. These results were confirmed by fluorescent emission of 1.0 mg / mL GLP-1 (FIG. 2C) at various NaCl concentrations (pH 4.0, 20 ° C.) after tryptophan excitation at 280 nm. The increase in ionic strength shifted the maximum fluorescence to lower wavelengths, which revealed that 1.0 mg / mL GLP-1 tertiary structure was produced and enhanced.

  Furthermore, by the tertiary structure analysis of 10 mg / mL GLP-1 at various ionic strengths (pH 4.0, 20 ° C.) using the near ultraviolet CD spectrum, the conformation in which GLP-1 self-associates is It was also found to increase with increasing strength (FIG. 2D).

  Therefore, the data suggest that ionic strength has a significant effect on the structure of GLP-1, thereby causing the protein to take an α-helical structure and associate with the oligomer. Furthermore, by increasing the ionic strength in the solution, the oligomerization of GLP-1 increases until it precipitates reversibly. This is evident with low concentrations of peptides that initially do not have tertiary structure and with high concentrations of peptides that already exhibit substantial secondary and tertiary structure. Thus, with increasing ionic strength, unstructured GLP-1 readily changes to the α-helix conformation and self-association conformation. Furthermore, the observed spectroscopic results were comparable to the previously demonstrated effect of increasing peptide concentration.

Effect of temperature and pH on GLP-1 A test was also conducted to determine whether the self-associated conformation of GLP-1 is sensitive to changes in either temperature or pH. FIG. 3A (near ultraviolet CD) reveals that the tertiary structure of 10 mg / mL GLP-1 significantly dissociates as the temperature increases. On the one hand, temperature does not affect low concentrations (0.05 mg / mL) GLP-1 at various temperatures and pH 4.0 (see FIGS. 3B and 3C (far UV CD)). Deep UV CD shows that the peptide is temperature insensitive. Therefore, increased molecular motion significantly prevents GLP-1 self-association.

  Conversely, FIG. 4A (far UV CD) shows that the solubility of the GLP-1 conformation of the α-helix is pH sensitive. Despite the structure of 10 mg / mL GLP-1 being relatively homogeneous at pH 4.4 or lower (ie, GLP-1 retains a helix), when the pH increases to or near neutral ( Some precipitation occurs between pH 6.3 and 7.6) and an irregular spectrum is produced. Samples with precipitation are less intense as a result of the presence of insoluble GLP-1 in the solution. This disordered structure is determined by one local minimum observed at 208 nm in FIG. 4A (far UV CD), which is further shown in FIG. 4B (near UV CD), indicating that GLP− in the solution after precipitation. This is considered to be caused by a decrease of 1. This precipitation can occur when the pH rises above pI5.5 for GLP-1. However, as the pH rose from near neutral to 11.7, most of the precipitate redissolved, indicating that the precipitation is reversible. As can be seen in FIG. 4A, it is believed that the undissolved precipitate of GLP-1 at pH 11.7 reduced the amount of peptide in the solution, thus reducing the intensity of the far ultraviolet CD spectrum. It is done. It was also found that GLP-1 was extremely insoluble when lyophilized GLP-1 powder was mixed with a pH 9 buffer to give a high concentration of GLP-1.

Stability of GLP-1 In addition to the effects of stirring and freeze-thaw cycles, the stability of GLP-1 peptide was examined by determining its resistance to amidolysis and oxidation.

GLP-1 (1 mg / mL) at pH 10.5 was incubated at 40 ° C. for 5 days, followed by reverse phase HPLC and electrospray mass spectrometry (MS) for analysis of amidolysis and oxidation. Oxidation tests were also performed on GLP-1 samples (1 mg / mL) incubated for 2 hours in the presence of 0.1% H ₂ O ₂ using HPLC and MS.

FIG. 5 shows the stability of GLP-1 under conditions of amidolysis and oxidation. The HPLC chromatogram shows that GLP-1 elutes with the same retention time and no degradation peak occurs for the unstable conditions analyzed. Furthermore, MS analysis produced a similar mass (3297 g / mol) for all samples, indicating that the mass was unchanged. The data also show that the peptide remains pure and unchanged when incubated under various conditions. Therefore, no amidolysis of GLP-1 was observed. GLP-1 was also shown to be stable against oxidative stress as observed in the presence of 0.1% H ₂ O ₂ , where GLP-1 purity and mass were Remained unchanged as determined by HPLC and MS, respectively. Overall, there was no change in retention time or mass values, and no degradation peak occurred, which revealed that the GLP-1 peptide is resistant to amidolysis and oxidation.

  The effects of agitation and continuous freeze-thaw cycles on various concentrations of GLP-1 were analyzed by near UV CD and internal fluorescence. Agitation of GLP-1 at 9.4 mg / mL and 1.5 mg / mL did not produce significant changes in the peptides as observed by near UV CD (FIG. 6A) and fluorescence emission (FIG. 6B). . Samples were stirred at room temperature for 30 and 90 minutes, and then the fluorescence emission spectrum was collected after tryptophan excitation at 280 nm. In an independent freeze-thaw test, solutions containing 1.6, 5.1 and 8.4 mg / mL GLP-1 (pH 4.0) were frozen at −20 ° C. and thawed at room temperature. The effect of 10 freeze-thaw cycles on GLP-1 was performed and analyzed by near ultraviolet CD (FIG. 7A) and fluorescence emission (FIG. 7B). Fluorescence emission spectra were collected after tryptophan excitation at 280 nm. Both analyzes showed that the tertiary structure of the peptide was not changed by multiple freeze-thaw cycles. In a similar experiment, the effect of 11 freeze-thaw cycles on the secondary structure of 10 mg / mL GLP-1 (pH 4.0) was analyzed (FIG. 7C). Deep UV CD shows that the secondary structure of the peptide does not change significantly as a result of multiple freeze-thaw cycles.

  Overall, biophysical analysis obtained from the above experiments showed that the structure of GLP-1 peptide is strongly influenced by its concentration in solution. As the GLP-1 concentration increased, the α-helix structure became more prominent. Furthermore, with the increase in ionic strength, the tertiary GLP-1 structure was strengthened and possibly generated.

Example 2
Adsorption test of GLP-1 / FDKP An adsorption test was performed to evaluate the interaction between GLP-1 and diketopiperazine (DKP) particles in suspension. The variables of the adsorption test were to investigate static, hydrogen bonding, water structure, protein flexibility effects, and specific salt binding interactions on GLP-1 / DKP interactions. In addition, several common protein stabilizers were tested for interference with GLP-1 adsorption on the DKP surface.

  Using pre-formed DKP suspended particles (ie, FDKP), the conditions under which GLP-1 was adsorbed on the surface of the pre-formed DKP particles were examined. A FDKP particle suspension in which FDKP particles were preformed was combined with a 3X solution of additive or excipient and 3X pH buffer. The final solution contained FDKP at a concentration of 5 mg / mL and GLP-1 at a concentration of 0.25 mg / mL (5% w / w). Unbound GLP-1 in the supernatant was filtered from the suspension. FDKP particles with associated GLP-1 protein were dissolved (reconstituted) with 100 mM ammonium bicarbonate and filtered to separate aggregate GLP-1 protein. The amount of GLP-1 in both the supernatant and the reconstituted fraction was quantified by HPLC. A series of experiments were performed, and the conditions used in the experiments included the use of additives such as salts, surfactants, ions, osmolytes, chaotropes, organics and various concentrations of GLP-1. The results of these tests are described below.

Salt test The effect of salt on GLP-1 binding to FDKP particles was observed by HPLC analysis. GLP-1 / FDKP particle loading was performed with 5 mg / mL FDKP and 0.25 mg / mL GLP-1 in the presence of 0, 25, 50, 100, 250, 500, 1000 and 1500 mM NaCl. (FIG. 8A). We also evaluated the amount of GLP-1 detected in reconstituted FDKP-free control samples as a function of pH and NaCl concentration (FIG. 8B). The pH of both data sets was controlled by a 20 mM phosphate / 20 mM acetate mixture.

  As observed in FIG. 8A, optimal binding (adsorption) of GLP-1 to FDKP particles was strongly influenced by the pH of the suspension. When the GLP-1 / FDKP ratio in the solution was 5% w / w at a pH of 4 or higher, about 3.2% to about 4% binding of GLP-1 to FDKP particles was observed. Adsorption of GLP-1 to FDKP particles in the presence of 0 and 25 mM NaCl was essentially apparent at pH 2.0, but some apparent loading was observed with increasing ionic strength. GLP-1 precipitation was observed in controls without FDKP with 1 M NaCl or more (FIG. 8B). This apparent loading at 1 M NaCl and above is the result of reversible precipitation (salting out) of the high ionic strength GLP-1 peptide. Also, the high salinity control of GLP-1 without FDKP particles showed a high GLP-1 concentration in the reconstituted sample, which allowed the GLP-1 to be filtered when the supernatant was collected. It was shown to be collected within. At NaCl below 1 M, there was no evidence of GLP-1 precipitation in the absence of FDKP particles.

Surfactant Testing The effect of surfactant on the binding of GLP-1 to FDKP particles was observed by HPLC analysis. Loading was performed with 5 mg / mL FDKP and 0.25 mg / mL GLP-1 in the presence of surfactant (FIG. 9A). We also assessed the amount of GLP-1 detected in the reconstituted FDKP-free control sample as a function of pH and surfactant concentration (FIG. 9B). The pH and control sample conditions were as described for the ionic strength test above. Surfactants used in this study were: 0.09 mM Brij78, 0.01 mM Tween 80, 0.2 mM Triton X, 0.12 mM Pluronic F68, 0.9 mM, H (CH ₂ ) ₇ SO ₄ Na, 0.9 mM CHAPS, 0.9 mM Cetrimide (cetrimide) were included. The loading curve for GLP-1 in the presence of each surfactant was shown for GLP-1 / FDKP as a function of pH.

  The data indicated that the pH-adsorption curves for GLP-1 / FDKP particles were near their critical micelle concentration (CMC), i.e., below the limit where virtually no aggregates / micelles were detected and below It can be seen that virtually all additional surfactant molecules were not affected by the presence of a surfactant in the vicinity of a small range of concentrations that divide the threshold for forming aggregates. Therefore, it further suggests that any of these surfactants can be used to optimize stability and / or pharmacokinetics (PK) as described below. As indicated above for the salt test, the interaction between GLP-1 and FDKP particles was affected by the pH of the suspension.

For this experiment, two different ions were tested to determine the effect of ions on the binding of GLP-1 to FDKP particles. In both tests, Cl ⁻ was the cation counterion and Na ⁺ was the anion counterion. GLP-1 / FDKP particle loading was performed as described for previous experiments. The pH was controlled as described above. Samples were either pH 3.0, 3.5, 4.0 or 5.0 pH buffer in the presence and absence of NaCl (used for better evaluation in the case of high ionic strength) Prepared by liquid. Additional ions were included in each sample as follows: 20 mM or 250 mM LiCl, 20 mM or 250 mM NH ₄ Cl, 20 mM or 250 mM NaF, and 20 mM or 250 mM NaCH ₃ COO.

  As shown in FIG. 10A, the data from the first ion test shows a GLP-1 / FDKP loading curve as a function of pH and ions. In the absence of NaCl, either 20 mM or 250 mM fluoride had a strong effect on adsorption at low pH (enhanced adsorption), and 250 mM NaF had maximum binding regardless of pH. showed that. This pattern was observed for the fluoride in solution, not the sodium salt, because the action of sodium bicarbonate was not the same at 20 mM and 250 mM. Furthermore, these effects were not the result of the sodium salt in the sample, as a similar concentration of salt did not show this effect as shown in FIG. In the presence of 1M NaCl, all ions imparted a high “apparent” load. The “apparent” loading for the 1M NaCl sample resulted from the GLP-1 peptide salting out of the solution at high ionic strength. This is further illustrated in FIG. 10B, which shows that GLP-1 is present in a reconstituted, FDKP-free control sample containing 1M NaCl. The amount of GLP-1 detected for these control samples increased with increasing ion concentration because the ion concentration increased the total ionic strength in the sample.

In the second ion experiment (FIG. 10C), GLP-1 / FDKP samples were prepared in the presence of 20 mM or 250 mM KCl, 20 mM or 250 mM imidazole, 20 mM or 250 mM NaI, or 20 mM or 250 mM NaPO ₄ . . The data shows that imidazole reduced loading in the presence of 1M NaCl at 250 mM, and that 250 mM phosphate and 250 mM produced a high “apparent” loading (FIG. 10C). From the amount of GLP-1 detected in the reconstituted FDKP-free control sample at 0 M and 1 M NaCl concentrations (FIG. 10D), these effects are directed against the interaction of the peptide with the FDKP particles. Rather, it resulted from the effect of ions on the GLP-1 peptide itself. Sodium phosphate and sodium iodide resulted in some salting out of GLP-1 in the absence of NaCl. Furthermore, imidazole promoted the solubilization of GLP-1 in a 1M NaCl sample, thereby reducing the “apparent” loading. Precipitation was also observed in a 0M NaCl control with 250 mM phosphate and iodide.

Test of osmolite The action of osmolite on the binding of GLP-1 to FDKP particles was observed by HPLC analysis. FIG. 11A shows the loading curve for GLP-1 / FDKP as a function of pH in the presence of a common stabilizer (osmolyte). GLP-1 / FDKP particle loading was performed as described for previous experiments. Similarly, the pH was controlled as described above. Samples were prepared at pH 3.0 and in the presence of 20, 50, 100, 150, 200 or 300 mM osmolite (stabilizer). Osmolyte is hexylene glycol (Hex-Gly), trehalose, glycine, PEG, TMAO, mannitol or proline, “N / A” indicates the absence of osmolite. In a similar experiment, the concentration of osmolyte (stabilizer) in the sample was kept constant at 100 mM and the pH was changed from 2.0 to 4.0.

  When the pH is maintained at pH 3.0 and the osmolyte concentration is changed (FIG. 11A; left curve), or when the osmolyte concentration is kept constant at 100 mM and the pH is changed (FIG. 11A). The osmolites (stabilizers) studied did not dramatically affect the adsorption of GLP-1 to the FDKP surface. GLP-1 precipitation was not detected in the reconstituted control sample without FDKP (FIG. 11B). These osmolites can be used to optimize stability and / or pharmacokinetics.

Chaotrope and liotrope tests Ionic species that affect water and protein structure (chaotrope and liotrope) were examined to determine the role these factors play in GLP-1 adsorption to FDKP. GLP-1 / FDKP particle loading was performed as described for previous experiments. Similarly, the pH was controlled as described above. Samples, pH 3.0, and 0,20,50,100,150,200 or 300mM of the following chaotropes or _{lyotropes: NaSCN, CsCl, Na 2 SO} 4, (CH 3) 3 N-HCl, Na 2 NO 3 , In the presence of sodium citrate and NaClO ₄ . In a similar experiment, the concentration of chaotrope or liotrope in the sample was kept constant at 100 mM and the pH was changed from 2.0 to 4.0.

FIG. 12A shows the loading curve for GLP-1 / FDKP as a function of pH and chaotrope and / or liotrope. Significant changes in loading at low pH (less than 3) occurred, especially at higher chaotrope concentrations for the different chaotropes analyzed. However, at pH 4, this change was not observed (FIG. 12C). Thus, although these agents promote GLP-1 binding to FDKP particles at undesirably low pH, it appears to be less effective at higher pH conditions favoring binding. According to data from a reconstituted FDKP-free control sample, the loading changes observed at pH 3.0 are, to varying degrees, on specific chaotropes that affect the salting out (precipitation) of GLP-1 peptide. This is suggested to be due in part (FIGS. 12B and 12D). This was observed for strong chaotropes such as NaSCN and NaClO ₄ .

Organic testing To determine the role that helical structures play in GLP-1 adsorption to FDKP, alcohols that are known to induce helical structures of unstructured peptides by increasing the strength of hydrogen bonds. Evaluated. GLP-1 / FDKP particle loading was performed as described for previous experiments. Similarly, the pH was controlled as described above. The action of each alcohol was observed at pH 2.0, 3.0, 4.0 and 5.0. The alcohol used was methanol (MeOH), ethanol (EtOH), trifluoroethanol (TFE) or hexafluoroisopropanol (HFIP). Each alcohol was evaluated at a concentration of 5%, 10%, 15% and 20% v / v.

  FIG. 13A shows the loading curve for GLP-1 / FDKP as a function of pH for each alcohol at each concentration. At pH 3.0, a low concentration of HFIP (5%) results in high adsorption as indicated by the mass ratio of GLP-1 to FDKP particles. Only the alcohol (HFIP) that enhances the strongest H bond (forms a helix) affected the adsorption in the buffered suspension. At higher concentrations of HFIP (20%), GLP-1 / FDKP adsorption was suppressed. FIG. 13B shows that at 20% alcohol concentration, no significant precipitation of GLP-1 was particularly observed in the reconstituted control sample without FDKP.

  This suggests that the structural flexibility of the drug (ie, the entropy and number of FDKP contacts that can be formed) can play a role in adsorption. The data suggest that H bonds may play a role in the interaction of GLP-1 with the FDKP surface under the conditions described above. Based on that data, more and stronger effects would have been expected if H-bonds acted as a major and general force in the FDKP-GLP-1 interaction. It is further speculated that.

Concentration test Adsorption of GLP-1 on the surface of FDKP particles at various concentrations was examined. FIG. 14A shows the loading curve from GLP-1 / FDKP as a function of GLP-1 concentration at various pHs. The GLP-1 concentration was 0.15, 0.25, 0.4, 0.5, 0.75, 1.0, 1.5, 2.0, 5.0 or 10 mg / mL. The sample pH was 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0.

  An increase in GLP-1 loading on FDKP particles was observed when the FDKP concentration was kept constant at 5 mg / mL and the concentration of GLP-1 was increased. When the concentration of GLP-1 was 10 mg / mL at pH 4, adsorption of about 20% GLP-1 to FDKP particles was observed. Surprisingly, saturation of GLP-1 loading adsorption to FDKP particles was not confirmed at high concentrations of GLP-1. This confirmation result is probably due to self-association of GLP-1 into the multilayer.

  Crystallinity of GLP-1 / FDKP particles that can form aggregates composed of two or more GLP-1 / FDKP particles by analysis of the morphology of the GLP-1 / FDKP formulation using a scanning electron microscope (SEM) Or it becomes clear that it exists as a flat structure (FIG. 14B). These formulations consisted of (Panel A) 0.5 mg / mL GLP-1 and 2.5 mg / mL FDKP; (Panel B) 0.5 mg / mL GLP-1 and FDKP in 10 mg / mL; C) 20 mM sodium chloride at pH 4.0, 20 mM potassium acetate and 20 mM potassium phosphate, 0.5 mg / mL GLP-1 and 10 mg / mL FDKP; and (Panel D) 20 mM sodium chloride at pH 4.0, 20 mM A solution containing 10 mg / mL GLP-1 and 50 mg / mL FDKP in potassium acetate and 20 mM potassium phosphate was prepared by lyophilization.

Summary of Results Overall, the adsorption test for the interaction of GLP-1 with FDKP particles indicated that GLP-1 binds to the surface of DKP particles in a pH-dependent manner at pH 4 or higher, high adsorption. Indicated. GLP-1 adsorption on the surface of DKP particles was found to be most strongly influenced by pH, but no substantial adsorption occurred at pH 2.0, and substantial interaction occurred at pH 4.0 or higher. It was. During observation, adsorption was enhanced at low pH by sodium ions and fluoride ions. Other additives such as surfactants and general stabilizers had only a minor effect on the adsorption of GLP-1 to the FDKP particle surface.

  Furthermore, the nature of GLP-1 itself also influenced the results of these experiments. The action of GLP-1 proved to be special and surprising in that no adsorption saturation was observed, which was due to GLP-1 self-association at high concentrations. there were. Self-association of GLP-1 at high concentrations allows coating of DKP particles with multiple layers of GLP-1 peptide, which results in a higher loading rate of GLP-1 peptide. This surprising self-association property proves beneficial for the preparation of stable dosage forms of GLP-1. Furthermore, the self-associated conformation of GLP-1 makes it possible to reduce or delay its degradation in the blood. However, it should be noted that the associated GLP-1 is sensitive to temperature and high pH, so care must be taken when using it.

Example 3
Integrity analysis of GLP-1 / FDKP formulations Based on the results from the experiments of Examples 1 and 2, a series of GLP-1 formulations having the properties described in Table 1 were transformed into cells as discussed herein. Selected for viability assay. Most formulations contained GRAS ("generally recognized as safe") excipients, but some were chosen so that the relationship between stability and adsorption could be examined.

In addition, based on the results obtained in Examples 1 and 2, a series of formulations was also selected for the Phase II integrity study of GLP-1 / FDKP. Table 2 below shows the 6 GLP-1 formulations selected for Phase II integrity. After powder preparation, they were mixed with blank FDKP to produce both GLP-1 peptide and FDKP of equal mass in each formulation.

The effect of stress on the GLP-1 / FDKP formulation in Table 2 was analyzed by HPLC (FIG. 15). Samples containing 5%, 10% or 20% GLP-1 / FDKP loaded in H ₂ O, or 5% or 10% GLP-1 / FDKP loaded in NaCl + pH 4.0 buffer The containing sample was incubated at 40 ° C. for 10 days. The HPLC chromatogram reveals that the GLP-1 peptide elutes with the same retention time and there is no degradation peak. Furthermore, MS analysis yields an equivalent mass (3297 g / mol) for all samples, which reveals that the mass is uniform for all samples analyzed. The data shows the mass to mass ratio of GLP-1 to FDKP particles and other components present in the solution prior to lyophilization. Overall, the GLP-1 / FDKP formulation was shown to be stable against stress.

Example 4
Dipeptidyl peptidase IV (DPP-IV) found in stable body fluids of GLP-1 incubated in lung lavage fluid cleaves and inactivates GLP-1 and thus in body fluids such as lung fluid and blood The stability of GLP-1 was analyzed.

Dipeptidyl peptidase IV (DPP-IV) is an outer membrane-bound serine protease that is expressed on the surface of several cell types, in particular CD4 ⁺ T cells. DPP-IV is also found in blood and lung fluid. DPP-IV is involved in the regulation of sugar metabolism because its substrate contains the insulinotropic hormone GLP-1, which is inactivated by removal of its two N-terminal amino acids (see FIG. 16A). . DPP-IV cleaves the major circulating form of the Ala-Glu bond of human GLP-1 (GLP-1 (7-36)), which releases the N-terminal two residues. DPP-IV negatively regulates glucose processing by degrading GLP-1 and thereby reducing incretin action on pancreatic β cells.

  A study was conducted to determine inhibition of GLP-1 degradation in rat blood and lung fluid in the presence of aprotinin or DPP-IV inhibitors. Aprotinin (natural serine protease inhibitor) known in the art to inhibit proteolysis was added to the collected samples at 1, 2, 3, 4 and 5 TIU / mL. DPP-IV activity was then measured by detecting cleavage of the luminescent substrate containing the Gly-Pro sequence recognized by DPP-IV. Bronchopulmonary lavage fluid was incubated with a proluminescent substrate for 30 minutes, and then the cleavage product was detected by luminescence.

  The data showed increased inhibition of DPP-IV activity as detected by inhibition of peptide degradation in various body fluids (those discussed herein) with increasing aprotinin concentration (FIG. 16B). . Similar results were observed with DPP-IV inhibitors added to samples after collection of 1.25, 2.5, 5, 10, and 20 μL / mL (FIG. 16C). By adding an inhibitor after sample collection, the sample could be evaluated more accurately.

  The stability of GLP-1 was also examined in lung lavage fluid using a capture ELISA mAb that recognizes GLP-1 amino acids 7-9. GLP-1 was incubated in lung lavage fluid (LLF) for 2, 5, 20 and 30 minutes. Incubation conditions were as follows: 1 μg or 10 μg (w / w) LLF and 1 μg or 10 μg (w / w) GLP-1 as shown in FIG. GLP-1 was not detected in LLF alone. In the combined use of various concentrations of LLF and GLP-1, the same high detection of GLP-1 as in the case of GLP-1 alone was observed, so that GLP-1 was stable in the lung lavage fluid over time. It becomes clear that there is (FIG. 17). The stability of GLP-1 in undiluted lung lavage fluid was confirmed in a similar study, showing 70-72% GLP-1 integrity at 20 minutes (data not shown).

  Furthermore, the stability of GLP-1 in rat plasma was examined. Plasma was obtained from various rats (indicated as plasma 1 and plasma 2 in the figure legend) and diluted 1: 2 or 1:10 (v / v). 1 μL of GLP-1 was added to 10 μL of plasma or PBS. Samples were incubated at 37 ° C. for 5, 10, 30 or 40 minutes. The reaction was stopped on ice and 0.1 U aprotinin was added. The data shows that GLP-1 in the 1: 2 and 1:10 plasma dilutions was at high concentrations across all time points tested (FIGS. 18A and 18B). Overall, the data reveals that GLP-1 is surprisingly stable in both lung lavage fluid and plasma where serine protease DPP-IV is observed.

Example 5
Effect of GLP-1 molecule on apoptosis and cell proliferation To examine whether GLP-1 inhibits apoptosis, a screening assay was performed to determine the effect of GLP-1 on the inhibition of β-cell death. Rat pancreatic epithelial (ARIP) cells (used as a pancreatic beta cell model; purchased from ATCC (purchased from Manassas, VA), USA) pre-treated with GLP-1 at concentrations of 0, 2, 5, 10, 15 or 20 nM for 10 minutes did. The cells were then dosed with nothing or 4.5 h staurosporine (apoptosis inducer) for 4.5 hours. Cell viability was assessed using Cell Titer-Glo® (Promega, Madison, Wis., USA). The rate of cell death reduction was shown by increasing GLP-1 concentration up to 10 nM in cells administered staurosporine (FIG. 19A).

  Further discussion of the effect of GLP-1 on apoptosis was done by FACS analysis using Annexin V staining. Annexin V staining is a useful tool in detecting apoptotic cells and is well known to those skilled in the art. Annexin V binding to the cell membrane allows analysis of changes in phospholipid (PS) asymmetry before morphological changes associated with apoptosis and before loss of membrane integrity. Therefore, the effect of GLP-1 on apoptosis is that 15 nm GLP-1, 1 μM staurosporine for 4 hours, or 1 μM staurosporine + 15 nm GLP-1 administered cells, or both staurosporine and GLP-1 Determined in cells not administered (experimental control). The data shows that GLP-1 inhibited apoptosis induced by staurosporine by about 40% (FIG. 19B).

  Similar results for inhibition of apoptosis were observed using a GLP-1 analog (Exendin-4) that binds to the GLP-1 receptor in a manner similar to that for GLP-1. 5 μM staurosporine was administered to ARIP cells for 16, 24 or 48 hours, respectively, in the presence of 0, 10, 20 or 40 nM exendin. The data (FIG. 20) shows that exendin was completely ineffective at suppressing apoptosis because 100% cell death occurred at 10 nM. At 20 and 40 nM, exendin inhibited apoptosis to some extent, with about 50% being inhibited in 48 hours in the presence of 40 nM exendin-4.

Example 6
Effects of candidate GLP-1 / FDKP preparation on cell death A cell-based assay was performed to assess the ability of the GLP-1 / FDKP preparation (as disclosed in Table 1 of Example 3 above) to inhibit cell death. . These GLP-1 / FDKP particle formulations are in the form of a suspension or lyophilized. The preparation was analyzed for its ability to suppress staurosporine-induced cell death in ARIP cells. ARIP cells pre-administered with GLP-1 samples were exposed to 5 μM staurosporine for 4 hours and analyzed with Cell Titer-Glo® (Promega, Madison, Wis., USA) to determine cell viability.

  Samples of various GLP-1 / FDKP formulations were either left unstressed or stressed at 4 ° C or 40 ° C for 4 weeks. Each GLP-1 / FDKP sample was used at 45 nM in a cell-based assay to determine its ability to suppress staurosporine-induced cell death. The control sample shown on the right shows cell viability in the case of medium alone, GLP-1 alone, staurosporine alone, or both GLP-1 and staurosporine are present. (Note: Graph legend does not apply to control samples. Each bar represents 3 separate sets). All results shown are the average of 3 replicates.

  The data shows that all stressed GLP-1 / FDKP lyophilized formulations suppressed cell death induced by staurosporine (FIG. 21). However, many of the GLP-1 / FDKP suspension formulations did not inhibit cell death.

Example 7
Pulmonary inhalation of GLP-1 / DKP particles To study the pharmacokinetics of GLP-1 / FDKP in plasma in female Sprague Dawley rats administered with various formulations of GLP-1 / FDKP by intravenous injection or intrapulmonary inhalation GLP-1 concentration was evaluated. In preliminary studies, about 4% and 16% (w / w) GLP-1 of the GLP-1 / FDKP particle formulation was used. Rats were randomly assigned to 12 groups including groups 1, 4, 7, and 10 that received GLP-1 solution administered by intrapulmonary fluid instillation or IV injection. Groups 2, 5, 8, and 11 received administration (disclosed in Table 2) related to GLP-1 / FDKP salt administered by pulmonary inhalation or IV injection. Groups 3, 6, 9, and 12 received a combined formulation related to GLP-1 / FDKP salt administered by pulmonary inhalation or IV injection. The GLP-1 / DKP formulation was a salt related formulation with a loading of about 16%. In order to achieve a loading of about 4%, 16% of the formulation was mixed with DKP particles in the form of a 3: 1 mixture. Intrapulmonary inhalation or intravenous injection was performed with 0.5 mg or 2.0 mg particles (16% or 4% GLP-1 loading, respectively) for a total dose of 0.08 mg GLP-1.

  In individual groups of animals (groups 7-12), administration was repeated on Day2. Groups 1, 4, 7 and 10 received 80 μg of GLP-1 solution. Groups 2, 5, 8, and 11 received a GLP-1 / DKP salt related formulation (approximately 16% GLP-1 loading). Groups 3, 6, 9, and 12 received a GLP-1 / DKP salt related mixed preparation (approximately 4% GLP-1 loading).

  The experiment was performed twice using the same formulation, with administration and blood collection for two consecutive days. Blood samples were taken on the day of dosing for each group before dosing (time 0) and 2, 5, 10, 20, 30, 60 and 120 minutes after dosing. At each time point, approximately 150 μL of whole blood was collected from the lateral tail vein into a cryovial tube containing approximately 3 U / mL aprotinin and 0.3% EDTA and stored upside down on ice. The blood sample is centrifuged at 4000 rpm and 40 μL of plasma is pipetted into a 96-well plate and the 96-well plate is GLP-1 by ELISA according to manufacturer's recommendations (Linco Research, St. Charles, Mo., USA). Stored at −80 ° C. until analyzed for concentration. The assay buffer was determined to be the optimal condition in the case of GLP-1 with only serum (5% FBS) and no matrix.

  Intravenous administration: Groups 5, 6, 10, 11 and 12 received intravenous (IV) administration of various GLP-1 / FDKP formulations and GLP-1 solutions (FIG. 22A). Groups 5 and 6 received 15.8% GLP-1 / FDKP, groups 11 and 12 received another 15.8% GLP-1 / FDKP on consecutive days, and group 10 received controls As a GLP-1 solution. The concentration of GLP-1 / FDKP was detected at 0, 2, 5, 10, 20, 40, 60, 80, 100 and 120 minutes. All groups showed a detectable increase in plasma GLP-1 concentration after intravenous administration, with a maximum concentration observed 2 minutes after administration. Plasma active GLP-1 levels returned to background levels by 20 minutes after dosing in all groups. When administered by intravenous injection, no significant difference was observed in the kinetics of these various formulations of GLP-1 / FDKP and GLP-1 solutions. In rats administered by intravenous injection, plasma GLP-1 concentrations returned to baseline concentrations 10-20 minutes after administration, suggesting physiological kinetics (ie, about 95% GLP-1 Was discharged within 10 minutes).

  Single inhalation administration: Groups 1, 2, 3, 7, 8, 9 and 12 received various GLP-1 / FDKP formulations or GLP-1 solutions by pulmonary inhalation (FIG. 22B). Group 1 receives 80 μg GLP-1 control by intrapulmonary fluid instillation (LIS), Group 2 receives 15.8% GLP-1 / FDKP by intrapulmonary inhalation (IS), Group 3 is administered 3.8% GLP-1 / FDKP by intrapulmonary inhalation (IS), Group 7 is administered 80 μg GLP-1 control by intrapulmonary fluid instillation (LIS), Group 8 is administered 15.8% GLP-1 / FDKP by pulmonary inhalation (IS), and Group 9 is 3.8% GLP-1 / FDKP by pulmonary inhalation (IS). Was administered. The concentration of GLP-1 / FDKP was measured at 0, 2, 5, 10, 20, 40, 60, 80, 100 and 120 minutes.

  All groups showed a detectable increase in plasma GLP-1 concentration after intrapulmonary administration. The maximum plasma GLP-1 concentration varied with the formulation / composition used. Groups 2 and 8 showed maximal GLP-1 plasma concentrations 10-20 minutes after administration, as indicated by AUC, while Groups 3 and 9 showed significant levels of active GLP after 5-10 minutes. -1 and groups 1 and 7 showed a rapid and temporary increase in plasma active GLP-1 concentration. In Groups 2, 3, 7 and 8, plasma active GLP-1 concentrations returned to background concentrations by 60 minutes after administration, while Groups 1 and 7 reached background concentrations by 20 minutes after administration. did.

  Eight nanomolar GLP-1 appears to be effective in a diabetic rat model, the GLP-1 dose is 80 μg (3000 times greater than the reported effective dose), and the plasma GLP-1 concentration is At 30 minutes, the pulmonary delivery was 10 times higher than the 3 hour infusion (Chelikani et al., 2005), and the bioavailability of GLP-1 / FDKP delivered by pulmonary inhalation was 71% . These results are further reported in Table 4 below. In most rats dosed by pulmonary delivery, plasma GLP-1 levels returned to baseline levels 30-60 minutes after dosing. All rats, with the exception of one rat in group 2, showed an increase in plasma GLP-1 concentration following intravenous administration or pulmonary inhalation of various GLP-1 / FDKP formulations.

  Conclusion: A difference was observed in the pharmacokinetic profile of the GLP-1 / FDKP formulation over the GLP-1 solution. Plasma GLP-1 concentrations were more sustained in rats administered the GLP-1 / FDKP formulation by pulmonary inhalation than in rats administered the GLP-1 solution. All animals showed a gradual decrease in plasma GLP-1 concentration between 20 and 60 minutes after dosing. Based on these results, relatively consistent results were observed in two experiments conducted for two consecutive days.

Example 8
GLP-1 / FDKP reduces food intake in rats GLP-1 is also known in the art to function in the brain to induce satiety and reduce food intake. Based on this role of GLP-1 in reducing satiety and food intake, the GLP-1 / FDKP formulation of the present invention can reduce feeding and thereby make it possible to control obesity Experiments were performed to determine if it was effective.

  Two groups of female Sprague Dawley rats were given a 15.8% GLP-1 / FDKP formulation at a dose of control (air) or 2 mg / day (0.32 mg / dose GLP-1) by intrapulmonary inhalation. Either was administered. The control group consisted of 5 rats and the test group consisted of 10 rats. Each rat received a single dose for 5 consecutive days, and food intake was 2 and 6 hours after each dose. Each rat was weighed daily.

  Preliminary data indicate that at 2 and 6 hours after dosing, the cumulative food intake of rats administered with the GLP-1 / FDKP formulation decreased overall (FIGS. 23A and 23B). The decrease became more pronounced 2 hours after Day4 administration (p = 0.01). The decrease in Day 1 and Day 2 at 6 hours was more significant (p <0.02). There was no effect on food intake 24 hours after administration.

Example 9
Toxicity studies After multiple doses were administered, repeated dose toxicity studies were conducted to assess the potential for GLP-1 / DKP's toxic effects and toxicokinetic profile. A 14-day study on rats and a 28-day study on monkeys were performed. GLP-1 / DKP is administered daily by the inhalation route. In studies where animals were administered for 28 days, some animals were sacrificed immediately after the dosing schedule, while others were given a one month recovery period prior to sacrifice. All animals are evaluated for clinical signs, various physiological parameters (GLP-1, glucose, insulin, organ weight, clinical pathology, etc.) and histopathology of various organs.

  A series of GLP mutagenicity tests were performed to evaluate the mutagenic potential of diketopiperazine particles. These tests include in vitro Ames tests and chromosomal aberration tests, both of which are well known to those skilled in the art. In addition, in vivo mouse micronucleus tests as known to those skilled in the art were also performed. Genotoxicity data showed no evidence of possible mutagenicity or genotoxicity from diketopiperazine particles.

  Tests were also conducted to evaluate the effect of diketopiperazine particles on reproductive toxicity. These tests included rat and rabbit reproduction tests, embryo-fetal development tests and postnatal development tests. Diketopiperazine particles administered by subcutaneous injection do not impair fertility or implantation in rats and there are no signs of teratogenicity in rats or rabbits. The diketopiperazine particles did not adversely affect fertility and early embryonic development, embryofetal development or prenatal or postnatal development.

Many drugs have been removed from the clinical market because of their tendency to cause long QT (LQT) syndrome (acquired LQTS or long QT syndrome is a heart that can occur in otherwise healthy people The hERG assay was used to study the efficacy of diketopiperazine particles. Most drugs that cause acquired LQTS cause it by blocking the potassium channel of the human ether-a-go-go related gene (hERG), which causes repolarization of the ventricular action potential, A hERG assay was utilized. Results from the hERG assay showed an IC ₅₀ of _greater than 100 μM for diketopiperazine particles. In addition, the results from a non-clinical study using diketopiperazine particles showed no prolongation in dogs (9 months or safety pharmacology cardiovascular study), so for QTc interval (heart rate corrected QT time interval) No effect was shown. When administered intravenously, there was no effect of diketopiperazine particles on the CNS or cardiovascular system evaluated in the safety pharmacology core battery.

Example 10
Effects of GLP-1 on β-cell populations GLP-1 is known to promote all steps in insulin biosynthesis and directly stimulate β-cell growth and survival and β-cell differentiation. The combination of these actions results in an increase in β cell population. Furthermore, GLP-1 receptor signaling results in a decrease in beta cell apoptosis, which further leads to an increase in the beta cell population. GLP-1 is known to regulate the β cell population by three possible pathways (increased cell proliferation of β cells, inhibition of apoptosis, differentiation of putative stem cells of ductal epithelium) ing.

  To show the effect of GLP-1 on the β cell population, GLP-1 / FDKP was administered to the cells at Day 1, 3 and 5 and compared to untreated cells. As suggested in the literature (Sturis et al., 2003), administration of active GLP-1 increased the beta cell population up to 2-fold. Furthermore, consideration of the action of various GLP-1 receptor (GLP-1R) agonists on diabetes has revealed that GLP-1R agonists prevent or delay the onset or progression of diabetes.

  The effects of GLP-1 / FDKP on β-cell proliferation, insulin and glucose were evaluated in male Zucker diabetic / obese (ZDF) rats (n = 8 / group). The animals received either control (air) or 2 mg GLP-1 / FDKP containing 15% (0.3 mg) GLP-1 daily for 3 consecutive days. An intraperitoneal (IP) glucose tolerance test was performed and blood samples were collected prior to dosing and 15, 30, 45, 60 and 90 minutes after dosing for plasma GLP-1 and glucose analysis. Pancreatic tissue was collected for analysis of insulin secretion, β cell population and apoptosis by immunohistochemistry.

  An IP glucose tolerance test (IPGTT, FIG. 24) was performed on Day 4 of administration. The animals received a bolus of glucose by intraperitoneal injection on Day 3, after an overnight fast, followed immediately by control (air) or GLP-1 / FDKP by intrapulmonary inhalation. Blood was collected at various time points before glucose administration and up to 90 minutes after administration. Thirty minutes after dosing, group 1 showed a 47% increase in glucose concentration over pre-dose, whereas group 2 (GLP-1 / FDKP) had a glucose concentration of 17% above the pre-dose value. An increase was shown. The glucose concentration was significantly lower in all animals after administration of glucose tolerance than in control animals (p <0.05).

  GLP-1 concentration was also measured at Day 3 of administration (Figure 25). The maximum plasma GLP-1 concentration in Group 2 was 10443 pM 15 minutes after dosing.

  Furthermore, the insulin concentration was measured at each time point of Day 3 together with the glucose measurement after the IP glucose tolerance test. Both Control (Air) Group 1 and Group 2 (GLP- / DKP) showed an initial decrease in insulin concentration from 46% and 30% pre-dose values, respectively, by 15 minutes post-dose (FIG. 26). ). However, 30 minutes after dosing, group 1 insulin concentrations continued to fall to 64% of pre-dose values, whereas group 2 insulin concentrations returned to baseline. In the administered animals, insulin concentrations at 45, 60 and 90 minutes were close to pre-dose values with a deviation of less than 1.5%.

  Slides were prepared for insulin immunostaining and microscopic evaluation of insulin expression. Based on quantitative assessment of insulin expression by light microscopy, statistical significance was not achieved as determined by the proportion of β-islet cells expressing insulin (p = 0.067), but dose related There was a dose-related increase in insulin expression in the pancreas of male ZDF rats.

  In addition, apoptosis analysis was performed on pancreatic tissues of ZDF rats. Pancreatic exocrine cells and pancreatic endocrine cells were evaluated by the TUNEL assay (Tornusciolo D.R. et al., 1995). Scores of approximately 10,000 cells (exocrine and endocrine) within the pancreas were recorded. Most TUNEL positive cells were exocrine. There was no difference in the apoptosis labeling rate of the administration group compared to the control group.

  In addition, β-cell proliferation was assessed in the pancreas of diabetic obese Zucker rats, where control (air) or GLP-1 / FDKP was administered by pulmonary inhalation once daily for 3 days. Slides were prepared for insulin co-localization using immunohistochemistry and Ki67 (proliferation marker). Microscopic evaluation of cell proliferation was performed in insulin positive islets and exocrine pancreas in a total of 17 ZDF rats. Based on a quantitative assessment of cell proliferation, no dose-related effects on cell proliferation in pancreatic islet β cells or exocrine cells in male ZDF rats were observed.

  Overall, according to this study, GLP-1 / FDKP administered with 2 mg or 0.3 mg of GLP-1 by pulmonary inhalation was able to improve blood glucose levels in diabetic hyperlipidemic rats (model of type 2 diabetes) after glucose tolerance test. Reduced and increased the number of insulin secreting cells per islet.

Example 11
Preparation of GLP-1 / FDKP particle formulation An alternative method for preparing GLP-1 / FDKP particle formulation was also used. The formulation was prepared as follows: 1% by weight of GLP-1 was added to 9 parts of deionized water, followed by the addition of a small amount of glacial acetic acid to give a clear solution of 10% by weight. A GLP-1 stock solution was prepared. The suspension stock solution of FDKP particles (about 10 wt% particles) was divided into three. Appropriate amount of GLP-1 stock solution was added to each suspension to obtain the desired composition having 5 wt% and 15 wt% GLP-1 in the dry powder. After the addition of the protein solution, the pH of the suspension was about 3.5. The suspension was then adjusted to about pH 4.4-4.5, after which the suspension was spheronized in liquid nitrogen and then lyophilized to remove the ice.

  Powder aerodynamics is characterized by a respirable fraction based on filling (RF based on filling), ie the percentage of respirable range powder normalized by the amount of powder in the cartridge, which is Determined as follows: Five cartridges were manually filled with 5 mg of powder and discharged by MannKind's MedTone® inhaler (described in US patent application Ser. No. 10 / 655,153).

  This method produced a formulation with good RF based on filling. The powder with 5 wt% GLP-1 was 48.8% RF / fill, while the powder containing about 15 wt% GLP-1 was 32.2% RF / fill.

Example 12
Pharmacokinetics of GLP-1 / FDKP with Various GLP-1 Concentrations To assess the pharmacokinetic properties of GLP-1 / FDKP with various GLP-1 concentrations, the body weight ranges from 192.3 grams to 211.5 grams. Body female SD rats were divided into 4 treatment groups: control group GLP-1 (group 1, n = 3) and GLP-1 / FDKP formulation group (groups 2-4, n = 5 / group). The animals received one of the following test substances: Control (air) by intrapulmonary instillation, 2.42 mg GLP-1 / FDKP containing 5% GLP-1 (0.12 mg GLP-1) by lung inhalation, 10% GLP-1 ( 1.85 mg GLP-1 / FDKP containing 0.19 mg GLP-1) or 2.46 mg GLP-1 / FDKP containing 15% GLP-1 (0.37 mg GLP-1). Blood samples were collected and tested for serum FDKP and plasma GLP-1 concentrations before and after administration (2, 5, 10, 20, 30, 40 and 60 minutes).

The maximum plasma GLP-1 concentration (C _max ) after administration of GLP-1 / FDKP (5% formulation) was 2,321 pM at T _max 5 minutes after administration, 10 minutes after administration (10% formulation) Of 4,887 pM at a T _max of 10, and 10,207 pM at a T _max 10 minutes after administration (15% formulation). As shown in FIG. 27, a significant GLP-1 concentration was observed until 30 minutes after administration. The values of the area under the curve (AUC) of GLP-1 were 10622, 57101, 92606, and 227873 pM ^* min for groups 1 to 4, respectively. The estimated half-life of GLP-1 was 10 minutes for GLP-1 / FDKP at 10% or 15% GLP-1 loading.

As shown in FIG. 28, the maximum FDKP concentration measurements were 8.5 μg / mL (Group 2), 4.8 μg for GLP-1 / FDKP formulations at 5%, 10% and 15% GLP-1, respectively. / ML (group 3) and 7.1 μg / mL (group 4). The time to reach the maximum concentration (T _max ) was 10 minutes. This data shows that FDKP and GLP-1 showed similar absorption kinetics, and equivalent amounts of FDKP were absorbed regardless of GLP-1 loading on the particles.

  Overall, this study revealed that plasma GLP-1 concentrations were detected at a significant level after a single dose of GLP-1 / FDKP by pulmonary inhalation in SD rats. A dose-related increase in plasma GLP-1 concentration was observed with the maximum concentration achieved approximately 10 minutes after administration and the observable GLP-1 concentration 40 minutes after administration. All animals survived until study completion.

Example 13
Pharmacodynamic properties of GLP-1 / FDKP administered by pulmonary inhalation To assess the pharmacodynamic properties of GLP-1 / FDKP, female Sprague Dawley rats were divided into two dose groups. The animals were inhaled once daily by intrapulmonary inhalation with 2 mg GLP-1 / FDKP (n = 10) containing control (air; n = 5) or 15% GLP-1 (0.3 mg GLP-1). ) For 4 consecutive days.

  Food intake was measured in the dark cycle for 4 consecutive days before dosing, 1, 2, 4 and 6 hours after dosing (FIG. 29). Food intake was reduced in treated animals in Day 1, 2 and 3 after daily administration of GLP-1 / FDKP by intrapulmonary inhalation compared to the control (air) group (p <0.05). Statistical significance of food intake in animals in the treated group over the control group (air) at Day 1 and 6 hours of Day 1, and at Day 4 and 6 hours of Day 2, and before Day 3 administration A significant decrease was observed.

  Body weight (Figure 30) was measured daily for 4 consecutive days before dosing. Body weight at the start of administration ranged from about 180 to 209 grams. Statistical significance between treated and control (air) animals was not reached, but body weight was smaller in treated animals. All animals survived until scheduled sacrifice.

Examples 14-16
Toxic Kinetics (TK) Tests Examples 14-16 below are repeated dose toxicity studies conducted in rats and monkeys to assess potential toxic effects and toxicokinetic profiles of GLP-1 / FDKP inhalation powders. Is shown. The data show that there is no apparent toxicity at a dose of GLP-1 / FDKP inhalation powder several times larger than that proposed for clinical use. Furthermore, there appears to be no difference between male and female animals within each species.

Example 14
Toxicology of GLP-1 / FDKP in monkeys administered by pulmonary inhalation for 5 days Once daily (30 minutes per day), nasal and oral administration to cynomolgus monkeys (Macaca fascicularis) for 5 consecutive days A study was conducted to determine the toxicity and toxicokinetic profile of GLP-1 / FDKP by the human therapeutic route of administration. For nasal and oral administration, the mouth and nose of monkeys were covered with a mask and the test preparation was inhaled for 30 minutes.

  For 14 days prior to the start of dosing, animals were accustomed to restraint and dosing methods. At the start of administration (Day 1), male animals are 30 to 56 months old and weigh 2.3 to 4.0 kg, female animals are 31 to 64 months old and weigh 1.6 to 3.4 kg. Met. Ten (5 male and 5 female) non-naive cynomolgus monkeys were assigned to 5 groups (2 animals per group) as shown in Tables 5 and 6 below. An administered monkey is a herd that has previously been administered the formulation to be tested. However, these formulations have a short half-life and are not expected to be present in the administration experiments disclosed herein or have any effect on monkeys. Animals were control (air), 2 mg / kg FDKP, or 0.3 (0.04 mg GLP-1), 1.0 (0.13 mg GLP-1) or 2.0 (0.26 mg GLP-1). -1) mg / kg GLP-1 / FDKP was administered.

Whole blood samples (1.4 mL / blood sample) were obtained at Day 5 at the following time points: pre-dose and 10, 30, 45, 60, 90, 120 minutes and 4 hours after administration. Blood was collected from the femoral vein by venipuncture. The blood sample was divided into two aliquots, one for plasma GLP-1 analysis (0.8 mL) and one (0.6 mL) for serum FDKP analysis. For plasma GLP-1 analysis, whole blood (0.8 mL) was collected in 1.3 mL EDTA tubes (0.1% EDTA) at each time point. Approximately 5-10 seconds after blood collection, DPP-IV inhibitor (Millipore, Billerica, Mass., USA) was added to the tube (10 μL / mL blood) (resulting in a concentration of 100 μM DPP-IV). The tube was turned upside down several times and immediately placed on ice with water. Whole blood samples were kept on ice with water until centrifugation (approximately 10 minutes, 4000 rpm (2-8 ° C.)) to obtain plasma. Plasma samples were transferred into appropriate vials, kept on dry ice, and then stored in a freezer at -70 (± 10) ° C. Plasma concentrations (C _max ), T _max , AUC and T _1/2 were determined for GLP-1.

After 4 days of inhalation administration of GLP-1 / FDKP, a detectable concentration of GLP-1 was detected in all pre-dose samples of Day5. On Day 5, the highest plasma concentration of GLP-1 (C _max ) was achieved within about 10 minutes after dose administration (FIG. 31).

The dose-related increase in GLP-1 C _max and AUC _last (area under the concentration curve from time zero to the final quantifiable concentration) as a function of dose is shown in Day 5 for both male and female monkeys. Recognized by As the dose was increased in both male and female monkeys over the dose range studied, with the exception of male monkeys at a dose concentration of 1 mg / kg / day, the ALP _{last of} GLP-1 increased proportionally with dose. There wasn't. The increase of 6.7 times the dose of 0.3 mg / kg / day to 2.0 mg / kg / day, 2.9 times the male _{AUC last} increase, and the female 1.1 times the _{AUC last} Only an increase occurred.

  When GLP-1 / FDKP was administered at dose concentrations of 0.3, 1.0, and 2.0 mg / kg / day, respectively, the mean maximum GLP-1 concentration was 17.2, 93.1 for males. 214 pg / mL, and 19.3, 67.9, 82.8 pg / mL for females. With a clear elimination half-life of 4 to 24 minutes, plasma GLP-1 concentrations declined rapidly.

When GLP-1 / FDKP was administered at doses of 0.3.1.0 and 2.0 mg / kg / day, respectively, the AUC values for GLP-1 were 21.6, 105, 62. 3 pg ^* h / mL and females were 33.4, 23.7, 35.4 pg ^* h / mL.

There was no apparent gender difference in GLP-1 TK parameters observed at the lowest dose concentration. However, male monkeys showed a consistently higher AUC _last value than female monkeys at the medium and high dose concentrations studied. Several samples from vehicle control and control (air) monkeys showed measurable concentrations of GLP-1. This may have been caused by contamination of the air inhaled by the animals, or it could be the amount of endogenous GLP-1 concentration in those particular monkeys. It should be noted that the control animals were exposed in a different room than the animals that received GLP-1 / FDKP.

  Since the biological half-life of GLP-1 is less than 15 minutes, GLP-1 from administration of GLP-1 / FDKP should be completely excreted within 24 hours. Therefore, the endogenous concentration of GLP-1 was consistent with that the concentration of GLP-1 in the zero time sample taken on Day 5 was consistently quantifiable in all GLP-1 / FDKP treated animals. It was an explanation. Subtraction of the zero value from the observed concentration of GLP-1 after administration reflects the change in GLP-1 due to administration of GLP-1 / FDKP.

For serum FDKP analysis, whole blood (0.6 mL) at each time point was collected in a tube containing no anticoagulant, allowed to clot at room temperature for at least 30 minutes, and separated by centrifugation to obtain serum. FDKP analysis and serum concentration (C _max ), T _max , AUC, T _1/2 were determined. After 4 days of inhalation administration of GLP-1 / FDKP, detectable concentrations of FDKP were observed in samples after all Day 5 administrations. On Day 5, the highest plasma concentration of FDKP (C _max ) was achieved approximately 10-30 minutes after administration.

A dose-related increase in FDKP AUC∞ (area extrapolated from zero to infinity) as a function of dose was observed in both male and female monkeys at Day 5. However, there was no difference in FDKP AUC∞ between 0.3 mg / kg / day and 1.0 mg / kg / day in females, but dose-related increases were 1 mg / kg / day and 2 mg / kg. / Indicated during the day. In all cases where an increase was observed, the increase was not proportional to dose. A 6.7-fold dose increase from 0.3 mg / kg / day to 2.0 mg / kg / day resulted in a 2.7-fold increase in male AUC _last and a 3.0-fold increase in female AUC∞. An increase occurred. When GLP-1F / DKP was administered at dose concentrations of 0.3, 1.0, and 2.0 mg / kg / day, the average value of the maximum FDKP concentration (C _max ) was 200, 451, 339 ng for males. / ML, 134, 161, and 485 ng / mL for females. When GLP-1 / FDKP was administered at dose concentrations of 0.3, 1.0, and 2.0 mg / kg / day, respectively, the AUC∞ value of FDKP was 307, 578, 817 ng. h / mL, 268, 235, 810 ng. h / mL. There is an exception that the concentrations of AUC∞ and _{Cmax in} animals receiving only FDKP at a dose of 2.1 mg / kg / day (Group 2) were slightly longer at T _max 30-45 minutes after administration of the dose. However, it was at the same digit position as the animals that received GLP-1 / FDKP at 2.13 mg / kg / day.

  Overall, GLP-1 / FDKP was well tolerated with no clinical signs and no effect on body weight, food consumption, clinical pathology parameters, macroscopic or microscopic evaluation. Also, GLP-1 / to cynomolgus monkeys at an estimated achieved dose of up to 2.13 mg / kg / day (corresponding to a dose of GLP-1 of 0.26 mg / kg / day) administered for 5 days for 30 minutes per day It is also observed that inhalation administration of FDKP was not associated with any dose limiting toxicity.

Example 15
Toxicology of GLP-1 / FDKP in rats administered by pulmonary inhalation for 14 days This study investigated the potential toxicity of GLP-1 / FDKP after daily administration by pulmonary inhalation for 14 consecutive days It has been evaluated. Rats received control (air), 10 mg / kg FDKP particles, 1 mg / kg GLP-1 / FDKP (0.15 mg GLP-1), 3 mg / kg GLP-1 / FDKP (0.45 mg GLP- Rats were administered 1) or 10 mg / kg GLP-1 / FDKP (1.5 mg GLP-1) as daily pulmonary inhalation for 14 consecutive days (n = 24 / sex / group). Animals were observed daily for clinical signs of toxicity and body weight and food intake were also recorded.

On Day 1 and 14, GLP-1 C _max was achieved within about 10-15 minutes after dose administration in all dose groups. The average maximum GLP-1 concentration at 10 mg / kg / day GLP-1 / FDKP is 6714 pg / mL and 6270 pg / mL for Day 1 and 27.9 pg / mL and 5834 pg / m for Day 14 in male and female, respectively. was. Plasma GLP-1 concentrations decreased with a clear elimination half-life of 0.7 to 4.4 hours. Mean AUC levels of GLP-1, in GLP-1 / FDKP for 10 mg / kg / day highest dose, male is 2187pM ^* h, female was 2703pM ^* h. GLP-1 showed minimal or no accumulation, and there was no gender difference in C _max , half-life and T _max . GLP-1 AUC values were slightly higher in female rats than in male rats across all doses. The NOAEL in rats administered GLP-1 / FDKP for 14 consecutive days by intrapulmonary inhalation was 10 mg / kg / day GLP-1 / FDKP (1.5 mg / kg / day GLP-1 )was.

  Approximately 24 hours after the last dose, animals (12 / sex / group) were sacrificed and clinical pathology, macroscopic and microscopic evaluations were performed. Toxic kinetics (TK) associated animals (12 / sex / group) were sacrificed on Day 14 of administration after final blood collection. There were no deaths and clinical findings associated with GLP-1 / DKP. There were no differences in body weight or food intake between control and treated animals. With only female 10 mg / kg GLP-1 / FDKP, the ratio of liver to liver weight and body weight was significantly lower than the control (air) group.

  There was no clear difference between rats receiving vehicle and rats receiving air control, as indicated by results for hematology, coagulation, chemistry, urinalysis, or urine chemistry . There were no gross or histopathological findings in the tissues measured to have potential toxicity due to administration of GLP-1 / FDKP.

Example 16
Toxicology of GLP-1 / FDKP administered for 28 days by pulmonary inhalation of monkeys This study evaluated the toxicity and toxicokinetics of GLP-1 / FDKP administered daily by inhalation for at least 4 weeks. is there. In order to assess the reversible, sustained or delayed onset of action, a 4 week recovery period was provided.

  The animals received one of the following doses: group 1: control (air), group 2: 3.67 mg / kg / day of FDKP particles, group 3: 0.3 mg / kg / day of GLP- 1 / FDKP (0.045 mg / kg / day GLP-1), group 4: 1 mg / kg / day GLP-1 / FDKP (0.15 mg / kg / day GLP-1), or group 5: 2 .6 mg / kg / day GLP-1 / FDKP (0.39 mg / kg / day GLP-1). Forty-two cynomolgus monkeys were divided into two groups: main study (n = 3 / sex / group) and recovery of groups 1, 2 and 5 (n = 2 / sex / group). Group 1: Air control, Group 2: FDKP (about 4 mg / kg / day), Group 3: 0.3 mg / kg / day GLP-1 / FDKP (low dose), Group 4:. 0 mg / kg / day GLP-1 / FDKP (medium dose), group 5: 2.6 mg / kg / day GLP-1 / FDKP (high dose). As usual, in the monkey study, only high doses and controls were evaluated during recovery.

  Animals were observed twice daily for mortality and morbidity, and at least once daily for abnormal and toxic signs 30 minutes after dosing. Body weight data was collected weekly and evaluated daily for qualitative feeding status. Blood was collected for toxicokinetics at Day 1, 28, 56. For three animals / sex / group, Day 29 was anesthetized, body weight was measured, whole blood was collected, and then necropsy was performed. For the remaining animals in Groups 1, 2, 5 (n = 2 / sex / group), Day 57 was anesthetized, weighed, whole blood was collected, and then necropsied. At necropsy, selected organs were weighed and selected tissues were collected and stored. All tissues from each animal were examined by microscopy.

  There were cases where body weight changes were observed across all groups, but there were no dose-related effects on body weight. In general, all animals maintained body weight throughout the study period or experienced a slight weight gain. The incidence and frequency of soft or liquid stool was found to be higher at higher doses. Significantly shown by clinical chemistry parameters considered relevant for administration, except for moderate increases in Day 29 (last day of administration) high dose female lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) There was no change (see Table 7). The concentration of LDH hardly increased in males. These changes disappeared by the end of the recovery period and did not correlate with any microscopic findings in the liver. The change in AST concentration in the high dose female group was primarily due to one of five animals.

At dose concentrations up to 2.6 mg / kg / day GLP-1 / FDKP, there was no evidence of any administration-related gross or histological changes. GLP-1 / FDKP has significant clinical signs, body weight, food intake, hematology, urine analysis, insulin analysis, ophthalmoscopic examination, ECG, GLP-1 / FDKP up to 2.6 mg / kg / day (0 It was well tolerated with no effects on gross or microscopic changes observed at a dose of .39 mg / kg / day GLP-1). Also, inhalation administration of FDKP at an estimated achieved dose of up to 3.67 mg / kg / day for 28 days for up to 30 minutes per day was not associated with any toxicity.

A dose related increase in C _max and AUC _last for GLP-1 and FDKP as a function of dose was observed in Day1 male and female monkeys. Over the considered dose _range, in the _{C max} of _AUC in _last without GLP-1, as increasing the dose of both male and female monkeys to Day 28, an increase in proportion to the dose was observed. The mean maximum GLP-1 blood concentration of 2.6 mg / kg / day GLP-1 / FDKP was 259 pg / mL for males and 164 pg / mL for females. Plasma GLP-1 concentrations decreased with elimination half-lives of 0.6 to 2.5 hours. For high doses, the mean AUC values for GLP-1, was 104pg ^* hr / mL at 103pg ^* hr / mL and female in male. At low doses, female monkeys showed higher values than male monkeys for AUC and _Cmax values. The mean maximum FDKP concentration at 2.6 mg / kg / day of GLP-1 / FDKP was 1800 ng / mL for males and 1900 pg / mL for females.

  In conclusion, GLP to cynomolgus monkeys with an estimated achieved dose of up to 2.6 mg / kg / day of GLP-1 / FDKP or 0.39 mg / kg / day of GLP-1 administered for 28 days up to 30 minutes per day The inhalation administration of -1 / FDKP was clinically well tolerated. The NOAEL was 2.6 mg / kg / day GLP-1 / FDKP (0.39 mg / kg / day GLP-1). As described in Example 19 below, the maximum human dose for Phase I studies is 1.5 mg GLP-1 / FDKP or about 0.021 mg / kg GLP-1 (70 Kg human per day). Suppose). Further study doses will be 3.0 mg GLP-1 / FDKP or about 0.042 mg / kg GLP-1 per day.

Example 17
Preparation of exendin / FDKP formulation Exendin-4 / FDKP was prepared by mixing an acidic exendin-4 peptide (SEQ ID NO: 3) solution with the FDKP particle suspension. In the acidic peptide solution, 10% (w / w) peptide was dissolved in 2% acetic acid. The FDKP suspension contained approximately 10% (w / w) FDKP particles. The acidic exendin-4 peptide solution was added to the FDKP particle suspension so as to mix gradually. The exendin-4 / FDKP mixture was slowly titrated to pH 4.50 with 25% ammonia solution. The mixture was then spheronized in liquid nitrogen and lyophilized.

  The percent respirable fraction based on filling (percent RF based on filling) for the 15% exendin-4 / FDKP powder was 36% and the cartridge discharge rate was 99%. A 15% GLP-1 / FDKP powder prepared at a similar ratio showed a percent RF based on a fill of 34% and a cartridge ejection rate of 100%.

Example 18
Pharmacokinetics of exendin / FDKP administered by pulmonary inhalation Pharmacokinetics and drugs of exendin-4 (GLP-1 analog) after multiple administrations via pulmonary route in various concentrations of exendin-4 / FDKP formulations Repeated dose preliminary toxicity studies are underway to investigate kinetic profiles.

  Rats and monkeys are tested for 28 days. Exendin / FDKP is administered daily by the inhalation route. In studies where animals are dosed for 28 days, some animals are sacrificed immediately after the regimen, while others have a recovery period of up to 1 month prior to sacrifice. All animals will be evaluated for clinical signs of toxicity, various physiological parameters including blood concentrations of exendin-4, glucose, insulin, organ weights, clinical pathology and histology of various organs.

  The first test group consists of 5 animals per group with 2 control groups, with air and exendin administered intravenously. There were 6 pulmonary inhalation groups, and an exendin / FDKP dose of approximately 2.0 mg was administered at 5%, 10%, 15%, 20%, 25% and 30% exendin load (w / w). Whole blood was collected for blood glucose and exendin concentrations until the 8th hour.

Data (C _max , T _1/2 , T _max ) are collected, thereby demonstrating that the exendin / FDKP formulation has comparable or better pharmacokinetics than GLP-1 / FDKP.

Example 19
GLP-1 / xDKP pharmacokinetics in rats administered by pulmonary inhalation . To determine whether different DKP can affect the pharmacokinetic profile of GLP-1 / FDKP formulations, various GLP-1 / xDKP formulations Is a US provisional patent application entitled “Asymmetrical FDKP Analogs for Use as Drug Delivery Agents” filed on the same date as this specification (incorporated herein in its entirety (US Attorney Docket No. 51300-00041). )).

  The study was performed on rats divided into 6 dose groups consisting of 5 animals per group. A control group (n = 3) was administered GLP-1 by liquid instillation. GLP-1 / FDKP (0.3 mg GLP-1) was administered by pulmonary inhalation and used as a second control. Each group of GLP-1 / xDKP administration groups was administered the GLP-1 / xDKP formulation by pulmonary inhalation at about 2.0 mg dose of xDKP loaded with 10% and 15% GLP-1. The xDKP used was ((E) -3- (4- (3,6-dioxopiperazin-2-yl) butylcarbamoyl) -acrylic acid), (3,6-bis (4-carboxypropyl) amidobutyl- 2,5-diketopiperazine) and ((E) -3,6-bis (4- (carboxy-2-propenyl) amidobutyl) -2,5-diketopiperazine disodium salt). Whole blood was collected for assessment of GLP-1 concentration at 5, 10, 20, 30, 45, 60 minutes and up to 90 minutes after administration.

Example 20
Phase 1a of GLP-1 / FDKP inhalation powder in healthy adult male subjects, single, open label, escalating dose, controlled safety and tolerability study GLP-1 was administered intravenously (iv) or subcutaneously ( sc) has been shown to control human hyperglycemia when administered by infusion or by multiple subcutaneous injections. Due to the extremely short half-life of the hormone, continuous subcutaneous injection or multiple daily subcutaneous injections are required. Neither of these routes is practical for long-term clinical use. Animal experiments have shown that therapeutic levels can be achieved when GLP-1 is administered by inhalation.

  Some of the effects of GLP-1 include decreased gastric emptying, increased satiety, inappropriate suppression of glucagon secretion, and are associated with a cluster of GLP-1 released at the start of the meal Seem. By supplementing this early surge in GLP-1 with GLP-1 / FDKP inhalation powder, a pharmacodynamic response in diabetic animals can be induced. Furthermore, the late surge in native GLP-1 associated with increased insulin secretion can be mimicked by postprandial administration of GLP-1 / FDKP inhalation powder.

  The Phase 1a clinical trial of GLP-1 / FDKP inhalation powder is designed to test the safety and tolerability of selected doses of the first novel inhaled glycemic control therapeutic product in human subjects. For administration, a previously tested MedTone® inhaler is used. The primary objective of this clinical trial is to provide a GLP-1 / FDKP inhalation powder by intrapulmonary inhalation that is safe, tolerable, and can be used for further clinical trials to establish evidence of efficacy and safety To identify a range of doses. The dose selected for the phase 1a clinical trial is based on the safety results in animals from the non-clinical trials of GLP-1 / FDKP inhalation powder described in the above examples in rats and primates.

  26 subjects were enrolled in 5 cohorts, of which up to 4 evaluable subjects in each group of cohorts 1 and 2 and up to 6 evaluable subjects in each group of cohorts 3-5 Subjects met eligibility criteria and completed clinical trials. Each subject received one dose of glucagon-like peptide 1 (GLP-1) as a GLP-1 / FDKP inhalation powder at the following dose concentrations: cohort 1: 0.05 mg, cohort 2: 0.45 mg, cohort 3: 0.75 mg, Cohort 4: 1.05 mg, and Cohort 5: 1.5 mg of GLP-1. The dropout was not replenished. For these doses, the body weight is assumed to be 70 kg. One of ordinary skill in the art can determine additional dosage concentrations based on the tests disclosed above.

  The purpose of this study is to determine the safety and tolerability of increasing doses of GLP-1 / FDKP inhalation powder in healthy adult male subjects. The tolerability of increasing doses of GLP-1 / FDKP inhalation powder as determined by monitoring pharmacological or adverse effects on the variables was assessed, including reported adverse events (AE), Examples include vital signs, physical examination, clinical examination, and electrocardiogram (ECG).

The second objective is to evaluate additional safety and pharmacokinetic parameters. These include additional safety parameters, including the incidence of lung and other AEs and changes in lung function between the first visit (screening) and the third visit (follow-up); AUC Plasma GLP-1 and serum fumaryl diketopiperazine (FDKP) following administration of GLP-1 / FDKP inhalation powder as measured by _{0-120 (min)} plasma GLP-1 and AUC _{0-480 min} serum FDKP Pharmacokinetic (PK) parameters are included, and additional PK parameters for plasma GLP-1 include t _max plasma GLP-1, C _max plasma GLP-1 and T _1/2 plasma GLP-1. Additional PK parameters for serum FDKP include T _max serum FDKP, C _max serum FDKP and T _1/2 serum FDKP.

Study endpoints are based on a comparison of the following pharmacological and safety parameters determined in the study population. The main evaluation items are as follows. Safety endpoints are assessed based on the reported incidence and severity of AEs, including cough and dyspnea, nausea and / or vomiting, and vital signs, clinical laboratory values from screening, A change in the physical examination value can be mentioned. The secondary endpoint, plasma GLP-1 and serum _{FDKP (AUC 0-120 (min)} plasma GLP-1 and _{AUC 0-480Min} serum FDKP of) PK predisposition; additional PK parameters of plasma GLP-1 ( T _max plasma GLP-1, C _max plasma GLP-1, T _1/2 plasma GLP-1); additional PK parameters of serum FDKP (T _max serum FDKP, C _max serum FDKP); additional safety parameters (pulmonary function test) (PFT)) and ECG.

  The Phase 1a single dose trial incorporates an open-label, incremental dose structure and design strategy, which includes 21 CFR 312 to determine the safety and tolerability of investigational drugs (IMPs) Good Clinical Practice: Conforms to guidance on Consolidated Guidance (ICH-E6) and Guidance on General Considerations for Clinical Trials (ICH-E8) ing.

  The clinical trial consists of 3 hospital visits: 1) one screening visit (first visit), 2) one administration visit (second visit), and 3) 8-14 days after Visit 2 1 follow-up visit (3rd visit). A single dose of GLP-1 / FDKP inhalation powder is administered at the second visit.

  This clinical trial will assess the safety parameters of each cohort. The cohort that will receive the next dose concentration will not be administered until all safety and tolerability data reviews for the first or previous dose have been performed by the investigator (PI). In order to ensure subject safety, a half hour dosing delay is provided between subjects in each cohort. Dosing can be discontinued when 3 or more subjects in the cohort develop severe nausea and / or vomiting or when the maximum dose is reached, or at the discretion of PI.

  Five doses of GLP-1 / FDKP inhalation powder (0.05, 0.45, 0.75, 1.05 and 1.5 mg GLP-1) are evaluated. To accommodate all doses, the prepared GLP-1 / FDKP is mixed with the FDKP inhalation powder. A single dose cartridge containing 10 mg dry powder consisting of GLP-1 / FDKP inhalation powder (15% by weight of GLP-1 / FDKP) as is or mixed with an appropriate amount of FDKP inhalation powder Used to obtain the desired dose of GLP-1 (0.05 mg, 0.45 mg, 0.75 mg, 1.05 mg, 1.5 mg). 1. The first two lowest dose concentrations are evaluated in 2 cohorts of 4 subjects each, and 3 high dose concentrations are evaluated in 3 cohorts of 6 subjects each. Each subject receives only one dose at one of the five dose concentrations to be evaluated. In addition to blood collection for GLP-1 (activity and total) and FDKP measurements, samples are taken for determination of glucagon, glucose, insulin and C-peptide.

  Numerous references to patents and printed publications are made throughout this specification. The above cited references and printed publications are hereby incorporated by reference in their entirety.

  Unless otherwise stated, all numbers representing the amount of ingredients, properties such as molecular weight, reaction conditions, etc. used in the specification and claims are modified in all cases by the term “about”. Understood. Accordingly, unless specified otherwise, the numerical parameters set forth in the following specification and appended claims are approximations that may vary depending on the desired properties of the soot obtained by the present invention. At a minimum, rather than trying to limit the application of the doctrine to the scope of the claims, each numerical parameter should at least take into account the number of significant figures reported and apply normal rounding techniques. Must be interpreted by. Although the numerical ranges and parameters describing the broad scope of the invention are approximate, the numerical values set forth in the examples are reported as accurately as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

  It will be readily apparent to those skilled in the art that various aspects and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention.

  As used herein, the use of the word “a” (“a” or “an”) is used in conjunction with the term “comprising” in the claims and / or specification. , May mean “one”, but it also matches the meaning of “one or more”, “at least one” and “one or more”.

  It is contemplated that any method or composition described herein can be implemented with respect to other methods or compositions described herein.

  The use of the term “or” in the claims is used to mean “and / or” unless the context clearly indicates that only the alternative is meant, or unless the alternatives contradict each other.

  Throughout this application, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being used to determine the value.

  Other objects, features and advantages of the present invention will be apparent from the above description and examples, and from the claims. However, since various modifications and changes within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description, the detailed description and specific examples, while indicating specific embodiments of the invention, are intended for purposes of illustration. It should be understood that it is provided as

References The following references are specifically incorporated herein by reference to the extent that they are illustrative procedures or other details supplements to those described herein.

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FIG. 1A: Far-UV circular dichroism (CD) of GLP-1 indicates that as the concentration increases, the secondary structure of the peptide changes from a largely unstructured conformation to a helical structure. Indicates. FIG. 1B: Near-UV CD shows that tertiary structure increases with increasing peptide concentration, suggesting that GLP-1 self-associates. FIG. 1C: Fluorescence emission of GLP-1 at various concentrations (pH 4, 20 ° C.) generated by tryptophan excitation at 280 nm. FIG. 1D: Transmission type FTIR of GLP-1 at various concentrations (pH 4, 20 ° C.). FIG. 2A: Far UV CD of 1.0 mg / mL GLP-1 shows that as the salt concentration increases, the disordered structure of GLP-1 changes to a more regular α-helix structure. FIG. 2B: Near UV CD of 1.0 mg / mL peptide shows that the tertiary structure of GLP-1 also increases with increasing NaCl concentration. FIG. 2C: Internal fluorescence emission of 1.0 mg / mL GLP-1 at various NaCl concentrations (pH 4, 20 ° C.) after tryptophan excitation at 280 nm. FIG. 2D: Tertiary structure analysis of 10 mg / mL GLP-1 at various ionic strengths (pH 4, 20 ° C.). FIG. 3A: Near-UV CD shows that GLP-1 oligomers separate with increasing temperature. FIG. 3B: Structural analysis of 10 mg / mL GLP-1 at various temperatures (pH 4). FIG. 3C: Structural analysis of 0.05 mg / mL GLP-1 at various temperatures (pH 4). FIG. 4A: Far UV CD of 10 mg / mL GLP-1 at various pH (20 ° C.). FIG. 4B: The increase in the spectrum at pH 7.6 reveals that the secondary structure of GLP-1 becomes irregular as a result of the decrease in concentration. FIG. Resistance of 1 mg / mL GLP-1 to amidolysis and oxidation as shown by HPLC. 6A-6B. Effect of stirring on the tertiary structure of 1.5 and 9.4 mg / mL GLP-1 (pH 4). Near-UV CD (FIG. 6A) and GLP-1 fluorescence emission (FIG. 6B) both indicate that the tertiary structure of the GLP-1 peptide does not change significantly due to agitation. Effect of 10 freeze-thaw cycles on the tertiary structure of GLP-1 (pH 4) at 1.6, 5.1 and 8.4 mg / mL. Near-UV CD (FIG. 7A) and GLP-1 fluorescence emission (FIG. 7B) both indicate that the tertiary structure of the peptide does not change significantly with multiple freeze-thaw cycles. A similar experiment was performed showing the effect of 11 freeze-thaw cycles on the secondary structure of 10 mg / mL GLP-1 (pH 4) by far UV CD (FIG. 7C). Salt test. Loading curve for GLP-1 / FDKP as a function of pH and NaCl concentration (FIG. 8A). FIG. 8B: Represents the amount of GLP-1 detected in a reconstituted FDKP-free control sample as a function of pH and NaCl concentration. Surfactant test. Loading curve for GLP-1 / FDKP as a function of pH and surfactant (FIG. 9A). FIG. 9B: Represents the amount of GLP-1 detected in a control sample without reconstituted FDKP as a function of pH and added surfactant. Ion test. Loading curve for GLP-1 / FDKP as a function of pH and ion. Loading was performed with 5 mg / mL FDKP and 0.25 mg / mL GLP-1 (FIGS. 10A and 10C). FIGS. 10B and 10D: Represents the amount of GLP-1 detected in a reconstituted, FDKP-free control sample as a function of pH, ion and 1M NaCl. Osmolite test. Loading curve for GLP-1 / FDKP as a function of pH in the presence of common stabilizers (osmolite; FIG. 11A). FIG. 11B: Represents the amount of GLP-1 detected in a reconstituted FDKP-free control sample as a function of pH and osmolyte. Chaotrope / liotrope test. Load curve for GLP-1 / FDKP as a function of chaotrope or lyotrope concentration at pH 3.0 (FIG. 12A) and pH 4.0 (FIG. 12C). 12B and 12D: Represents the amount of GLP-1 detected in a reconstituted FDKP-free control sample as a function of pH in the presence of various chaotropes or liotropes. Alcohol test. Loading curve for GLP-1 / FDKP as a function of pH and alcohol. Four alcohol concentrations (5% v / v, 10% v / v, 15% v / v and 20% v / v alcohol each) were evaluated (FIG. 13A). FIG. 13B: Represents the amount of GLP-1 detected for a reconstituted FDKP free control sample as a function of pH and alcohol (20%). FIG. 14A. Load from GLP-1 / FDKP concentration test. FIG. 14B. Scanning electron microscope (SEM) images (10000 × magnification) of multiple GLP-1 / FDKP formulations. FIG. Figure 6 represents the effect of stress on multiple GLP-1 / FDKP formulations. FIG. 16A. Represents the glycylated form (SEQ ID NO: 1) and amidated form (SEQ ID NO: 2) of GLP-1. FIG. 16B: Inhibition of DPPIV activity by aprotinin. FIG. 16C: Inhibition of DPPIV activity by DPPIV inhibitors. FIG. Detection of GLP-1 after incubation in lung lavage fluid. FIG. 6 represents quantification of GLP-1 in plasma. FIG. 18A shows quantification at a 1: 2 dilution of plasma. FIG. 18B shows quantification at a 1:10 dilution of plasma. FIG. 19A. Effect of GLP-1 and GLP-1 analogs on cell survival. Effect of GLP-1 on rat pancreatic epithelial (ARIP) cell death. FIG. 19B. Annexin V staining representing inhibition of apoptosis in the presence of GLP-1 and staurosporine (Stau) as a single agent and in combination. FIG. Effect of exendin-4, a GLP-1 analog, on cell viability. FIG. Effect of multiple GLP-1 / FDKP formulations on staurosporine-induced cell death. Pharmacokinetic study showing a single intravenous injection (IV; FIG. 22A) and pulmonary inhalation (IS; FIG. 22B) in rats using various concentrations of GLP-1 / FDKP formulation. Reduction in cumulative food intake of rats administered GLP-1 / FDKP formulation at 2 hours (FIG. 23A) and 6 hours (FIG. 23B) after administration. FIG. Pharmacodynamic study of GLP-1 / FDKP administered by intrapulmonary inhalation in male obese Zucker rats. FIG. Pharmacodynamic study of GLP-1 / FDKP administered by intrapulmonary inhalation in male obese Zucker rats. FIG. Pharmacodynamic study of GLP-1 / FDKP administered by intrapulmonary inhalation in male obese Zucker rats. FIG. Pharmacokinetic study of GLP-1 / FDKP at various GLP-1 concentrations administered by pulmonary inhalation in female rats. FIG. Pharmacokinetic study of GLP-1 / FDKP at various GLP-1 concentrations administered by pulmonary inhalation in female rats. Figure 29 in female rats (n = 10) administered GLP-1 / FDKP containing 15% GLP-1 (0.3 mg GLP-1) by pulmonary inhalation once a day for 4 consecutive days Pharmacodynamic test of GLP-1 / FDKP. Figure 30 in female rats (n = 10) administered GLP-1 / FDKP containing 15% GLP-1 (0.3 mg GLP-1) by pulmonary inhalation once daily for 4 consecutive days Pharmacodynamic test of GLP-1 / FDKP. Figure 31: GLP-1 / FDKP toxicokinetic study in monkeys administered GLP-1 / FDKP by nasal or oral administration once daily for 30 consecutive days (30 minutes per day).

Claims (32)

Including natural GLP- 1 adsorbed on the surface of diketopiperazine microparticles, wherein GLP- 1 is the only therapeutic agent, and the diketopiperazine has the formula: 2,5-diketo-3,6-di ( 4-X-aminobutyl) piperazine having a diketopiperazine, wherein X is selected from the group consisting of succinyl, glutaryl, maleyl and fumaryl.
An inhalable dry powder composition for use in the treatment of diabetes.   The dry powder composition of claim 1, wherein the diketopiperazine is 2,5-diketo-3,6-di (4-fumaryl-aminobutyl) piperazine. Providing native GLP- 1 ;
Step of mixing the GLP 1 Contact and said diketopiperazine in the form of and co-solution; particle forming diketopiperazine, step provides a diketopiperazine in a form selected from diketopiperazine microparticles and combinations thereof Including,
Particles containing natural GLP- 1 adsorbed on the surface of diketopiperazine microparticles are formed, said GLP- 1 being the only therapeutic agent, said diketopiperazine having the formula: 2,5-diketo-3, A diketopiperazine having 6-di (4-X-aminobutyl) piperazine, wherein X is selected from the group consisting of succinyl, glutaryl, maleyl and fumaryl;
A method of forming inhalable particles for use in the treatment of diabetes comprising natural GLP- 1 adsorbed on the surface of diketopiperazine microparticles. 4. The method of claim 3 , further comprising removing the solvent from the common solution by lyophilization, filtration or spray drying. The method of claim 4 , wherein the particles are formed prior to removing the solvent. The GLP 1 is provided in the form of a solution containing native GLP-1 at a concentration of 1 [mu] g / to 50 mg / mL, process of claim 3. The GLP 1 is provided in the form of a solution containing native GLP-1 at a concentration of 0.1 mg / to 10 mg / mL, process of claim 3. The GLP 1 is provided in the form of a solution containing native GLP-1 at a concentration of 0.25 mg / mL, process of claim 3. 4. The method of claim 3 , wherein the diketopiperazine is provided in the form of a suspension of diketopiperazine microparticles. 4. The method of claim 3 , wherein the diketopiperazine is provided in the form of a solution comprising particle-forming diketopiperazine and further comprises adjusting the pH of the solution to form diketopiperazine microparticles. Further comprising adding an agent to the solution or suspension, wherein the agent is sodium chloride, surfactant, hexylene glycol, trehalose, glycine, polyethylene glycol, trimethylamine N-oxide, mannitol, proline, chloride. The method according to claim 9 or 10 , which is cesium, sodium citrate, sodium sulfate, methanol, ethanol, trifluoroethanol or hexafluoroisopropanol. 12. The method of claim 11 , wherein the agent promotes association between the GLP- 1 and the diketopiperazine microparticles or the particle-forming diketopiperazine. 12. The method of claim 11 , wherein the agent improves the stability or efficacy of the GLP- 1 . The method of claim 11 , wherein the agent is sodium chloride. 11. A method according to claim 9 or claim 10 , further comprising adjusting the pH of the suspension or solution. The method according to claim 15 , wherein the pH is adjusted to 4 or more. The GLP 1 natural adsorbed on the surface of diketopiperazine microparticles have a greater stability, the method according to claim 3. 4. The method of claim 3 , wherein the common solution comprises natural GLP-1 at a concentration of 1 [mu] g / mL to 50 mg / mL. 4. The method of claim 3 , wherein the common solution comprises natural GLP-1 at a concentration of 0.1 mg / mL to 10 mg / mL. 4. The method of claim 3 , wherein the common solution comprises natural GLP-1 at a concentration of 0.25 mg / mL. Adding an agent to the common solution, the agent comprising sodium chloride, surfactant, hexylene glycol, trehalose, glycine, polyethylene glycol, trimethylamine N-oxide, mannitol, proline, cesium chloride, 4. A process according to claim 3 which is sodium citrate, sodium sulfate, methanol, ethanol, trifluoroethanol or hexafluoroisopropanol. 23. The method of claim 21 , wherein the agent promotes association between the GLP- 1 and the diketopiperazine microparticles or the particle-forming diketopiperazine. 24. The method of claim 21 , wherein the agent improves the stability or efficacy of the GLP- 1 . The method of claim 21 , wherein the agent is sodium chloride. The method of claim 3 , further comprising adjusting a pH of the common solution. 26. The method of claim 25 , wherein the pH is adjusted to 4 or higher. Comprising microparticles comprising natural GLP- 1 adsorbed on the surface of diketopiperazine microparticles, said GLP- 1 being the only therapeutic agent, said diketopiperazine having the formula: 2,5-diketo-3, A diketopiperazine having 6-di (4-X-aminobutyl) piperazine, wherein X is selected from the group consisting of succinyl, glutaryl, maleyl and fumaryl;
In the manufacture of a pharmaceutical composition for pulmonary administration to a subject for the treatment of diabetes natural GLP 1 in an effective amount, characterized in that, the use of natural GLP 1. Administration of the particles, results in improved native GLP-1 pharmacokinetic half-life and than pharmaceutical composition without diketopiperazine include native GLP 1 bioavailability claim 27 Use as described in. Providing native GLP- 1 ;
Providing the particle-forming diketopiperazine in the form of a solution;
Forming diketopiperazine microparticles;
Mixing said GLP- 1 with said solution to form a common solution; and microparticles comprising native GLP- 1 adsorbed on the surface of diketopiperazine microparticles, said powder in a powder composition GLP- 1 is the only therapeutic agent, and the diketopiperazine is a diketopiperazine having the formula 2,5-diketo-3,6-di (4-X-aminobutyl) piperazine, wherein X Removing the solvent from the common solution by spray drying to form a powder having an improved native GLP-1 pharmacokinetic profile selected from the group consisting of succinyl, glutaryl, maleyl and fumaryl. A method of forming an inhalable powder composition for use in the treatment of diabetes with improved native GLP-1 pharmacokinetic profile. 30. The method of claim 29 , wherein the improved natural GLP-1 pharmacokinetic profile comprises a higher natural GLP-1 half-life. 32. The method of claim 30 , wherein the higher natural GLP-1 half-life is 7.5 minutes or greater. GLP-1 pharmacokinetic profile of the improved natural comprises the bioavailability of GLP-1 improved natural than powder composition without diketopiperazine include natural GLP 1, wherein Item 30. The method according to Item 29 .