Classifications

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Definitions

• the present invention relates to the field of pharmaceutical formulations.
• the present invention discloses dry powder formulations comprising diketopiperazine (DKP) particles in combination with glucagon- like peptide 1 (GLP-I).
• DKP diketopiperazine
• GLP-I glucagon- like peptide 1
• the present invention has utility as a pharmaceutical formulation for treating diseases such as diabetes, cancers, and obesity but is not limited to such diseases. More particularly, the present invention has utility as a pharmaceutical formulation for pulmonary delivery.
• Glucagon-like peptide 1 as disclosed in the literature is a 30 or 31 amino acid incretin, released from the intestinal endocrine L-cells in response to fat, carbohydrate ingestion, and protein from a meal. Secretion of this peptide hormone is found to be impaired in individuals with type 2 diabetes mellitus making it a potential candidate for the treatment of this and other related diseases.
• GLP-I is secreted from the intestinal L-cell in response to orally ingested nutrients, (particularly sugars), stimulating meal-induced insulin release from the pancreas, inhibiting glucagon release from the liver, as well as other effects on the gastrointestinal tract, and brain.
• GLP-I effect in the pancreas is glucose dependent, minimizing the risk of hypoglycemia during exogenous peptide administration.
• GLP-I also promotes all steps in insulin biosynthesis and directly stimulates ⁇ -cell growth and survival as well as ⁇ -cell differentiation. The combination of these effects results in increased ⁇ -cell mass.
• GLP-I receptor signaling results in a reduction of ⁇ -cell apoptosis, which further contributes to increased ⁇ -cell mass.
• GLP-I In the gastrointestinal tract, GLP-I inhibits GI motility, increases the secretion of insulin in response to glucose, and decreases the secretion of glucagon, thereby contributing to a reduction of glucose excursion.
• Central administration of GLP-I has been shown to inhibit food intake in rodents, suggesting that peripherally released GLP-I may directly affect the brain. This is feasible since it has been shown that circulating GLP-I can access GLP-I receptors in certain brain areas; namely the subfornical organ and the area postrema. These areas of the brain are known to be involved in the regulation of appetite and energy homeostasis.
• GLP-I 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 given as a continuous intravenous infusion (Nauck et al, 1993).
• infusion of GLP-I has been shown to lower glucose levels in patients previously treated with non-insulin oral medication and in patients requiring insulin therapy after failure on sulfonylurea therapy (Nauck et al, 1993).
• the effects of a single subcutaneous injection of GLP-I provided disappointing results, as is noted in the art and discussed herein below.
• GLP-I analogs e.g., Liraglutide (Novo Nordisk, Copenhagen, Denmark)); exenatide (exendin-4; Byetta®) (Amylin Inc., San Diego, CA) and (exenatide-LAR, Eli Lilly, Indianapolis, IN)) that are resistant to degradation, called “incretin mimetics,” have been investigated in clinical trials.
• Dipeptidyl peptidase IV inhibitors e.g., Vildagliptin (Galvus) developed by Novartis, Basel, Switzerland) and Januvia (sitagliptin) developed by Merck, Whitehouse Station, New Jersey
• the multiple modes of action of GLP-I e.g., increased insulin release, delayed gastric emptying, and increased satiety
• hypoglycemia e.g., increased insulin release, delayed gastric emptying, and increased satiety
• GLP-I Stable, inhalable glucagon-like peptide 1
• DKP diketopiperazine
• the dry powder composition of the present invention comprises a GLP-I molecule selected from the group consisting of a native GLP-I, a GLP-I metabolite, a GLP-I analog, a GLP-I derivative, a dipeptidyl-peptidase-IV (DPP-IV) protected GLP-I, a GLP-I mimetic, an exendin, a GLP-I peptide analog, or a biosynthetic GLP-I analog.
• a GLP-I molecule selected from the group consisting of a native GLP-I, a GLP-I metabolite, a GLP-I analog, a GLP-I derivative, a dipeptidyl-peptidase-IV (DPP-IV) protected GLP-I, a GLP-I mimetic, an exendin, a GLP-I peptide analog, or a biosynthetic GLP-I analog.
• the dry powder composition comprises 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.
• the dry powder composition comprises a diketopiperazine salt.
• the diketopiperazine is 2,5-diketo-3,6-di(4-fumaryl- aminobutyl)piperazine.
• the present invention further contemplates a dry powder composition wherein the
• GLP-I molecule is native GLP-I, or an amidated GLP-I molecule wherein the amidated GLP-I molecule is GLP-I (7-36) amide.
• a process for preparing a particle comprising a GLP-I molecule and a diketopiperazine comprising the steps of: providing a GLP-I solution comprising a GLP-I molecule; providing a solution of a particle-forming diketopiperazine or a suspension of particles of a diketopiperazine; and combining the GLP-I solution with the diketopiperazine solution or suspension.
• the process for preparing a particle comprising a GLP-I molecule and a diketopiperazine further comprises removing solvent from the solution or suspension by lyophilization, filtration, or spray drying.
• the particle of the invention is formed by removing solvent or is formed prior to removing solvent.
• GLP-I molecule and a diketopiperazine there is provided a GLP-I molecule selected from the group consisting of a native GLP-I, a GLP-I analog, a GLP-I derivative, a dipeptidyl-peptidase- IV (DPP-IV) protected GLP-I, a GLP-I mimetic, an exendin, a GLP-I peptide analog, or a biosynthetic GLP-I analog.
• the process for preparing a particle having a GLP-I molecule and a diketopiperazine comprises a diketopiperazine provided as a suspension of particles.
• the diketopiperazine is provided in solution and the process includes adjusting the pH of the solution to precipitate the diketopiperazine and form particles.
• the GLP-I solution is at a concentration of about 1 ⁇ g/ml-50 mg/ml, more preferably about O.lmg/ml-10 mg/ml. In yet another particular embodiment of the invention, the GLP-I solution is at a concentration of about 0.25 mg/ml.
• the process further comprises adding an agent to the solution, wherein the agent is selected from salts, surfactants, ions, osmolytes, chaotropes and lyotropes, acid, base, and organic solvents.
• the agent promotes association between the GLP-I and the diketopiperazine particle and also improves the stability and/or pharmacodynamics of the GLP-I molecule.
• 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, and Brij, H(CH 2 ) V SO 4 Na.
• the agent may be an ion, for example, a cation or anion.
• the agent may be an osmolyte (stabilizer), such as, but not limited to Hexylene-Glycol (Hex-Gly), trehalose, glycine, polyethylene glycol (PEG), trimethylamine n-oxide (TMAO), mannitol, and proline.
• stabilizer such as, but not limited to Hexylene-Glycol (Hex-Gly), trehalose, glycine, polyethylene glycol (PEG), trimethylamine n-oxide (TMAO), mannitol, and proline.
• the agent may be a chaotrope or lyotrope, such as, but not limited to, cesium chloride, sodium citrate, and sodium sulfate.
• the agent may be an organic solvent for example, an alcohol selected from methanol (MeOH), ethanol (EtOH), trifluoroethanol (TFE), and hexafluoroisopropanol (HFIP).
• a process for preparing a particle comprising a GLP-I molecule and a diketopiperazine, wherein the process comprises adjusting the pH of the particle suspension to about 4 or greater.
• the process for preparing a particle comprises a GLP-I molecule and a diketopiperazine, wherein the GLP-I molecule in the particle has greater stability.
• a method of administering an effective amount of a GLP-I molecule to a subject in need thereof comprising providing to the subject a GLP-1/diketopiperazine particle.
• the method of administering may be intravenously, subcutaneously, orally, nasally, buccally, rectally, or by pulmonary delivery but is not limited to such. In one embodiment, the method of administering is by pulmonary delivery.
• the method of administering comprises treating a condition or disease selected from the group consisting of diabetes, ischemia, reperfused tissue injury, dyslipidemia, diabetic cardiomyopathy, myocardial infarction, acute coronary syndrome, obesity, catabolic changes after surgery, hyperglycemia, irritable bowel syndrome, stroke, neurodegenerative disorders, memory and learning disorders, islet cell transplant and regenerative therapy.
• a condition or disease selected from the group consisting of diabetes, ischemia, reperfused tissue injury, dyslipidemia, diabetic cardiomyopathy, myocardial infarction, acute coronary syndrome, obesity, catabolic changes after surgery, hyperglycemia, irritable bowel syndrome, stroke, neurodegenerative disorders, memory and learning disorders, islet cell transplant and regenerative therapy.
• 1/diketopiperazine particle composition results in improved pharmacokinetic half- life and bioavailability of GLP- 1.
• a method of preparing a dry powder composition with an improved pharmacokinetic profile comprising the steps of: providing a solution of a GLP-I molecule; providing a particle- forming diketopiperazine; forming particles; and combining the GLP-I and the diketopiperazine; and thereafter removing solvent by a method of drying to obtain a dry powder, wherein the dry powder has improved pharmacokinetic profile.
• the improved pharmacokinetic profile comprises increased half- life of GLP-I and/or improved bioavailability of GLP-I.
• the increased half- life of GLP-I is greater than or equal to 7.5 minutes.
• a dry powder composition comprising a GLP-I molecule and a diketopiperazine or a pharmaceutically acceptable salt thereof.
• the GLP-I molecule is selected from the group consisting of native GLP-Is, GLP-I metabolites, GLP-I analogs, GLP-I derivatives, dipeptidyl-peptidase-IV (DPP-IV) protected GLP-Is, GLP-I mimetics, GLP-I peptide analogs, or biosynthetic GLP-I analogs.
• 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.
• the diketopiperazine is a diketopiperazine salt.
• the diketopiperazine is 2,5-diketo-3,6-di(4-fumaryl-aminobutyl)piperazine.
• the GLP-I molecule is native GLP-I.
• the GLP-I molecule is an amidated GLP-I molecule.
• the amidated GLP-I molecule is GLP-l(7-36) amide.
• a process for forming a particle comprising a GLP-I molecule and a diketopiperazine comprising the steps of: providing a GLP-I molecule; providing a diketopiperazine in a form selected from particle-forming diketopiperazine, diketopiperazine particles, and combinations thereof; and combining the GLP- 1 molecule and the diketopiperazine in the form of a co-solution, wherein the particle comprising the GLP-I molecule and the diketopiperazine is formed.
• the process further comprises removing a solvent from said co-solution by lyophilization, filtration, or spray drying.
• the particle comprising said GLP-I molecule and the diketopiperazine is formed by removing the solvent.
• the particle comprising the GLP-I molecule and the diketopiperazine is formed prior to removing the solvent.
• the GLP-I molecule is selected from the group consisting of a native GLP-I, a GLP-I analog, a GLP-I derivative, a dipeptidyl-peptidase-IV (DPP-IV) protected GLP-I, a GLP-I mimetic, a GLP-I peptide analog, or a biosynthetic GLP-I analog.
• the GLP-I molecule is provided in the form of a solution comprising a GLP-I concentration of about l ⁇ g/ml -50 mg/ml.
• the GLP-I molecule is provided in the form of a solution comprising a GLP-I concentration of about 0.1 mg/ml - 10 mg/ml. In another embodiment, the GLP-I molecule is provided in the form of a solution comprising a GLP-I concentration of about 0.25 mg/ml.
• the diketopiperazine is provided in the form of a suspension of diketopiperazine particles.
• the diketopiperazine is provided in the form of a solution comprising particle-forming diketopiperazine, the process further comprising adjusting the pH of the solution to form diketopiperazine particles.
• the process further comprises adding an agent to said solution or suspension, wherein the agent is selected from the group consisting of salts, surfactants, ions, osmolytes, chaotropes and lyotropes, acids, bases, and organic solvents.
• the agent promotes association between the GLP-I molecule and the diketopiperazine particles or the particle-forming diketopiperazine. In another embodiment, the agent improves the stability or pharmacodynamics of the GLP-I molecule. In another embodiment, the agent is sodium chloride.
• the process further comprises adjusting the pH of the suspension or solution.
• the pH is adjusted to about 4.0 or greater.
• the GLP-I molecule in the particle has greater stability than native GLP-I .
• the co-solution comprises a GLP-I concentration of about 1 ⁇ g/ml-50 mg/ml. In another embodiment, the co-solution comprises a GLP-I concentration of about 0.1 mg/ml- 10 mg/ml. In another embodiment, the co-solution comprises a GLP-I concentration of about 0.25 mg/ml.
• the process further comprises adding an agent to the co-solution, wherein the agent is selected from the group consisting of salts, surfactants, ions, osmolytes, chaotropes and lyotropes, acids, bases, and organic solvents.
• the agent promotes association between the GLP-I molecule and the diketopiperazine particles or the particle-forming diketopiperazine.
• the agent improves the stability or pharmacodynamics of the GLP-I molecule.
• the agent is sodium chloride.
• the process further comprises adjusting the pH of the co- solution.
• the pH is adjusted to about 4.0 or greater.
• a method is provided of administering an effective amount of a GLP-I molecule to a subject in need thereof the method comprising providing to the subject a particle comprising GLP-I and diketopiperazine.
• the providing is carried out intravenously, subcutaneously, orally, nasally, buccally, rectally, or by pulmonary delivery.
• the providing is carried out by pulmonary delivery.
• the need comprises the treatment of a condition or disease selected from the group consisting of diabetes, ischemia, reperfused tissue injury, dyslipidemia, diabetic cardiomyopathy, myocardial infarction, acute coronary syndrome, obesity, catabolic changes after surgery, hyperglycemia, irritable bowel syndrome, stroke, neurodegenerative disorders, memory and learning disorders, islet cell transplant and regenerative therapy.
• a condition or disease selected from the group consisting of diabetes, ischemia, reperfused tissue injury, dyslipidemia, diabetic cardiomyopathy, myocardial infarction, acute coronary syndrome, obesity, catabolic changes after surgery, hyperglycemia, irritable bowel syndrome, stroke, neurodegenerative disorders, memory and learning disorders, islet cell transplant and regenerative therapy.
• the provision of the particle results in improved pharmacokinetic half-life and bioavailability of GLP-I as compared to native GLP-I.
• a method is provided of forming a powder composition with an improved GLP-I pharmacokinetic profile, comprising the steps of: providing a GLP-I molecule; providing a particle-forming diketopiperazine in a solution; forming diketopiperazine particles; combining the GLP-I molecule and the solution to form a co-solution; and, removing solvent from the co-solution by spray-drying to form a powder with an improved GLP-I pharmacokinetic profile.
• the improved GLP-I pharmacokinetic profile comprises an increased GLP-I half- life. In another embodiment, the increased GLP-I half-life is greater than or equal to 7.5 minutes. In another embodiment, the improved GLP-I pharmacokinetic profile comprises improved bioavailability of GLP-I as compared to native GLP-I.
• FIGs. 1A-1D Structural analysis of GLP-I at various concentrations (pH 4, 20 0 C).
• FIG. IA The far-UV circular dichroism (CD) of GLP-I illustrates that as the concentration increases, the secondary structure of the peptide is transformed from a predominantly unstructured conformation to a helical conformation.
• FIG. IB The near-UV CD illustrates that the tertiary structure increases with increasing concentration of peptide suggesting that GLP-I self-associates.
• FIG. 1C Fluorescence emission of GLP-I at various concentrations (pH 4, 20 0 C) resulting from tryptophan excitation at 280 nm.
• FIG. ID Transmission FTIR of GLP-I at various concentrations (pH 4, 20 0 C). The amide I band at 1656 cm "1 indicates that GLP-I has a ⁇ -helical structure at concentrations > 2 mg/mL.
• FIGs. 2A-2D Structural analysis of low concentration GLP-I at varying ionic strength (pH 4, 20 0 C).
• FIG. 2A The far-UV CD of 1.0 mg/mL GLP-I illustrates that increasing the concentration of salt converts the unordered structure of GLP-I into more ordered ⁇ -helical structures.
• FIG. 2B The near-UV CD of 1.0 mg/mL peptide demonstrates that increasing the NaCl concentration also enhances the tertiary structure of GLP-I.
• FIG. 2C Intrinsic fluorescence emission of 1.0 mg/mL GLP-I at varying NaCl concentrations (pH 4, 20 0 C) following tryptophan excitation at 280 nm. At high peptide concentrations, the maxima decreases in intensity and shifts to a lower wavelength, which is indicative of a well-defined tertiary structure.
• FIG. 2D Tertiary structural analysis of 10 mg/mL GLP-I at varying ionic strength (pH 4, 20 0 C). The near-UV CD spectra demonstrate that increased ionic strength enhances the tertiary structure of self-associated GLP-I.
• FIGs. 3A-3B Structural analysis of 10 mg/mL GLP-I at various temperatures (pH
• FIG. 3A The near-UV CD illustrates that GLP-I oligomers dissociate with increasing temperature.
• FIG. 3B Structural analysis of 10 mg/mL GLP-I at various temperatures (pH 4).
• FIG. 3C Structural analysis of 0.05 mg/mL GLP-I at various temperatures (pH 4).
• the far-UV CD illustrates that the peptide is insensitive to temperature.
• FIGs. 4A-4B Structural analysis of GLP-I at varying pH (20 0 C).
• FIG. 4A The far-UV CD of 10 mg/mL GLP-I at varying pH (20 0 C). As the pH is increased, self-associated GLP-I precipitates between pH 6.3 and 7.6 but retains a helical structure at pH 1.5 and 11.7.
• FIG. 4B Enlarging the spectrum at pH 7.6 reveals that the secondary structure of GLP-I is unordered as a result of the concentration decrease.
• FIG. 5 Resistance of 1 mg/mL GLP-I to both deamidation and oxidation as demonstrated by HPLC. Deamidation conditions were achieved by incubating GLP-I at pH 10.5 for 5 days at 40° C. Oxidative conditions were achieved by incubating GLP-I in 0.1 % H 2 O 2 for 2 hours at room temperature.
• FIGs. 6A-6B The effect of agitation on the tertiary structure of 1.5 and 9.4 mg/mL
• GLP-I (pH 4).
• Samples were agitated for both 30 and 90 min at room temperature and the fluorescence emission spectra were collected after tryptophan excitation at 280 nm.
• FIGs. 7A-7C The effect of 10 freeze-thaw cycles on the tertiary structure of 1.6
• FIGs. 8A-8B Salt Studies. Loading curves for GLP-I /FDKP as a function of pH and NaCl concentration (FIG. 8A). Loading was performed at 5 mg/mL FDKP and 0.25 mg/mL GLP-I. NaCl concentrations are expressed as mM.
• FIG. 8B - Depicts the amount of GLP-I detected in the reconstituted FDKP-free control samples as a function of pH and NaCl concentration.
• FIGs. 9A-9B Surfactant Studies. Loading curves for GLP-I /FDKP as a function of pH and surfactant (FIG. 9A). Loading was performed at 5 mg/mL FDKP and 0.25 mg/mL GLP-I.
• FIG. 9B - Depicts the amount of GLP-I detected in the reconstituted FDKP-free control samples as a function of pH and surfactant added.
• FIGs. 1 OA-I OD Ion Studies. Loading curves for GLP-I /FDKP as a function of pH and ions. Loading was performed at 5 mg/mL FDKP and 0.25 mg/mL GLP-I (FIGs. 1OA and HC). Ion concentrations are indicated in the legend (mM). Right-hand curves depicts the results for IM NaCl.
• FIGs. 1OB and 1OD - Depict the amount of GLP-I detected in the reconstituted FDKP-free control samples as a function of pH, ions and IM NaCl.
• FIGs. 11-1 IB Osmolyte Studies. Loading curves for GLP-1/FDKP as a function of pH and in the presence of common stabilizers (osmolytes; FIG. HA). Loading was performed at 5 mg/mL FDKP and 0.25 mg/mL GLP-I.
• FIG. HB - Depicts the amount of GLP- 1 detected in the reconstituted FDKP-free control samples as a function of pH and osmolyte. "N/ A" indicates no osmolyte was present in the sample.
• FIGs. 12A-12B Chaotrope/lyotrope Studies.
• FIGs. 12B and 12D Depict the amount of GLP-I detected in the reconstituted FDKP-free control samples as a function of pH in the presence of the various chaotropes or lyotropes. "N/A" indicates no chaotropes or lyotropes were present in the sample.
• FIG. 13B - Depicts the amount of GLP-I detected for reconstituted FDKP-free control samples as a function of pH and alcohol (20%).
• FIGs. 14A-14B Loading from GLP-1/FDKP concentration studies (FIG. 14A).
• FIG. 14B Scanning Electron Microscopy (SEM) images of multiple GLP-1/FDKP formulations (at 1000Ox magnification) depicts clusters of spherical and rod-like GLP-1/FDKP particle formulations.
• FIG. 15. Depicts the effect of stress on multiple GLP-1/FDKP formulations.
• the legend indicates the mass-to-mass percentage of GLP-I to FDKP particles and the other components that were present in solution, prior to lyophilization. The samples were incubated for 10 days at 4O 0 C.
• FIGs. 16A-16C Structure of GLP-I.
• FIG. 16A Depicts the glycine-extended form of GLP-I (SEQ ID NO. 1) and the amidated form (SEQ ID NO. 2).
• FIG. 16B Inhibition of DPPIV activity by aprotinin.
• FIG. 16C Inhibition of DPPIV activity by DPPIV inhibitor.
• FIG. 17 Detection of GLP-I after incubation in lung lavage fluid.
• FIGs. 18A-18B Depicts the quantitation of GLP-I in plasma.
• FIG. 18A shows quantitation in 1 :2 dilution of plasma.
• FIG. 18B shows quantitation in 1 :10 dilution of plasma.
• FIGs. 19A-19B Effect of GLP-I and GLP-I analogs on cell survival. Effect of
• GLP-I on rat pancreatic epithelial (ARIP) cell death FIG. 19A.
• Annexin V staining depicting inhibition of apoptosis in the presence of GLP-I and staurosporine (Stau) as single agents and in combination (FIG. 19B).
• the concentration of GLP-I is 15nM and the concentration of stauropsorine is 1 ⁇ M
• FIG. 20 Effect of the GLP-I analog exendin-4 on cell viability.
• ARIP cells were treated with 0, 10, 20 and 40 nM exendin 4 for 16, 24 and 48 hours.
• FIG. 21 The effect of the multiple GLP-1/FDKP formulations on staurosporine- induced cell death.
• ARIP cells pre-treated with GLP-I samples were exposed to 5 ⁇ M staurosporine for 4 hours and were analyzed with Cell Titer-GloTM to determine cell viability. Samples were stressed at 4° and 40 0 C for 4 weeks.
• Control samples shown on the right (Media, GLP-I, STAU, GLP+STAU), illustrate the viability of cells in media (without GLP-I or stauroporine), with GLP-I, with stauropsorine and with GLP-I and staurosporine (note: the graph legend does not apply to the control samples). All of the results shown are averages of triplicate runs.
• FIGs. 22A-22B Pharmacokinetic studies depicting single intravenous injection
• FIGs. 23A-23B Decrease in the cumulative food consumption in rats dosed with
• FIG. 24 Pharmacodynamic study of GLP-1/FDKP administered via pulmonary insufflation in male obese Zucker rats.
• the data depicts the glucose measurements at 0, 15, 30, 45, 60 and 90 minutes for the control (air; group 1) and the GLP-1/FDKP treated (group 2).
• FIG. 25 Pharmacodynamic study of GLP-1/FDKP administered via pulmonary insufflation in male obese Zucker rats. The data depicts the GLP-I measurements at 0, 15, 30, 45, 60 and 90 minutes for the control (air; group 1) and the GLP-1/FDKP treated (group 2).
• FIG. 26 Pharmacodynamic study of GLP-1/FDKP administered via pulmonary insufflation in male obese Zucker rats. The data depicts the insulin measurements at 0, 15, 30, 45, 60 and 90 minutes for the control (air; group 1) and the GLP-1/FDKP treated (group 2).
• FIG. 27 Pharmacokinetic study of GLP-1/FDKP with various GLP-I concentrations administered via pulmonary insufflation in female rats.
• the data depicts the GLP-I measurements at 0, 2, 5, 10, 20, 30, 40 and 60 minutes for the control (air; group 1) and GLP-1/FDKP treated groups 2, 3 and 4 administered 5%, 10% and 15% GLP-I respectively.
• FIG. 28 Pharmacokinetic study of GLP-1/FDKP with various GLP-I concentrations administered via pulmonary insufflation in female rats.
• the data depicts the FDKP measurements at 0, 2, 5, 10, 20, 30, 40 and 60 minutes for the control (air; group 1) and GLP-1/FDKP treated groups 2, 3 and 4 administered 5%, 10% and 15% GLP-I respectively.
• FIG. 29 Pharmacodynamic study of GLP-1/FDKP in female rats administered
• the data depicts average food consumption measured at predose, 1, 2, 4 and 6 hours post dose for 4 consecutive days.
• FIG. 30 Pharmacodynamic study of GLP-1/FDKP in female rats administered
• the data depicts average body weight measured at predose, 1, 2, 4 and 6 hours post dose for 4 consecutive days.
• FIG. 31 Toxicokinetic study of GLP-1/FDKP in monkeys administered GLP-
• GLP-I Stable, inhalable glucagon-like peptide 1
• Stable, inhalable glucagon-like peptide 1 (GLP-I) formulations for use as pharmaceutics are deficient in the art. This is due to the instability of GLP-I peptide in vivo. GLP-I compounds tend to remain in solution under a number of conditions, and have a relatively short in vivo half-life when administered as a solution formulation. Further, dipeptidyl-peptidase IV (DPP-IV,) which is found to be present in various biological fluids such as the lung and blood, greatly reduces the biological half-life of GLP-I molecules. For example, the biological half-life of GLP-l(7-37) has been shown to be 3 to 5 minutes; see U.S. Patent No.
• GLP-I has also been shown to undergo rapid absorption in vivo following parenteral administration.
• amide GLP- 1(7-36) has a half- life of about 50 minutes when administered subcutaneously; see also U. S. Patent No. 5,118,666.
• the rapid clearance and short half-life of GLP-I compositions in the art present a deficiency that the current invention overcomes.
• the present invention overcomes the deficiencies in the art by providing an optimized native GLP-I /FDKP (fumaryl diketopiperazine) formulation especially suited for pulmonary delivery.
• the present invention provides formulations of a native GLP-I molecule that can elicit a GLP-I response in vivo. Use of variants of native GLP-I in such formulations is also contemplated.
• the present invention provides formulations of GLP-I in combination with diketopiperazine (DKP) particles.
• DKP diketopiperazine
• the GLP-I /DKP formulations are provided for administration to a subject.
• the GLP-1/DKP formulations comprise fumaryl diketopiperazine (FDKP), but are not limited to such, and can include other DKPs (asymmetrical DKPs, xDKPs) such as 2,5-diketo-3,6-di(4-succinyl-aminobutyl)piperazine (SDKP), asymmetrical diketopiperazines including ones substituted at only one position on the DKP ring (for example "one armed" analogs of FDKP), and DKP salts.
• FDKP fumaryl diketopiperazine
• other DKPs asymmetrical DKPs, xDKPs
• SDKP 2,5-diketo-3,6-di(4-succinyl-aminobutyl)piperazine
• SDKP 2,5-diketo-3,6-di(4-succinyl-aminobutyl)piperazine
• Adsorption of GLP-I to FDKP particles was also observed under a variety of conditions which included variation in pH, GLP-I concentration, and in the concentration of various surfactants, salts, ions, chaotropes and lyotropes, stabilizers, and alcohols.
• the absorption of GLP-I to FDKP particles was found to be affected strongly by pH, specifically, binding occurred at about pH 4.0 or greater. Other excipients were found to have a limited effect on the absorption of GLP-I to FDKP particles.
• GLP-I /DKP formulations of the present invention a number of parameters that would affect or impact its deliverability and absorption in vivo were evaluated. Such parameters included, for example, the structure of the GLP-I peptide, the surface charges on the molecule under certain formulation conditions, solubility and stability as a formulation, as well as susceptibility to serine protease degradation and in vivo stability; all of which play a critical role in generating a formulation that can be readily absorbed which exhibits an extended biological half-life.
• GLP-1/FDKP formulations obtained was tested under a variety of conditions both in vitro and in vivo.
• the stability of GLP-I was analyzed by HPLC analysis and cell-based assays.
• stability of GLP-I was examined in lung lavage fluid (which contains DPP-IV). It was also found that the stability of native GLP-I was concentration dependent in solution.
• the present invention provides optimized formulations comprising native human GLP-I combined with fumaryl diketopiperazine (FDKP) that are stable and resistant to degradation.
• FDKP fumaryl diketopiperazine
• GLP-I native human glucagon- like peptide 1
• FDKP fumaryl diketopiperazine
• GLP-I Human GLP-I is well known in the art and originates from the preproglucagon polypeptide synthesized in the L-cells in the distal ileum, in the pancreas and in the brain.
• GLP- 1 is a 30-31 amino acid peptide that exists in two molecular forms, 7-36 and 7-37, with the 7-36 form being dominant. Processing of preproglucagon to GLP- 1(7-36) amide and GLP- 1(7-37) extended form occurs mainly in the L-cells. It has been shown in the art that, in the fasted state, plasma levels of GLP-I are about 40 pg/ml. After a meal, GLP-I plasma levels rapidly increase to about 50-165 pg/ml.
• GLP-I molecules refers to GLP-I proteins, peptides, polypeptides, analogs, mimetics, derivatives, iso forms, fragments and the like. Such GLP-I molecules may include naturally occurring GLP-I polypeptides (GLP-1(7-37)OH, GLP-1(7- 3O)NH 2 ) and GLP-I metabolites such as GLP-l(9-37).
• GLP-I molecules include: a native GLP-I, a GLP-I analog, a GLP-I derivative, a dipeptidyl-peptidase-IV (DPP-IV) protected GLP-I, a GLP-I mimetic, a GLP-I peptide analog, or a biosynthetic GLP-I analog.
• DPP-IV dipeptidyl-peptidase-IV
• an "analog” includes compounds having structural similarity to another compound.
• the anti-viral compound acyclovir is a nucleoside analogue and is structurally similar to the nucleoside guanosine which is derived from the base guanine.
• acyclovir mimics guanosine (is biologically analogous with) and interferes with DNA synthesis by replacing (or competing with) guanosine residues in the viral nucleic acid and prevents translation/transcription.
• compounds having structural similarity to another (a parent compound) that mimic the biological or chemical activity of the parent compound are analogs.
• Analogs can be, and often are, derivatives of the parent compound (see “derivative" infra). Analogs of the compounds disclosed herein may have equal, lesser or greater activity than their parent compounds.
• a “derivative” is a compound made from (or derived from), either naturally or synthetically, a parent compound.
• a derivative may be an analog (see “analog” supra) and thus may possess similar chemical or biological activity. However, unlike an analog, a derivative does not necessarily have to mimic the biological or chemical activity of the parent compound.
• the antiviral compound ganclovir is a derivative of acyclovir
• ganclovir has a different spectrum of anti-viral activity and different toxicological properties than acyclovir.
• Derivatives of the compounds disclosed herein may have equal, less, greater or even no similar activity when compared to their parent compounds.
• a "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. A primary metabolite is directly involved in normal growth, development, and reproduction of living organisms. A secondary metabolite is not directly involved in those processes, but typically has important ecological function (e.g., an antibiotic).
• biosynthetic refers to any production of a chemical compound by a living organism.
• particle-forming refers to chemical, biosynthetic, or biological entities or compounds that are capable of forming solid particles, usually in a liquid medium.
• the formation of particles typically occurs when a particle-forming entity is exposed to a certain condition(s) such as, for example, changes in pH, temperature, moisture, and/or osmolarity/osmolality. Exposure to the condition(s) may result in, for example, binding, coalescence, solidification and/or dehydration such that a particle is formed.
• a precipitation reaction is one example of a particle-forming event.
• co-solution is any medium comprised of at least two chemical, biological and/or biosynthetic entities.
• a co-solution may be formed by combining a liquid comprising at least one chemical, biological and/or biosynthetic entity with a solid comprising a chemical, biological and/or biosynthetic entity.
• a co-solution may be formed by combining a liquid comprising at least one chemical, biological and/or biosynthetic entity with another liquid comprising a chemical, biological and/or biosynthetic entity.
• a co-solution may be formed by adding at least two solids, each comprising at least one chemical, biological and/or biosynthetic entity, into a single solution.
• Native GLP-I is a polypeptide having the amino acid sequence of SEQ ID NO. 1 or SEQ ID NO. 2. Native GLP-I peptide undergoes rapid cleavage and inactivation within minutes in vivo.
• GLP-I analogs of the present invention may include the exendins, which are peptides found to be GLP-I receptor agonists; such analogs may further include exendins 1 to 4. Exendins are found in the venom of the Gila-monster and share about 53% amino acid homology with mammalian GLP-I. Exendins also have similar binding affinity for the GLP-I receptor.
• Exendin-3 and exendin-4 were reported to stimulate cAMP production in, and amylase release from, pancreatic acinar cells (Malhotra et al, 1992; Raufman et al, 1992; Singh et al, 1994).
• the use of exendin-3 and exendin-4 as insulinotrophic agents for the treatment of diabetes mellitus and the prevention of hyperglycemia has been proposed (U.S. Patent No. 5,424,286).
• exendin[9-39] Carboxyl terminal fragments of exendin such as exendin[9-39], a carboxyamidated molecule, and fragments 3-39 through 9-39 have been reported to be potent and selective antagonists of GLP-I (Goke et al, 1993; Raufman et al, 1991; Schepp et al, 1994; Montrose- Raf ⁇ zadeh et al, 1996).
• the literature has also demonstrated that exendin[9-39] blocks endogenous GLP-I in vivo, resulting in reduced insulin secretion (Wang et al, 1995; D'Alessio et al, 1996).
• Exendin-4 potently binds to GLP-I receptors on insulin-secreting ⁇ -TCl cells, to dispersed acinar cells from pancreas, and to parietal cells from stomach. Exendin-4 peptide also plays a role in stimulating somatostatin release and inhibiting gastrin release in isolated stomachs (Goke et al, 1993; Schepp et al, 1994; Eissele et al, 1994).
• exendin-4 is reportedly an agonist, i.e., it increases cAMP, while exendin[9-39] is identified as an antagonist, i.e., it blocks the stimulatory actions of exendin-4 and GLP-I. exendin has also been found to be resistant to degradation.
• Peptide mimetics are peptides that biologically mimic active determinants on hormones, cytokines, enzyme substrates, viruses or other bio- molecules, and may antagonize, stimulate, or otherwise modulate the physiological activity of the natural ligands. Peptide mimetics are especially 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 GLP-I molecules of the invention will have at least one biological activity of native GLP-I such as the ability to bind to the GLP-I receptor and initiate a signal transduction pathway resulting in insulinotropic activity.
• a GLP-I molecule may be a peptide, polypeptide, protein, analog, mimetic, derivative, isoform, fragment and the like, that retains at least one biological activity of a naturally-occurring GLP-I.
• GLP-I molecules may also include the pharmaceutically acceptable salts and prodrugs, and salts of the prodrugs, polymorphs, hydrates, solvates, biologically-active fragments, biologically active variants and stereoisomers of the naturally-occurring human GLP-I as well as agonist, mimetic, and antagonist variants of the naturally-occurring human GLP-I, the family of exendins including exendins 1 through 4, and polypeptide fusions thereof.
• a GLP-I molecule of the invention may also include a dipeptidyl- peptidase-IV (DPP-IV) protected GLP-I that prevents or inhibits the degradation of GLP-I.
• DPP-IV dipeptidyl- peptidase-IV
• GLP-I molecules of the present invention include peptides, polypeptides, proteins and derivatives thereof that contain amino acid substitutions, improve solubility, confer resistance to oxidation, increase biological potency, or increase half-life in circulation.
• GLP-I molecules as contemplated in the present invention comprise amino acid substitutions, deletions or additions wherein the amino acid is selected from those as are well known in the art.
• the N- or C- termini of the molecule may also be modified such as by acylation, acetylation, amidation, but is not limited to such.
• amino acid refers to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids.
• Naturally encoded amino acids are the 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) and pyrolysine and selenocysteine.
• Amino acid analog refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an ⁇ carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, such as, homoserine, norleucine, norvaline, methionine sulfoxide, methionine methyl sulfonium, citrulline, hydroxyl glutamic acid, hydroxyproline, and praline.
• Such analogs have modified R groups (such as norleucine), but retain the same basic chemical structure as a naturally occurring amino acid.
• Amino acids contemplated in the present invention also include ⁇ -amino acids which are similar to ⁇ -amino acids in that they contain an amino terminus and a carboxyl terminus. However, in ⁇ -amino acids two carbon atoms separate these functional termini, ⁇ -amino acids, with a specific side chain, can exist as the R or S isomers at either the alpha (C2) carbon or the beta (C3) carbon. This results in a total of four possible diastereoisomers for any given side chain.
• GLP-I molecules of the present invention may also include hybrid GLP-I proteins, fusion proteins, oligomers and multimers, homologues, glycosylation pattern variants, and muteins thereof, wherein the GL-P-I molecule retains at least one biological activity of the native molecule, and further regardless of the method of synthesis or manufacture thereof including, but not limited to, recombinant (whether produced from cDNA, genomic DNA, synthetic DNA or other form of nucleic acid), synthetic, and gene activation methods.
• Recombinant DNA technology is well known to those of ordinary skill in the art (see Russell, D. W., et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 2001.
• Diketopiperazines are well known in the art for their ability to form microparticles that are useful for drug delivery and stabilization.
• diketopiperazines are employed to facilitate the absorption of GLP-I molecules thereby providing a stable formulation that is resistant to degradation.
• diketopiperazines can be formed into particles that incorporate GLP-I molecules, or particles onto which GLP-I molecules can be adsorbed. This may involve mixing of the diketopiperazine solutions with solutions or suspensions of GLP-I molecules followed by precipitation and subsequent formation of particles comprising diketopiperazine and GLP-I. Alternatively, the diketopiperazine can be precipitated to form particles and subsequently mixed with a solution of GLP-I molecules. Association between the diketopiperazine particle and the GLP-I molecule can be driven by solvent removal or a specific step, such as a pH adjustment, can be included prior to drying in order to promote the association.
• diketopiperazines of the present invention include but are not limited 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 (which may also be referred to as fumaryl diketopiperazine or FDKP).
• diketopiperazines contemplated in the present invention include, without limitation, derivatives of 3,6-di(4-aminobutyl)-2,5-diketopiperazine such as: 3,6-di(succinyl-4- aminobutyl)-2,5-diketopiperazine (also referred to herein as 3,6-bis(4- carboxypropyl)amidobutyl-2,5-diketopiperazine; succinyl diketopiperazine or SDKP); 3,6- di(maleyl-4-aminobutyl)-2, 5 -diketopiperazine; 3,6-di(citraconyl-4-aminobutyl)-2-5- diketopiperazine; 3,6-di(glutaryl-4-aminobutyl)-2,5-diketopiperazine; 3,6-di(malonyl-4- aminobutyl)-2,5-diketopiperazine;
• the present invention contemplates the use of diketopiperazine salts.
• Such salts may include, for example, any pharmaceutically acceptable salt such as the Na, K, Li, Mg, Ca, ammonium, or mono-, di- or tri-alkylammonium (as derived from triethylamine, butylamine, diethanolamine, triethanolamine, or pyridines, and the like) salts of diketopiperazine.
• the salt may be a mono-, di-, or mixed salt. Higher order salts are also contemplated for diketopiperazines in which the R groups contain more than one acid group.
• a basic form of the agent may be mixed with the diketopiperazine in order to form a drug salt of the diketopiperazine, such that the drug is the counter cation of the diketopiperazine.
• a salt as contemplated herein includes in a non-limiting manner FDKP diNa.
• Drug delivery using DKP salts is taught in U.S. Patent Application No: 11/210,710, incorporated herein by reference for all it contains regarding DKP salts.
• the present invention also employs novel asymmetrical analogs of FDKP, xDKPs such as: (£)-3-(4-(3,6-dioxopiperazin-2- yl)butylcarbamoyl)-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)acrylic acid and disclosed in U.S. Provisional Patent Application entitled "Asymmetrical FDKP Analogs for Use as Drug Delivery Agents" filed on even date herewith and incorporated herein in its entirety (Atty Docket No. 51300-00041)
• Diketopiperazines can be formed by cyclodimerization of amino acid ester derivatives, as described by Katchalski, et ah, (J. Amer. Chem. Soc. 68:879-80; 1946), by cyclization of dipeptide ester derivatives, or by thermal dehydration of amino acid derivatives in high-boiling solvents, as described by Kopple, et al., (J. Org. Chem. 33:862-64;1968), the teachings of which are incorporated herein.
• the loaded diketopiperazine particles of the present invention are dried by a method of spraying drying as disclosed in, for example, U.S. Patent Application Serial No. 11/678,046 filed on February 22, 2006 and entitled "A Method For Improving the Pharmaceutic Properties of Microparticles Comprising Diketopiperazine and an Active Agent.”
• U.S. Patent Application Serial No. 11/678,046 filed on February 22, 2006 and entitled "A Method For Improving the Pharmaceutic Properties of Microparticles Comprising Diketopiperazine and an Active Agent.”
• the present invention further provides a GLP-I /FDKP formulation for administration to a subject in need of treatment.
• a subject as contemplated in the present invention may be a household pet or human.
• the treatment is for Type II diabetes, obesity, cancer or any related diseases and/or conditions therefrom. Humans are particularly preferred subjects.
• diseases or conditions contemplated in the present invention include, but are not limited to, irritable bowel syndrome, myocardial infarction, ischemia, reperfused tissue injury, dyslipidemia, diabetic cardiomyopathy, acute coronary syndrome, metabolic syndrome, catabolic changes after surgery, neurodegenerative disorders, memory and learning disorders, islet cell transplant and regenerative therapy or stroke.
• Other diseases and/or conditions contemplated in the present invention are inclusive of any disease and/or condition related to those listed above that may be treated by administering a GLP-I /FDKP dry powder formulation to a subject in need thereof.
• the GLP-I /FDKP dry powder formulation of the present invention may also be employed in the treatment of induction of beta cell differentiation in human cells of type-II diabetes and hyperglycemia.
• the subject may be a household pet or animal, including rats, rabbits, hamsters, guinea pigs, gerbils, woodchucks, cats, dogs, sheep, goats, pigs, cows, horses, monkeys and apes (including chimpanzees, gibbons, and baboons).
• the GLP-I /FDKP particle formulations of the invention can be administered by various routes of administration known to persons of ordinary skill in the art and for clinical or non-clinical purposes.
• the GLP-1/FDKP compositions of the invention may be administered to any targeted biological membrane, preferably a mucosal membrane of a subject. Administration can be by any route, including but not limited to oral, nasal, buccal, systemic intravenous injection, subcutaneous, regional administration via blood or lymph supply, directly to an affected site or even by topical means.
• administration of GLP-1/FDKP composition is by pulmonary delivery.
• Other alternative routes of administration may include: intradermal, intraarterial, intraperitoneal, intralesional, intracranial, intraarticular, intraprostatic, intrapleural, intratracheal, intravitreal, intravaginal, rectal, intratumoral, intramuscular, intravesicular, mucosally, intrapericardial, bronchial administration local, using aerosol, injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the foregoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 1990, incorporated herein by reference for all it contains regarding methods of administration).
• the GLP-1/DKP particles of the present invention can be delivered by inhalation to specific areas of the respiratory system, depending on the particle size. Additionally, the 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 a suspension, tablets or capsules.
• the GLP-1/DKP composition may be delivered from an inhalation device, such as a nebulizer, a metered-dose inhaler, a dry powder inhaler, and a sprayer.
• an "effective amount” of a GLP-1/DKP formulation to a patient in need thereof is contemplated.
• An "effective amount" of a GLP- 1/DKP dry powder formulation as contemplated in the present invention refers to that amount of the GLP-I compound, analog or peptide mimetic or the like, which will relieve to some extent one or more of the symptoms of the disease, condition or disorder being treated.
• an "effective amount" of a GLP-1/DKP dry powder formulation would be that amount of the GLP-I molecule for treating diabetes by increasing plasma insulin levels, reducing or lowering fasting blood glucose levels, and increasing pancreatic beta cell mass by at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50%, or greater, but not limited to such.
• the present invention contemplates treating obesity by administering to a subject in need of such treatment a pharmaceutically effective amount of the GLP-I molecule.
• an "effective amount" of a GLP-1/DKP dry powder formulation would be that amount of the GLP-I molecule for treating obesity by reducing or lowering body weight by at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50%, or greater, but is not limited to such.
• the present invention also contemplates administering an "effective amount" of a GLP-1/DKP dry powder formulation for controlling satiety, by administering to a subject in need of such treatment a pharmaceutically effective amount of the GLP-I molecule.
• the GLP-I molecule can be an exendin molecule such as exendin-1 or -4.
• an "effective amount" of a GLP-1/DKP dry powder formulation would be that amount of the GLP-I molecule that reduces the perception of hunger and food intake (as measured by mass or caloric content, for example) by 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, 50%, or greater, but not limited to such.
• An "effective amount” of a GLP-1/DKP dry powder formulation may be further defined as that amount sufficient to detectably and repeatedly ameliorate, reduce, minimize or limit the extent of the disease or condition or symptoms thereof. Elimination, eradication or cure of the disease or condition may also be possible utilizing an "effective amount" of the inventive formulation..
• the actual dosage amount of the composition can be determined on the basis of physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and the route of administration. A skilled artisan would be able to determine actual dosages 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 the disease or condition to be treated. Administration of the GLP- 1/DKP formulation can be provided to the subject at intervals ranging over minutes, hours, days, weeks or months. In some instances, timing of the therapeutic regimen may be related to the half- life of the GLP-I molecule upon administration. In further embodiments, in treating particular or complex diseases or conditions such as cancer, for example, it may be desirable to administer a GLP-1/DKP formulation of the present invention with a pharmaceutical excipient or agent. In such cases, an administration regimen may be dictated by the pharmaceutical excipient or agent.
• GLP-I To analyze both the structure and behavior of GLP-I a number of biophysical and analytical techniques were employed. These techniques included far-ultraviolet circular dichroism (far-UV CD), near-ultraviolet circular dichroism (near-UV CD), intrinsic fluorescence, fourier transform infrared spectroscopy (FTIR), high pressure liquid chromatography (HPLC), and mass spectroscopy (MS); all of which are well know to one of ordinary skill in the art. A wide range of conditions were employed to investigate the effects of concentration, ionic strength, temperature, pH, oxidative stress, agitation, and multiple freeze- thaw cycles on the GLP-I peptide; all of which are described in further detail below. These analyses were also employed to characterize the major routes of degradation and to establish conditions that manipulate peptide structure of GLP-I in order to achieve certain GLP-I /DKP formulations.
• far-UV CD far-ultraviolet circular dichroism
• near-UV CD near-ultraviol
• GLP-I was purchased either from American Peptide (Sunnyvale, CA) or AnaSpec (San Jose, CA), or prepared in house (MannKind Corporation, Valencia, CA). Aqueous GLP-I samples, of varying concentration, were analyzed at pH 4.0 and 2O 0 C (unless otherwise noted). Samples were generally prepared fresh and were mixed with the appropriate additive (e.g., salt, pH buffer, H 2 O 2 etc., if any), prior to each experiment. Secondary structural measurements of GLP-I under various conditions were collected with far-UV CD and transmission fourier transform infrared spectroscopy (FTIR). In addition, both near-UV CD and intrinsic fluorescence were employed to analyze the tertiary structure of GLP-I by monitoring the environments surrounding its aromatic residues, namely tryptophan.
• FTIR transmission fourier transform infrared spectroscopy
• Circular dichroism (CD) spectra was used to analyze the ⁇ -helix, random coil, ⁇ - pleated sheet, ⁇ -turns and random coil that may be displayed by a molecule such as a protein or peptide.
• a molecule such as a protein or peptide.
• far-UV CD was used to determine the type of secondary structure, for example pure ⁇ -helix, ⁇ -sheet, etc., in proteins and peptides.
• near-UV CD was used to analyze the tertiary structures of a molecule.
• far- and near- UV CD techniques were employed.
• GLP-I forms two distinct structures which include ⁇ -helices and random coils, over a wide range of concentrations (for example: 1.8, 4.2, 5.1, 6.1, 7.2 and 8.6 mg/mL).
• concentrations for example: 1.8, 4.2, 5.1, 6.1, 7.2 and 8.6 mg/mL.
• GLP-I is primarily unstructured, as determined by the large single minima at 205 nm.
• the peptide adopts an ⁇ -helical structure as determined by the two minima at 208 nm and 224 nm (FIG. IA).
• FIG. 2 A (far-UV-CD) illustrates that increasing the concentration of salt (from 100 mM to 1000 mM) converts the unordered structure of GLP-I into a ⁇ -helical conformation, as revealed by the minimas at 208 and 224 nm.
• IM Upon raising the NaCl concentration to IM, much of the peptide (at 1.0 mg/mL) precipitates out of solution (FIG. 2A). Nevertheless, this type of precipitate was shown to dissolve upon dilution with water, thus establishing that at high ionic strength GLP-I can be reversibly precipitated.
• FIG. 3 A near-UV-CD
• FIG. 3B and 3C far-UV-CD
• the far-UV CD illustrates that the peptide is insensitive to temperature. Therefore, increased molecular motion significantly hinders self-association of GLP-I.
• GLP-I (1 mg/mL) at pH 10.5, was incubated for 5 days at 4O 0 C following which reverse-phase HPLC and electrospray mass spectrometry (MS) were performed for deamidation and oxidation analyses. Oxidation studies were also conducted on GLP-I samples (1 mg/mL) incubated for 2 hours in the presence of 0.1% H 2 O 2 using both HPLC and MS.
• FIG. 5 depicts the stability of GLP-I under conditions of deamidation and oxidation.
• the HPLC chromatograms illustrate that GLP-I elutes at the same retention time and that no degradation peaks result for the destabilizing conditions analyzed. Additionally, MS analyses yielded a similar mass for all the samples, 3297 g/mol, indicating that the mass is unaltered. The data also illustrates that the peptide remains pure and intact when incubated under various conditions. Thus, deamidation of GLP-I was not observed. GLP-I was also shown to be stable to oxidative stress as observed in the presence of 0.1% H 2 O 2 , where the purity and mass of GLP-I remained intact, as determined by HPLC and MS respectively. Overall, there were no changes in the retention times or the mass values and no degradation peaks resulted, thereby demonstrating that GLP-I peptide is resistance to both deamidation and oxidation.
• FDKP DKP suspension particles
• FDKP particle suspension in which the FDKP particles are pre-formed, was combined with 3X pH buffer and 3X solution of an additive or excipient.
• the final solution contained a FDKP concentration of 5,mg/ml and a GLP-I concentration of 0.25,mg/ml (5% w/w). Unbound GLP-I in the supernatant was filtered off the suspension.
• the FDKP particles with the associated GLP-I protein were dissolved (reconstituted) with 100,mM ammonium bicarbonate and filtered to separate out any aggregated GLP-I protein.
• the amount of GLP-I in both the supernatant and reconstituted fractions was quantitated by HPLC.
• a series of experiments were conducted in which conditions employed included use of additives such as salts, surfactants, ions, osmolytes, chaotropes, organics, and various concentrations of GLP-I . The results from these studies are described below.
• the optimal binding (adsorption) of GLP-I to FDKP particles was strongly influenced by the pH of the suspension. At a pH of 4 and above, about 3.2% to about 4% binding of GLP-I to FDKP particles was observed where the GLP-1/FDKP ratio in solution was 5% w/w. Essentially no adsorption of GLP-I to FDKP particles was evident at pH 2.0 in the presence of 0 and 25 mM NaCl, but some apparent loading was observed with increased ionic strength. GLP-I precipitation was observed in the FDKP-free controls with >1M NaCl FIG. 8B.
• Surfactants employed in this study included: Brij 78 at 0.09 mM, Tween 80 at 0.01 mM, Triton X at 0.2 mM, Pluronic F68 at 0.12 mM, H(CH 2 ) 7 SO 4 Na at 0.9 mM, CHAPS at 0.9 mM, Cetrimide at 0.9 mM. Loading curves for GLP-I in the presence of each surfactant are shown are for GLP-1/FDKP as a function of pH.
• FIG. HA shows the loading curves for GLP- 1/FDKP as a function of pH in the presence of common stabilizers (osmolytes). Loading of the GLP-1/FDKP particles was performed as described for the previous experiment. Similarly, the pH was controlled as described supra. The samples were prepared at pH 3.0 and in the presence of 20, 50, 100, 150, 200 or 300 mM of an osmolyte (stabilizer).
• the osmolytes were Hexylene- Glycol (Hex-Gly), trehalose, glycine, PEG, TMAO, mannitol or proline; N/A indicates no osmolyte.
• concentration of the osmolyte (stabilizer) in the samples was held constant at 100 mM and the pH varied from 2.0 to 4.0.
• chaotropes and lyotropes Ionic species that affect the structure of water and proteins (chaotropes and lyotropes) were studied to determine the role that these factors play in GLP-I adsorption to FDKP. Loading of the GLP-I /FDKP particles was performed as described for the previous experiments. Similarly, the pH was controlled as described supra. The samples were prepared at pH 3.0 and in the presence of 0, 20, 50, 100, 150, 200 or 300 mM of the following chaotropes or lyotropes: NaSCN, CsCl, Na 2 SO 4 , (CH 3 ) 3 N-HC1, Na 2 NO 3 , Na Citrate, and NaClO 4 . In a similar experiment, the concentration of the chaotrope or lyotrope in the samples was held constant at 10OmM and the pH varied from 2.0 to 4.0.
• FIG. 12A shows the loading curves for GLP-1/FDKP as a function of pH and chaotrope and/or lyotrope.
• ⁇ 3 the loading curves for GLP-1/FDKP as a function of pH and chaotrope and/or lyotrope.
• FIG. 13A shows the loading curves for GLP-1/FDKP as a function of pH for each alcohol at each concentration.
• pH 3.0 low concentration of HFIP (5%) results in a high adsorption, as demonstrated by the mass ratio of GLP-I to FDKP particles.
• Only the strongest H-bond strengthening (helix-forming) alcohol, HFIP had an effect on adsorption in the buffered suspensions.
• HFIP H-bond strengthening (helix-forming) alcohol
• FIG. 13B shows that at 20% alcohol concentration, no significant precipitation of GLP-I was noted in the reconstituted FDKP-free control samples.
• FIG. 14A shows loading curves from GLP- 1/FDKP as a function of GLP-I concentration at various pHs. GLP-I concentrations were at 0.15, 0.25, 0.4, 0.5, 0.75, 1.0, 1.5, 2.0, 5.0, or 10 mg/mL. The pH of the samples was at 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0.
• GLP-1/FDKP particles are present as crystalline or plate like structures which can form aggregates comprising of more than one GLP-1/FDKP particles (FIG. 14B).
• GLP-I itself influenced the results of these experiments.
• the behavior of GLP-I was found to be atypical and surprising in that there was no saturation of adsorption observed, which was attributed to GLP-I self-association at high concentrations.
• the self-association of GLP-I at high concentration allows for the possible coating of DKP particles with multiple layers of the GLP-I peptide thereby promoting higher percent load of the GLP-I peptide.
• This surprising self-association quality proves to be beneficial in preparing stable GLP-I administration forms.
• the self-associated conformation of GLP-I may be able to lessen or delay its degradation in blood. However, it is noted that care must be taken when working with associated GLP-I since it is sensitive to temperature and high pH.
• GLP-1/FDKP Formulations Based on the results from the experiments in Examples 1 and 2, a series of GLP-I formulations having the characteristics described in Table 1 were selected for the cell viability assay as discussed herein. Most of the formulations contained GRAS ("generally recognized as safe") excipients, but some were selected to allow the relationship between stability and adsorption to be studied.
• GRAS generally recognized as safe
• GLP-1/FDKP formulations chosen for phase II integrity.
• the formulation made from 10 mg/ml GLP-I in 2OmM NaCl and pH 4.0 buffer is desribed as the salt-associated formulation.
• DPP-IV dipeptidyl-peptidase IV
• Dipeptidyl-peptidase IV is an extracellular membrane-bound serine protease, expressed on the surface of several cell types, in particular CD4 + T-cells. DPP-IV is also found is blood and lung fluids. DPP-IV has been implicated in the control of glucose metabolism because its substrates include the insulinotropic hormone GLP-I which is inactivated by removal of its two N-terminal amino acids; see FIG. 16A. DPP-IV cleaves the Ala-Glu bond of the major circulating form of human GLP-I (GLP-I (7-36)) releasing the N- terminal two residues. DPP-IV exerts a negative regulation of glucose disposal by degrading GLP-I thus lowering the incretin effect on ⁇ cells of the pancreas.
• aprotinin or DPP-IV inhibitor Studies were conducted to determine inhibition of GLP-I degradation in rat blood and lung fluid in the presence of aprotinin or DPP-IV inhibitor.
• Aprotinin a naturally occurring serine protease inhibitor, which is known in the art to inhibit protein degradation was added to the samples post collection at 1, 2, 3, 4 and 5 TIU/ml.
• DPP-IV activity was then measured by detecting the cleavage of a luminescent substrate containing the DPP-IV recognized Gly-Pro sequence. Bronchial lung lavage fluid was incubated with proluminescent substrate for 30 min and cleavage product was detected by luminescence.
• GLP-I The stability of GLP-I was also examined in lung lavage fluid using a capture ELISA mAb that recognizes GLP-I amino acids 7-9.
• GLP-I was incubated in lung lavage fluid (LLF) for 2, 5, 20 and 30 mins. The incubation conditions were: 1 or 10 ⁇ g (w/w) of LLF and 1 or 10 ⁇ g (w/w) GLP-I as depicted in FIG. 17. No GLP-I was detected in LLF alone. With the combination of LLF and GLP-I at various concentrations there was a high detection of GLP-I comparable to that of GLP-I alone, indicating that GLP-I is stable, over time, in lung lavage fluid (FIG. 17). Stability of GLP-I in undiluted lung lavage fluid was confirmed in similar studies; at 20 minutes 70-72% GLP-I integrity was noted (data not shown).
• Rat pancreatic epithelial (ARIP) cells (used as a pancreatic ⁇ -cell model; purchased from ATCC, Manassas, VA) were pretreated with GLP-I at 0, 2, 5, 10, 15 or 20 nM concentration for 10 minutes. The cells were then left untreated or were treated with 5 ⁇ M staurosporine (an apoptosis inducer) for 4.5 hours. Cell viability was evaluated using Cell Titer-GloTM (Promega, Madison, WI). A decrease in the percent of cell death was noted with an increase in GLP-I concentration of up to 10 nM in the staurosporine treated cells (FIG. 19A).
• Annexin V staining is a useful tool in detecting apoptotic cells and is well known to those of skill in the art. Binding of Annexin V to the cell membrane, allows for the analysis of changes in phospholipids (PS) asymmetry before morphological changes associated with apoptosis occurred and before membrane integrity is lost.
• PS phospholipids
• GLP-1/FDKP formulations (as disclosed in Example 3, Table 1 above), to inhibit cell death.
• These GLP- 1/FDKP particle formulations were either in a suspension or lyophilized. The formulations were analyzed for their ability to inhibit staurosporine-induced cell death in ARIP cells.
• ARIP cells pre-treated with GLP-I samples were exposed to 5 ⁇ M staurosporine for 4 hours and were analyzed with Cell Titer-GloTM (Promega, Madison, WI) to determine cell viability.
• GLP-1/FDKP plasma concentrations of GLP- 1 were evaluated in female Sprague Dawley rats administered with various formulations of GLP- 1/FDKP via intravenous injections or pulmonary insufflation.
• GLP-I at approximately 4% and 16% (w/w) of the GLP-1/FDKP particle formulations was used.
• Rats were randomized into 12 groups with groups 1, 4, 7 and 10 receiving GLP-I solution administered via pulmonary liquid instillation or IV injection.
• Groups 2, 5, 8, and 11 received GLP-1/FDKP salt-associated formulation (as disclosed in Table 2), administered via pulmonary insufflation or IV injection.
• Groups 3, 6, 9, 12 received the GLP-1/FDKP salt-associated blended formulation administered via pulmonary insufflation or IV injection.
• the GLP-1/DKP formulation was a salt-associated formulation at approximately 16% load. To achieve an approximate 4% load, the 16% formulation was blended with DKP particles in a 3:1 mixture. Pulmonary insufflation or intravenous injection was at 0.5 or 2.0 mg of particles (16% or 4% GLP-I load, respectively) for a total GLP-I dose of 0.08 mg.
• Groups 7-12 administration was repeated on Day 2.
• Groups 1, 4, 7, and 10 were administered 80 ⁇ g of a GLP-I solution.
• Groups 2, 5, 8, and 11 were administered a GLP-1/DKP salt-associated formulation (-16% GLP-I load).
• Groups 3, 6, 9, 12 received the GLP-1/DKP salt-associated blended formulation (-4% GLP-I load).
• Groups 5, 6, 10, 11 and 12 received various GLP- 1/FDKP formulations and GLP-I solution intravenously (IV); (FIG. 22A).
• Groups 5 and 6 were administered 15.8% GLP-1/FDKP and groups 11 and 12 were administered another dose of 15.8% GLP-1/FDKP on a consecutive day; group 10 was administered GLP-I solution as a control.
• the concentration of GLP-1/FDKP was detected at time points of 0, 2, 5, 10, 20, 40, 60, 80, 100, and 120 mins. All groups showed a detectable increase in GLP-I plasma levels after intravenous administration, with maximal concentrations observed at 2 minutes post treatment. Plasma levels of active GLP-I returned to background levels by 20 minutes post treatment for all groups.
• Groups 1, 2, 3, 7, 8 and 9 12 received various GLP-1/FDKP formulations or GLP-I solution by pulmonary insufflation (FIG. 22B).
• Group 1 was administered 80 ⁇ g of a GLP-I control by pulmonary liquid instillation (LIS);
• group 2 was administered 15.8% GLP-1/FDKP by pulmonary insufflation (IS);
• group 3 was administered 3.8% GLP-1/FDKP by pulmonary insufflation (IS);
• group 7 was administered 80 ⁇ g of a GLP-I control by pulmonary liquid instillation (LIS);
• group 8 was administered 15.8% GLP-1/FDKP by pulmonary insufflation (IS); and
• group 9 was administered 3.8% GLP-1/FDKP by pulmonary insufflation (IS).
• the concentration of GLP-1/FDKP was measured at time points of 0, 2, 5, 10, 20, 40, 60, 80, 100, and 120 mins. [00181] All groups showed a detectable increase in plasma GLP-I concentration following pulmonary administration. Maximum plasma concentration of GLP-I varied with the formulation/composition used. Groups 2 and 8 showed maximal plasma levels of GLP-I at 10-20 minutes post treatment as indicated by the AUC, while groups 3 and 9 showed significant levels of active GLP-I at 5-10 minutes, and groups 1 and 7 showed a more rapid and transient increase in plasma levels of active GLP-I. Plasma levels of active GLP-I returned to background levels by 60 minutes post treatment in groups 2, 3, 7 and 8, while groups 1 and 7 reached background levels by 20 minutes post treatment.
• GLP-I is also known in the art to work in the brain to trigger a feeling of satiety and reduce food intake. Based on this role of GLP-I in satiety and reduction of food intake, experiments were conducted to determine whether GLP-1/FDKP formulations of the present invention were effective as agents to reduce feeding and thereby have potential for controlling obesity.
• Diketopiperazine particles were also conducted to assess the effect of diketopiperazine particles on reproductive toxicity. These studies included fertility, embryo-fetal development and postnatal development studies in rats and rabbits. Diketopiperazine particles administered via subcuteanous injection does not impair fertility or implantation in rats and there is no evidence of teratogenicity in rats or rabbits. Diketopiperazine particles did not adversely affect fertility and early embryonic development, embryo fetal development, or prenatal or postnatal development.
• an hERG assay was employed to examine the pharmacology of diketopiperazine particles.
• the hERG assay was utilized given that the vast majority of pharmaceuticals that cause acquired LQTS do so by blocking the human ether-a-go-go related gene (hERG) potassium channel that is responsible for the repolarization of the ventricular cardiac action potential.
• Results from the hERG assay indicated an IC 50 >100 ⁇ M for diketopiperazine particles.
• GLP-I is known to promote all steps in insulin biosynthesis and directly stimulate ⁇ -cell growth and survival as well as ⁇ -cell differentiation. The combination of these effects results in increased ⁇ -cell mass. Furthermore, GLP-I receptor signaling results in a reduction of ⁇ -cell apoptosis, which further contributes to increased ⁇ -cell mass. GLP-I is known to modulate ⁇ -cell mass by three potential pathways: enhancement of ⁇ -cell proliferation; inhibition of apoptosis of ⁇ -cells; and differentiation of putative stem cells in the ductal epithelium.
• GLP-I GLP-I receptor
• IPGTT IP glucose tolerance test
• GLP-I levels were also measured on day 3 of dosing (FIG. 25).
• the maximum concentration of plasma GLP-I levels in Group 2 was 10,643 pM at 15 minutes post-dose.
• insulin levels were measured at various timepoints on day 3 along with glucose measurements followng the IP glucose tolerance test.
• Both control (air) Group 1 and Group 2 demonstrated an initial decrease in insulin concentration from pre-dose levels, 46% and 30%, respectively, by 15 minutes post-dose (FIG. 26).
• insulin levels in Group 2 returned to baseline whereas insulin levels in Group 1 continued to decrease to 64% of pre-dose values.
• insulin levels at 45 minutes, 60 minutes, and 90 minutes were near pre-dose values with deviations of less than 1.5%.
• Apoptosis analysis was also conducted on the pancreatic tissue of ZDF rats. Exocrine and endocrine pancreas cells were evaluated by the TUNEL assay (Tornusciolo D. R. et al., 1995). Approximately 10,000 cells in the pancreas (exocrine and endocrine) were scored. Most TUNEL-positive cells were exocrine. There were no differences in apoptosis labeling index in treated versus control groups.
• ⁇ cell proliferation was evaluated in the pancreas of Zucker Diabetic obese rats dosed once daily for 3 days with control (air) or GLP-I /FDKP via pulmonary insulfflation. Slides were prepared for co-localization of insulin and Ki67 (a proliferation marker) using immunohistochemistry. Microscopic evaluation of cell proliferation was conducted within insulin-positive islets and in the exocrine pancreas in a total of 17 ZDF rats. Based on quantitative assessment of cell proliferation, there were no treatment-related effects on cell proliferation within the islet beta cells or exocrine cells of the pancreas in male ZDF rats.
• Example 11 Preparation of GLP-1/FDKP particle formulations
• a 10 wt% GLP-I stock solution was prepared by adding 1 part GLP-I (by weight) to 9 parts deionized water and adding a small amount of glacial acetic acid to obtain a clear solution.
• a stock suspension of FDKP particles (approximately 10 wt% particles) was divided into three portions.
• An appropriate amount of GLP-I stock solution was added to each suspension to provide target compositions of 5 and 15 wt% GLP-I in the dried powder.
• the pH of the suspensions was approximately 3.5.
• the suspensions were then adjusted to approximately pH 4.4-4.5, after which the suspensions were pelletized in liquid nitrogen and lyophilized to remove the ice.
• the aerodynamics of the powders is characterized in terms of respirable fraction on fill (RF Based on Fill), i.e., the percentage (%) of powder in the respirable range normalized by the quantity of powder in the cartridge, which was determined as follows: five cartridges were manually filled with 5 mg of powder and discharged through MannKind's MedTone® inhaler (described in U.S. Patent Application No. 10/655,153).
• This methodology produced a formulation with a good RF on fill.
• the powder with 5 wt% GLP-I was measured at 48.8 %RF/fill while the powder containing approximately 15 wt% GLP-I was 32.2 %RF/fill.
• the maximum plasma GLP-I concentrations (Cmax) following the administration of GLP-1/FDKP (5% formulation) were 2321 pM at a Tmax of 5 minutes post dose; 4,887 pM at a Tmax of 10 minutes post dose (10% formulation); and 10,207 pM at a Tmax of 10 minutes post dose (15% formulation).
• Cmax The maximum plasma GLP-I concentrations (Cmax) following the administration of GLP-1/FDKP (5% formulation) were 2321 pM at a Tmax of 5 minutes post dose; 4,887 pM at a Tmax of 10 minutes post dose (10% formulation); and 10,207 pM at a Tmax of 10 minutes post dose (15% formulation).
• AUC area under the curve
• the area under the curve (AUC) levels for GLP-I were 10622, 57101, 92606, 227873 pM*min for Groups 1-4, respectively.
• Estimated half-life of GLP-I was 10 min for GLP-1/FDKP at 10% or
• maximum FDKP concentrations were determined to be 8.5 ⁇ g/mL (Group 2), 4.8 ⁇ g/mL (Group 3) and 7.1 ⁇ g/mL (Group 4) for the GLP-1/FDKP formulations at 5%, 10% and 15% GLP-I, respectively.
• the time to maximum concentrations (Tmax) was 10 minutes. This data shows that, FDKP and GLP-I exhibited similar absorption kinetics and similar amounts of FDKP were absorbed independent of the GLP-I load on the particles.
• Body weights were measured daily at predose for 4 consecutive days. Body weights at the initiation of dosing ranged from about 180 to 209 grams. Although statistical significance between treated and control (air) animals was not reached, body weight were lower in treated animals. All animals survived until scheduled sacrifice.
• Examples 14 to 16 below disclose repeat-dose toxicity studies performed in rats and monkeys to evaluate the potential toxic effects and toxicokinetic profile of GLP-I /FDKP inhalation powder. The data indicates no apparent toxicity with GLP-I /FDKP inhalation powder at doses several fold higher than those proposed for clinical use. Additionally, there appeared to be no differences between the male and female animals within each species.
• Gravimetric analysis is performed by weighing the filter papers in the inhalation chamber both before, during and after dosing to calculate the aerosol concentration in the chamber and to determine the duration of dosing.
• Gravimetric analysis is performed by weighing the filter papers in the inhalation chamber both before, during and after dosing to calculate the aerosol concentration in the chamber and to determine the duration of dosing.
• GLP-I C max and AUCi ast areas under the concentration- time curve from time zero to the time of the last quantifiable concentration
• GLP-I AUCi ast areas under the concentration- time curve from time zero to the time of the last quantifiable concentration
• the peak concentration (Cmax) of FDKP averaged 200, 451 and 339 ng/niL in males and 134, 161 and 485 ng/mL in females administered GLP-1F/DKP at dose levels 0.3, 1.0 and 2.0 mg/kg/day respectively.
• the AUC ⁇ values for FDKP were 307, 578 and 817 ng.h/niL in males and 268, 235 and 810 ng.h/niL in females administered GLP-1/FDKP at dose levels of 0.3, 1.0 and 2.0 mg/kg/day respectively.
• AUC ⁇ and C max levels in animals administered FDKP only at a dose of 2.1 mg/kg/day (Group 2) were of the same order of magnitude as animals receiving GLP-1/FDKP at 2.13 mg/kg/day, with the exception that the T max was slightly longer at 30 to 45 minutes following dose administration.
• GLP-1/FDKP was well tolerated with no clinical signs or effects on body weights, food consumption, clinical pathology parameters, macroscopic or microscopic evaluations. It is also noted that inhalation administration of GLP-1/FDKP to cynomolgus monkeys at estimated achieved doses of up to 2.13 mg/kg/day (corresponding to a dose of 0.26 mg/kg/day GLP-I) administered for 30 minutes a day for 5 days is not associated with any dose limiting toxicity.
• GLP-I C max was achieved within approximately 10 to 15 minutes following dose administration in all dose groups. Peak concentrations of GLP-I at 10 mg/kg/day GLP-1/FDKP averaged 6714 and 6270 pg/mL on Day 1 and 2979 and 5834 pg/mL on Day 14 in males and females, respectively. Plasma levels of GLP-I declined with apparent elimination half-lives ranging from 0.7 hours to 4.4 hours. Mean AUC levels of GLP-I were 2187 pM*h in males and 2703 pM*h in females at the highest dose of 10 mg/kg/day GLP-1/FDKP.
• Group 1 air control
• Group 2 FDKP ( ⁇ 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 (mid dose);
• Group 5 2.6 mg/kg/day GLP-1/FDKP (high dose).
• FDKP air control
• Group 3 0.3 mg/kg/day GLP-1/FDKP (low dose)
• Group 4 .0 mg/kg/day GLP-1/FDKP (mid dose)
• Group 5 2.6 mg/kg/day GLP-1/FDKP (high dose).
• GLP-I and FDKP C max and AUCi ast as a function of dose were observed in both male and female monkeys on Day 1. Over the dose range studied, less than dose-proportional increases in GLP-I C max but not AUCi ast were observed with increasing doses in both male and female monkeys on Day 28. Peak concentrations of GLP-I at 2.6 mg/kg/day GLP-1/FDKP averaged 259 pg/mL in males and 164 pg/mL in females. Plasma levels of GLP-I declined with elimination half lives varying from 0.6 to 2.5 hours.
• Mean AUC values for GLP-I were 103 pg*hr/mL in males and 104 pg*hr/mL in females at the high dose.
• Female monkeys displayed higher AUC and C max values at the low dose compared to males.
• Peak concentrations of FDKP at 2.6 mg/kg/day GLP-1/FDKP averaged 1800 ng/mL in males and 1900 pg/mL in females.
• the NOAEL was 2.6 mg/kg/day GLP-1/FDKP (0.39 mg/kg/day GLP-I).
• the maximum human dose in the Phase I study will be 1.5 mg GLP-1/FDKP per day or -0.021 mg/kg GLP-I (assuming 70 Kg human). Additional studies will dose to 3.0 mg GLP-1/FDKP per day or -0.042 mg/kg GLP-I.
• Exendin-4/FDKP was prepared by combining an acidic exendin-4 peptide (SEQ ID No. 3) solution with a FDKP particle suspension.
• the acidic peptide solution was 10% (w/w) of peptide 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 as it gently mixed.
• the exendin-4/FDKP mixture was slowly titrated with a 25% ammonia solution to pH 4.50. The mixture was then pelleted into liquid nitrogen and lyophilized.
• Exendin/FDKP is dosed daily, via the inhalation route.
• a proportion of the animals are sacrificed immediately after the dosing regimen while other animals are allowed up to a one month recovery period prior to sacrifice. All animals are evaluated for clinical signs of toxicity; various physiological parameters including blood levels of Exendin-4, glucose, and insulin; organ weights, and clinical pathology and histopathology of various organs.
• the intial study groups consisted of five animals per group with two control groups: air and Exendin administered intravenously. There were six pulmonary insufflation groups which received approximately 2.0 mg doses of Exendin/FDKP at 5%, 10%, 15%, 20% and 25%, and 30% Exendin load (w/w). Whole blood was collected for blood glucose and Exendin concentrations out to an 8 hour time point. [00240] The data (C max , T 1/2 and T ma ⁇ ), are collected, demonstrating that Exendin/FDKP formulations have comparable or better pharmacokinetics than GLP-1/FDKP.
• GLP-1/FDKP 0.3mg GLP-I
• pulmonary insufflation a second control.
• Each of the GLP-1/xDKP treated groups received GLP-1/xDKP formulations via pulmonary insufflation at ⁇ 2.0mg doses of xDKP loaded with GLP-I at 10% and 15%.
• the xDKPs used were (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) loads.
• Whole blood was collected for evaluation of GLP-I concentrations at 5, 10, 20, 30, 45, 60 and up to 90 minutes post dose.
• GLP-I has been shown to control elevated blood glucose in humans when given by intravenous (iv) or subcutaneous (sc) infusions or by multiple subcutaneous injections. Because of the extremely short half-life of the hormone, continuous subcutaneous infusion or multiple daily subcutaneous injections would be required. Neither of these routes is practical for prolonged clinical use. Experiments in animals showed that when GLP-I was administered by inhalation, therapeutic levels could be achieved. [00244] Several of the actions of GLP-I, including reduction in gastric emptying, increased satiety, and suppression of inappropriate glucagon secretion appear to be linked to the burst of GLP-I released as meals begin.
• GLP-I /FDKP inhalation powder By supplementing this early surge in GLP-I with GLP-I /FDKP inhalation powder a pharmacodynamic response in diabetic animals can be elicited. In addition, the late surge in native GLP-I linked to increased insulin secretion can be mimicked by postprandial administration of GLP-I /FDKP inhalation powder.
• the Phase Ia clinical trial of GLP-1/FDKP inhalation powder is designed to test the safety and tolerability of selected doses of a new inhaled glycemic control therapeutic product for the first time in human subjects. Administration makes use of the MedTone® Inhaler device, previously tested.
• the primary intent of this clinical trial is to identify a range of doses for GLP- 1/FDKP inhalation powder by pulmonary inhalation that are safe, tolerable and can be used in further clinical trials to establish evidence of efficacy and safety.
• the doses selected for the phase Ia clinical trial are based on animal safety results from non-clinical trials of GLP-1/FDKP inhalation powder described in above Examples, in rats and primates.
• GLP-I Glucagon-Like Peptide- 1
• Each subject is dosed once with Glucagon-Like Peptide- 1 (GLP-I) as GLP-1/FDKP Inhalation Powder at the following dose levels: 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-I. Dropouts will not be replaced. These dosages assume a body mass of 70 kg. Persons of ordinary skill in the art can determine additional dosage levels based on the studies disclosed above.
• the objectives of this trial are to determine the safety and tolerability of ascending doses of GLP-1/FDKP inhalation powder in healthy adult male subjects.
• the tolerability of ascending doses of GLP-1/FDKP inhalation powder as determined by monitoring pharmacological or adverse effects on variables, including reported adverse events (AE), vital signs, physical examinations, clinical laboratory tests and electrocardiograms (ECG) will be evaluated.
• the secondary objectives are to evaluate additional safety and pharmacokinetic parameters. These include additional safety parameters, as expressed by the incidence of pulmonary and other AEs and changes in pulmonary function between Visit 1 (Screening) and Visit 3 (Follow-up); pharmacokinetic (PK) parameters of plasma GLP-I and serum fumaryl diketopiperazine (FDKP) following dosing with GLP-I /FDKP inhalation powder, as measured via AUCo-i2o(mm) plasma GLP-I and AUCo-4so mm serum FDKP; and additional PK parameters of plasma GLP-I include: tmaxplasma GLP-I; Cmaxplasma GLP-I; and GLP-I. Additional PK parameters of serum FDKP include: Tmax serum FDKP; Cm 3x serum FDKP; and Ty 2 serum FDKP.
• additional PK parameters of serum FDKP include: Tmax serum FDKP; Cm 3x serum FDKP; and Ty
• Trial Endpoints are based on a comparison of the following pharmacological and safety parameters determined in the trial subject population.
• Primary endpoints will include: Safety endpoints will be assessed based on the incidence and severity of reported AEs, including cough and dyspnea, nausea and/or vomiting, as well as changes from screening in vital signs, clinical laboratory tests and physical examinations.
• Secondary endpoints will include: PK disposition of plasma GLP-I and serum FDKP (AUC0-120 min plasma GLP-I and AUCo-480 min serum FDKP); additional PK parameters of plasma GLP-I (Tmax plasma GLP-I, Cmax plasma GLP-I Ty 2 plasma GLP-I; additional PK parameters of serum FDKP (Tmax serum FDKP, Cmax serum FDKP); and additional safety parameters (pulmonary function tests (PFTs)) and ECG.
• PK disposition of plasma GLP-I and serum FDKP AUC0-120 min plasma GLP-I and AUCo-480 min serum FDKP
• additional PK parameters of plasma GLP-I Tmax plasma GLP-I, Cmax plasma GLP-I Ty 2 plasma GLP-I
• additional PK parameters of serum FDKP Tmax serum FDKP, Cmax serum FDKP
• additional safety parameters pulmonary function tests (PFTs)
• the Phase Ia, single-dose trial incorporates an open-label, ascending dose structure and design strategy that is consistent with 21 CFR 312, Good Clinical Practice: Consolidated Guidance (ICH-E6) and the Guidance on General Considerations for Clinical Trials (ICH-E8) to determine the safety and tolerability of the investigational medicinal product (IMP).
• ICH-E6 Consolidated Guidance
• ICH-E8 Guidance on General Considerations for Clinical Trials
• the clinical trial will consist of 3 clinic visits: 1) One screening visit (Visit 1); 2) One treatment visit (Visit 2); and 3) One follow-up visit (Visit 3) 8-14 days after Visit 2.
• Administration of a single dose of GLP-I /FDKP inhalation powder will occur at Visit 2.
• GLP-1/FDKP will be mixed with FDKP inhalation powder.
• Single-dose cartridges containing 10 mg dry powder consisting of GLP-1/FDKP inhalation powder (15% weight to weight GLP-1/FDKP) as is or mixed with the appropriate amount of FDKP inhalation powder will be used to obtain the desired dose of GLP-I (0.05 mg, 0.45 mg, 0.75 mg, 1.05 mg and 1.5 mg).: 1.
• the first 2 lowest dose levels will be evaluated in 2 cohorts of 4 subjects each and the 3 higher dose levels will be evaluated in 3 cohorts of 6 subjects each. Each subject will receive only 1 dose at 1 of the 5 dose levels to be assessed.
• samples will be drawn for glucagon, glucose, insulin and C-peptide determination.
• Deacon CF Therapeutic strategies based on glucagon-like peptide 1. Diabetes. Sep;53(9):2181-9, 2004.
• Mentlein R et al., Dipeptidyl peptidase IV hydro lyses gastric inhibitory polypeptide, glucagon-like peptide-1 (7-36) amide, peptide histidine methionine and is responsible for their degradation in human serum. Eur J Biochem., 214:829 -835, 1993.
• Nauck MA et al., Normalization of fasting hyperglycemia by exogenous GLP-I (7- 36 amide) in type 2 diabetic patients. Diabetologia, 36:741 -744, 1993.

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