Methods for preparing stable peptide formulations

Abstract

An improved method for preparing a powder formulation containing a peptide or an improved method for preparing a powder formulation containing glucagon or a glucagon analogue is provided. A method for preparing a powder formulation includes forming a mixture of an acid, a phospholipid surfactant, and a cyclodextrin in an aqueous carrier, subjecting the mixture to a filtration step, adding a peptide to the filtered product to form a second mixture, subjecting the filtered product to a second filtration step, drying the filtered product to form a solid formulation, and processing the solid formulation to produce a final powder formulation suitable for nasal administration.

Description

[Technical field]

[0001] The present invention relates to the field of medicine. More specifically, the present invention relates to a powder formulation containing a peptide. The present invention further provides an improved method for preparing glucagon or glucagon. The present invention provides an improved method for preparing a powder formulation containing a steroid analog, the powder formulation being Suitable for nasal administration. [Background technology]

[0002] Peptides are prone to physical instability, such as aggregation, during and after the manufacturing process. Aggregation is a complex process that occurs through several different mechanisms. Induced by nucleation of some peptides or proteins, forming soluble aggregates These then serve as nucleation centers for the subsequent growth of larger insoluble aggregates. The nucleation-growth process depends on time, temperature, protein concentration, and other factors. During production, proteins can be purified by ultrafiltration, affinity cloning, etc. Chromatography, selective absorption chromatography, ion exchange chromatography, freezing It is purified and concentrated using a variety of means, including drying, dialysis, and precipitation or "salting out." Such concentration processes can lead to aggregation (Maggio, BioProcess s International 2008;6(10):58-65). These Additional processing steps are required to remove or solubilize the bodies, which can be expensive. and can reduce the overall product yield. The effects of agglomeration include loss of material, reduced efficacy, altered pharmacokinetics, reduced stability and shelf life of the product, and undesirable immune responses. This includes induction of epidemiology.

[0003] In particular, the current trend towards highly concentrated solutions increases the likelihood of protein-protein interactions. This promotes aggregation, which is a major problem for biopharmaceutical manufacturers. (Maggio, BioProcess International 2 008;6(10):58-65). The following may be considered, including but not limited to, pH, buffer conditions, ionic strength, etc. Aggregation of peptides, including adjustment and / or addition of other excipients such as cyclodextrins Various approaches have been investigated to limit

[0004] Glucagon is known to have a tendency to aggregate in aqueous solution (Pedersen JS.,J Diabetes Sci Technol.2010;4(6):13 57-1367;Beaven et al.,The European J.Bio chem.1969;11(1):37-42;Matilainen et al., European Journal of Pharmaceutical Sciences 20 09;(36):412-420), which may cause problems during the manufacture of powdered glucagon formulations. Previous methods for preparing glucagon powder formulations suitable for nasal administration are disclosed in WO2016 / 13 This is disclosed in US Pat. No. 3,863.

[0005] For preparing a peptide powder formulation, particularly a glucagon or glucagon analogue powder formulation There is a need for alternative methods, particularly methods that reduce or eliminate aggregation of peptides in aqueous solutions. A method is needed to reduce or preferably eliminate agglomeration to improve the final powder production. This is highly advantageous as the formulation retains a very high percentage of the active peptide. Preferably, the method involves subjecting the material to physical and chemical treatment for an extended period of time, for example up to 24 hours, prior to drying. This extended stability makes the process more suitable for large-scale manufacturing. Furthermore, the final product has a long shelf life, preferably up to about 24 months. A method is needed that results in a powder formulation.

[0006] Thus, the present invention provides an improved cost-effective method for reducing peptide aggregation during the manufacture of powder formulations. This method provides a highly efficient method for the purification of septic and septic septic fluids. One such peptide that may be used in the present invention is glucagon or a glucagon analog. The powder formulations prepared according to the methods of the present invention are particularly suitable for intranasal administration. Summary of the Invention

[0007] According to one aspect of the present invention, a method for preparing a peptide powder formulation is provided. The method includes the following steps: a. First mixing an acid, a phospholipid surfactant, and a cyclodextrin in an aqueous carrier. forming an article; b. subjecting the first mixture to a first filtration step, wherein the filter filters out about a membrane having a pore size of about 0.4 μm to about 0.5 μm; c. adding a peptide to the first filtered product to form a second mixture; and subjecting the second mixture to a first 2. A step of subjecting the mixture to a filtration step, the filter being about 0.4 μm to about 0.5 μm. a membrane having a pore size of m; d. Drying the second filtered product to form a solid formulation and processing the solid formulation to produce a final powder. A step of manufacturing the agent.

[0008] In one embodiment, the peptide is glucagon or a glucagon analogue. It is glucagon.

[0009] In one embodiment, the acid is citric acid or acetic acid. In particular, it is acetic acid. More specifically, the acetic acid is at a concentration of 1M.

[0010] In one embodiment, the surfactant, cyclodextrin and peptide together In certain embodiments, the second mixture comprises about 1.5% to about 3% by weight of the second mixture. These comprise about 2% by weight of the second mixture. In a further embodiment, they are It constitutes about 2.5% by weight of the second mixture.

[0011] In one embodiment, the surfactant is dodecylphosphocholine (DPC), didecylphosphocholine (DPC), Phosphatidylcholine (DDPC), Lysolauroylphosphatidylcholine (LLPC), Di Octanoylphosphatidylcholine (D8PC), or dilauroylphosphatidylcholine Glycerol (DLPG). In particular, the surfactant is DPC.

[0012] In one embodiment, the cyclodextrin is α-cyclodextrin, β-cyclodextrin, xylitol, hydroxypropyl β-cyclodextrin, or γ-cyclodextrin In particular, the cyclodextrin is a β-cyclodextrin.

[0013] In one embodiment, greater than 98% of the peptide in the final powder formulation is purified by reverse phase HPLC. Preferably, more than 99% of the peptides are non-aggregated peptides. More preferably, 100% of the peptides are non-aggregated peptides.

[0014] According to another aspect of the present invention, a method for preparing a peptide powder formulation comprising the steps of: Methods are provided: a. forming a first mixture of a phospholipid surfactant and a cyclodextrin in an aqueous carrier; and b. subjecting the first mixture to a first filtration step, wherein the filter filters out about a membrane having a pore size of about 0.4 μm to about 0.5 μm; c. adding a peptide to the first filtered product to form a second mixture; and subjecting the second mixture to a first 2. A step of subjecting the mixture to a filtration step, the filter being about 0.4 μm to about 0.5 μm. a membrane having a pore size of m; d. Drying the second filtered product to form a solid formulation and processing the solid formulation to produce a final powder. A step of manufacturing the agent.

[0015] In one embodiment, the surfactant, cyclodextrin and peptide together In certain embodiments, the second mixture comprises about 1.5% to about 3% by weight of the second mixture. These comprise about 2% by weight of the second mixture. In a further embodiment, they are It constitutes about 2.5% by weight of the second mixture.

[0016] In one embodiment, the surfactant is DPC, DDPC, LLPC, D8PC, or DLPG. In particular, the surfactant is DPC.

[0017] In one embodiment, the cyclodextrin is α-cyclodextrin, β-cyclodextrin, xylitol, hydroxypropyl β-cyclodextrin, or γ-cyclodextrin In particular, the cyclodextrin is a β-cyclodextrin.

[0018] In one embodiment, greater than 98% of the peptide in the final powder formulation is purified by reverse phase HPLC. Preferably, more than 99% of the peptides are non-aggregated peptides. More preferably, 100% of the peptides are non-aggregated peptides.

[0019] According to another aspect of the present invention, a method for preparing a glucagon powder formulation comprising the steps of: A method is provided. a. forming a first mixture of acetic acid, DPC, and β-cyclodextrin in an aqueous carrier; and b. subjecting the first mixture to a first filtration step, wherein the filter filters out about a membrane having a pore size of about 0.4 μm to about 0.5 μm; c. adding glucagon to the first filtered product to form a second mixture; and A step of subjecting the mixture to a second filtration step, the filter being between about 0.4 μm and about 0.5 μm. a membrane having a pore size of 100 μm; d. Drying the second filtered product to form a solid formulation and processing the solid formulation to produce a final powder. A step of manufacturing the agent.

[0020] In one embodiment, glucagon, DPC and β-cyclodextrin are combined. In certain embodiments, the second mixture comprises about 1.5% to about 3% by weight of the second mixture. They comprise about 2% by weight of the second mixture. In a further embodiment, they , constituting about 2.5% by weight of the second mixture.

[0021] In one embodiment, the acetic acid is at a concentration of 1M.

[0022] In one embodiment, greater than 98% of the glucagon in the final powder formulation is determined by reverse phase HPLC. Preferably, more than 99% of the glucagon is non-aggregated glucagon. More preferably, 100% of the glucagon is non-aggregated glucagon.

[0023] The present invention further provides a powder formulation prepared according to the method of the present invention.

[0024] In certain embodiments, drying of the second filtered product is by freeze-drying (lyophilization) or Alternatively, the process may be carried out by spray drying.

[0025] In certain embodiments, the filter membranes in both the first and second filtration steps Examples of the material include, but are not limited to, polyvinylidene fluoride (PVDF), cellulose acetate, and cellulose nitrate. Polytetrafluoroethylene (PTFE, Teflon (registered trademark)), polyvinyl chloride Filter media include fluororesin, polyethersulfone, or other filter media suitable for use in a cGMP manufacturing environment. In a preferred embodiment, the filter membrane comprises PVDF.

[0026] In certain embodiments, the filter membranes in both the first and second filtration steps is a membrane having a pore size of about 0.45 μm. In a preferred embodiment, the filter membrane is a PVDF membrane with a pore size of 0.45 μm.

[0027] In one embodiment, the pH of the solution is maintained at 2-3 during the method of the invention.

[0028] In one embodiment, the liquid phase of the process of the present invention is maintained at a temperature of 15 to 30° C., preferably 18 to 25° C. , more preferably at a temperature of about 20°C.

[0029] The method of the present invention can be used for peptides that tend to aggregate during the manufacture of powder formulations. In particular, the method can be used to treat, but is not limited to, amylin, amylin analogs, recombinant human factor V, Factor III (rfVII), calcitonin gene-related peptide (CGRP), calcitonin GLP-1 analogs, GLP-1-GLP dual agonists, GIP agonists, Recombinant human growth hormone (rhGH), octapeptide CCR5 inhibitor D-Ala-peptide T-amylin, recombinant human insulin, insulin analogs, PTH 1-31 cyclic peptide Interferon analogs, interferon-β, interferon-β-1a and β-1b, interferon -leukin-2 (IL-2), erythropoietin (EPO), pramlintide acetate, and and peptides containing enzymes such as urokinase.

[0030] In particular, the methods of the present invention can be used to prepare glucagon powder formulations. Lucagon is a highly effective treatment for severe hypoglycemia in both the hospital and out-of-hospital setting. Glucagon is available as a powder formulation that must be mixed with a diluent immediately prior to administration by injection. Liquid formulations of glucagon are also known (Pontiroli et al. al.,Br Med J(Clin Res Ed)1983;287:462-46 3) A nasal glucagon powder for the treatment of severe hypoglycemia was developed and published in WO20 16 / 133863, which has recently been marketed under the name Baqsimi (trademark). Approved in the US and Europe.

[0031] The glucagon or glucagon analog formulations produced according to the methods of the present invention can be administered intranasally. In a preferred embodiment, the formulations produced according to the methods of the present invention are , having one or more of the following: A low percentage of small particles that can reach the lungs Provides the total dose of drug needed to achieve therapeutic effect as a single dose into one nostril Contains enough drug to - To deliver a total dose of tens of milligrams, or the maximum amount possible by the delivery device Adequate drug content - To be effective despite nasal congestion, which can accompany allergies or colds Adequate drug content and absorption characteristics Stability during storage under ambient conditions for an extended period of time, preferably at least 24 months -Good safety and tolerability profile

[0032] As used herein, the term "aggregation" refers to the aggregation of small oligomers such as peptides. Accumulation, aggregation, agglomeration, dimerization, polymerization, of precursor seed nuclei, nucleation centers, fibrils, or gels; The size of the aggregates refers to the size of soluble dimers and other multimers (apparent spherical diameter). Approximately 5-10 nm) and can be identified as particles that are invisible to the naked eye and visible particles. These range from the larger insoluble species (approximately 20-50 μm in apparent diameter) to the soluble From the aggregate population, the larger ones, such as high molecular weight species, may lead to adverse clinical outcomes. It may be possible to induce an immunogenic response that is

[0033] As used herein, the term "seed nucleus" or "nucleation center" refers to a larger It refers to the minimum aggregate size at which aggregates are formed.

[0034] Reverse phase HPLC can be used to determine the amount of unaggregated peptide in the final powder formulation. Using standard conditions known to those skilled in the art, such as those described in the Examples below. It is possible.

[0035] As used herein, "glucagon" refers to the compound of the sequence His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln- Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr (SEQ ID NO: 1) refers to the polypeptide. Glucagon can be chemically synthesized, produced by recombinant DNA technology, or naturally occurring. The term "glucagon analogue" refers to a compound that inhibits the in vivo regulation of blood glucose levels. It refers to variants of this sequence that retain the ability to stimulate proliferation.

[0036] Glucagon analogs in which one amino acid of the native sequence is replaced by alanine, Examples of analogs having the substitutions are described in Chabenne et al., Molecular Metabolism 2014;3:293-300. Exemplary three amino acid modifications resulting in biologically active glucagon analogs are shown in Table 1. A suitable analogue is [Lys 17,18 ,G1u 21 ]Glucagon is a Zealand Ph Arma is disclosed in, for example, U.S. Patent Publication Nos. 20140080757, 2014001733 No., 20130316941, 20130157935, 20130157929 , No. 20120178670, No. 20110293586, No. 20110286982, No. 20110286981 and No. 20100204105 contain numerous glucagon-like These analogs have higher binding to the GLP receptor than to the glucagon receptor. It has been reported that it has a lower binding affinity to glucagon but still retains glucagon activity. Zealand Pharma has developed a compound called ZP4207 for the treatment of hypoglycemia. Clinical trials of glucagon analogs have also begun. U.S. Patent Publication No. 2013 / 0053310 No. 6,393,633 discloses other glucagon analogs useful for treating hypoglycemia.

[0037] Phospholipid surfactants are widely distributed in biological membranes that are part of the cells and tissues of the human body, including the nasal mucosa. The most common phospholipid surfactant in cells is phosphatidylcholine. Phosphorus and phosphocholine (PC), but phosphatidylglycerol (PG) is It is an important component of membranes. It can be converted to diacyl PC or PG by removing one of the acyl groups. Lysophosphospholipids derived therefrom can also be used.

[0038] An exemplary phospholipid surfactant that can be used in the present invention is dodecylphosphocholine. (DPC), didecylphosphatidylcholine (DDPC or 1,2-didecyl-sn- glycero-3-phosphocholine), lysolauroylphosphatidylcholine (LLPC or 1-Didecanoyl-sn-glycero-3-phosphocholine, dioctanoyl phosphatidyl Alcohol (D8PC or 1,2-dioctanoyl-sn-glycero-3-phosphocholine) ) and dilauroylphosphatidylglycerol (DLPG or 1,2-dilauroyl It is 1'-sn-glycero-3-phospho(1'-rac-glycerol).

[0039] Preferred phospholipid surfactants are those that, at the concentrations used during manufacture of the powder formulation, form a bilayer but not These include DPC, DDPC, LLPC, and D8 PC is included, but DLPG is not included. Most preferred is DPC.

[0040] In certain embodiments of the present invention, a single type of phospholipid surfactant is used. In embodiments, the phospholipid surfactant component may be, for example, any of the surfactants identified above. A mixture of phospholipid surfactants containing any two, three, or four combinations of It can be configured.

[0041] As used herein, the term "cyclodextrin" refers to a cyclodextrin having six, seven or more rings. , or cyclodextrins that contain eight glucose residues and form a cone shape, i.e. , which refers to: α-Cyclodextrin: A six-membered sugar ring molecule ●β-Cyclodextrin: a seven-membered sugar ring molecule ●γ (gamma)-cyclodextrin: An eight-membered sugar ring molecule.

[0042] α-CD was developed in clinical trials by Novo Nordisk in a powder formulation (HypoGon (Stenniger et al., Diab etologia 1993;36:931-935;Rosenfalck AM, e t al., Diabetes Res Clin Pract 1992;17:43 -50) The aqueous solubility of α-CD is reported to be approximately 5% by weight.

[0043] One has a lower aqueous solubility than α-CD (β-CD, 1.85 wt%), and the other has a lower aqueous solubility than α- Two other cyclodextrins with higher aqueous solubility than CD (HP-β-CD) were also used in the present invention. as well as gamma-cyclodextrin, which is freely soluble in water and is suitable for use in be.

[0044] The cyclodextrin in the formulation acts as a bulking agent and also adheres to the nasal mucosal surface to promote glucan transport. When delivered to the nostrils, the main ingredient (90% to 70% by weight), i.e. Thus, the cyclodextrin helps the powder adhere to the mucosal surface.

[0045] The cyclodextrins may be used individually or any two or more cyclodextrins may be used in combination. A mixture of quinolins may also be used.

[0046] In certain embodiments, the glucagon powder formulation prepared according to the present method comprises glucagon Preferably, the powder formulation comprises 10:10 Glucagon, DPC in a weight ratio of 80 (glucagon:DPC:β-cyclodextrin) and β-cyclodextrin. Preferably, the glucagon is administered as a single dose into one nostril. In one embodiment, the glucagon is present in a therapeutic amount that is effective when administered in an amount The dose is approximately 3 mg.

[0047] Mixing can be accomplished by methods including static and dynamic mixing. Dynamic mixing is achieved by using a axial This can be done using a blade inserted into the liquid that is attached to the Static mixing is achieved by forcing the liquid through a tortuous path in a static mixer. The presence of an air-water interface during mixing under high speed mixing conditions can lead to foaming. High speed mixing may also lead to protein destabilization due to shear stress. To minimize and preferably eliminate foaming, slow mixing conditions are preferred. For dynamic mixing, the speed is determined by the number of revolutions per minute (rpm) of the stirrer. The rpm value is preferably 50 to 300, more preferably 50 to 250, and even more preferably 50 to 300. The number is 100.

[0048] The second filtered product is dried to remove the solvent and leave a solid product. Drying can be performed using a method such as lyophilization. This can be done by spray drying, tray drying or other techniques. The physical properties vary depending on the drying technique, resulting in flaky or dry solids from freeze-drying. It may be in the form of a cake.

[0049] Powders with excessive moisture content can be sticky and form clumps, This results in a powder that is difficult to manipulate for filling into a dosing device. The level of residual moisture directly affects stability. A residual moisture content above 5% reduces the stability compared to powders with a residual moisture content below 5%. Thus, in certain embodiments, the powder formulations prepared according to the present invention preferably contain 5 % residual moisture content.

[0050] In certain embodiments, the amount of acid in a powder formulation prepared according to the present invention is 10% w % w / w, preferably less than 6% w / w.

[0051] Powders suitable for nasal administration are advantageous in that they can be loaded into nasal delivery devices. They need physical properties that allow for sufficient flow. Flowability is determined by particle size, shape, density, surface area, etc. Texture, surface area, density, cohesion, adhesion, elasticity, porosity, moisture absorption, and friability It is determined by various parameters including:

[0052] Powders with the right particle size and flow properties eliminate particles that are too small or too large can be produced by processing the bulk powder so that it is too small or The method of treating the bulk powder to remove oversized particles is to mill the bulk powder. This may include breaking down larger particles and isolating particles of the desired size range by sieving. Vertical vibration sieve, horizontal sieve, tap type sieve, ultrasonic sieve, and air circulation jet Various sieving methods can be implemented, such as a single sieve of constant nominal aperture. or with gradually smaller openings to obtain the desired particle size distribution. The bulk powder may be processed through a series of sieves having nominal openings of 25 to 10. The sieve may be a wire mesh sieve in the range of 0.100 μm. EXAMPLES

[0053] Example 1. Preparation of Glucagon Powder Formulation (Double Filtration Step) DPC is dissolved in 1M acetic acid solution by stirring. Next, β-cyclodextrin is added to DPC. The first solution is added to the solution and stirred until dissolved to form a first solution. m The first filtration step is carried out through a PVDF filter. The filtered product (excipient solution ) was collected in a new clean tank, and the temperature of the tank was adjusted to 20°C ± 2°C to allow for the Ensure solubility of materials. Once the target temperature is reached in the tank, add glucan while stirring the solution. Add glucagon or a glucagon analog to the tank. When the glucagon appears to have dissolved, Immediately (by visual confirmation), stirring is immediately terminated. The glucagon solution is then added to the second Filter through a 0.45 μm PVDF filter and transfer the filtered material to a second cleaner. This second filtered material (second filtration product) was collected in a 97.5% w / w 1M acetic acid in water, 0.25% w / w DPC, 2% w / w β-cyclodextrin, and 0.25% w / w glucagon (total 2.5% w / w solids weight). The material is then freeze-dried and undergoes a densification step to produce the final glucagon powder formulation. .

[0054] Comparative Example. Preparation of Glucagon Powder Formulation (Single Filtration Step) Dissolve DPC in 1M acetic acid solution (8 L) by stirring. Add glucagon. As soon as the glucagon appears to have dissolved (by visual inspection), Add β-cyclodextrin while stirring. Wait until all added solids are dissolved. Once visible, filter the solution through a 0.45 μm PVDF filter. - If membrane clogging or fouling occurs, multiple filters may be required. The filtered material contained 0.3% w / w DPC, 2.4% w / w β-cyclohexyl rhodextrin, and 0.3% w / w glucagon (total 3% w / w solids). The filtered material is collected and lyophilized.

[0055] Stability of excipient solutions after the first filtration The excipient solutions (acetic acid, DPC and β-cyclodextrin) were prepared essentially as described in Example 1. The solution is prepared at 2.5% w / w solids concentration for testing. The solution is kept at 25°C for the duration of the test. The data are summarized in Table 1. No significant changes in content occurred over the 22-hour period. The material balance was confirmed. [Table 1]

[0056] Stability of aqueous solutions containing glucagon. Solution Assays Glucagon solution was prepared essentially as described in Example 1 (second filtered product). After preparation, the glucagon solution is allowed to stand without stirring. Solution samples are taken at the designated times. The cells were then passed through a 0.45 μm filter prior to assay. Because kagon is removed by this filtration step, this assay provides an estimate of the extent of aggregation. provide.

[0057] Fluorescence assay The basis of the fluorescence method is the shift in the emission wavelength of a single tryptophan residue in the glucagon molecule. (Pedersen JS., J Diabetes Sci Tech hnol.2010;4(6):1357-1367). The glucagon molecule is Changing the conformation from random coil or α-helical to aggregated forms The local environment of the phasant molecule changes in the form of a blue shift in the emission spectrum. Using a backscattering fluorescent probe optically coupled to a fiber, the luminescent fluorescent signal of glucagon was detected. By monitoring the change in wavelength of the light over time, the researchers were able to distinguish between non-aggregated glucagon and aggregated forms of the molecule. Calculating the ratio of the emission peaks of the two molecules can be used as a tool to monitor aggregation in real time. It can be used as such.

[0058] Glucagon solution was prepared essentially as described in Example 1 (second filtered product). The fluorescence probe is used to monitor the change in the emission spectrum over time. Monitor temperature for 24 hours.

[0059] In small-scale experiments (100 mL), no change in glucagon fluorescence ratio was observed during this 24-hour period. It was not done.

[0060] In a further experiment, a glucagon solution was prepared essentially as in Example 1 (second filtered product). The samples were prepared as described and kept at different temperatures. For comparison, this was followed by a second filtration step. The results are summarized in Table 2. [Table 2]

[0061] The results of the study were obtained by passing the glucagon solution through a second filtration step and stirring at either 5°C or 20°C. This indicates that no glucagon is lost due to aggregation from the system when kept without stirring. However, unless the solution is filtered, the glucagon content remains constant over a 24-hour period. About 8% is lost.

[0062] The chemical stability of the glucagon solution prepared essentially as described in Example 1 was also They can be tested using reverse phase HPLC essentially as described below.

[0063] Dual filtration of a 100 liter (2.5% w / w solids) scale volume yielded essentially the above In the preparation carried out in this way, it was surprising to find that the solution collected after the second filtration step was , and was found to be physically and chemically stable for up to 24 hours with no detectable aggregation. (determined by one or more of the methods above). On the other hand, The glucagon solution material provided (comparative example, 8 liters and 3% w / w solids) was Visible aggregation was observed within about 15 minutes of addition of the agonist.

[0064] Chemical stability analysis of nasal glucagon powder formulations by HPLC. Nasal glucagon prepared according to Example 1 against a well-defined external reference standard The stability of the powder formulation is determined using routine RP-HPLC techniques. A HPLC reverse phase column C18 (inner diameter 3.0 mm × 150 mm, particle size 2.6 μm) was used. A UV detection wavelength of 20 nm was used with a potassium phosphate buffer:acetonitrile mobile phase. The gradient mobile phase consisted of 54%, 80:20 150 mM potassium phosphate buffer:acetonite. Start with a 3 min hold in Tril, then 70%, 60:40 potassium phosphate buffer:acetamide The composition of acetonitrile is completed in 8 minutes.

[0065] As shown in Table 3, in experiments carried out essentially as described above, Example 1 (100 Representative samples from three different batches of nasal glucagon powder formulations prepared according to The samples retained approximately 100% glucagon activity within experimental precision.

[0066] Potency bioassay of nasal glucagon powder formulations. Stably express both the glucagon cell surface receptor and the CRE-luciferase reporter gene The final nasal glucagon was produced using the embryonic kidney cell line HEK293 engineered to express This cell-based assay determines the relative potency of CRE-promoted Transcription of luciferase from the promoter is regulated by endogenous cyclic AMP (cAMP) signal transduction. glucagon is regulated by triggering responses along the delivery pathway. Binding to cell surface receptors induces cAMP production, which activates the cAMP response element. This leads to phosphorylation and activation of CREB, which results in the Expression of luciferase by the E-luciferase reporter gene occurs. Luciferase production was measured by adding luciferin substrate to the reaction mixture and measuring the luciferase activity using a luminometer. The luminescence signal is determined by quantifying the oxidation of luciferin. The amount of glucagon used to induce the cells is directly proportional to the amount of enzyme that is used to induce the cells. The relative potency of the test samples was determined by sampling a typical 8-point dose-response curve of the reference standard. Response data are compared to those of a four-parameter logistic regression. The EC standard was fitted to the EC 50 and sample EC 50 Determine (these E-commerce 50 The ratio of values ​​represents the relative efficacy of the test materials).

[0067] HEK293 cells were cultured in growth medium (1.0 mg / mL Genetecin® and Dulbecco's modified Eagle's medium (DME) containing 125 μg / ml hygromycin B. 10% fetal bovine serum (FBS) in 100 mM M. Penicillin and streptomycin were added at 100 mM NaCl. units / mL penicillin and 100 μg / mL streptomycin at a final concentration The cells are seeded on a 30-well plate (optionally dried) and allowed to attach at 37°C for 30 minutes to 2.5 hours. The growth medium is then washed off. DMEM with 0.25% FBS, 0.5% bovine serum albumin, 1x penicillin / sulfate Leptomycin and Glucocorticoids at concentrations ranging from 0.00032 ng / mL to 25 ng / mL The plate was then incubated at 37°C for 4.5 hours. Add 100 μL of SteadyGlo® per well, then The wells are continuously agitated for 30 minutes at ambient temperature. The plates are read in a luminometer.

[0068] In experiments carried out essentially as described above, cell-based assays were performed as shown in Table 3. The relative efficacy of glucagon as measured by It was found that no aggregation occurred during the preparation of the formulation in Example 1 (100 L). These results are consistent with those of a glucagon chemical-based assay using the same reference standard. The results were equivalent to those of the previous study.

[0069] Impurity analysis of nasal glucagon powder formulations Identification and quantification of potential impurities in nasal glucagon powder formulations was performed using routine RP-HP This is done using LC techniques. Impurities can be due to the manufacturing process or the chemical composition of the materials in the final drug product. The method is based on the conditions outlined in USP41-NF36. This assay provides an indication of the stability of the glucagon powder formulation.

[0070] As shown in Table 3, experiments conducted essentially as described above showed that the total impurities at the time of batch release The additive levels range from about 0.4% to about 0.56%. The proposed shelf life specification analysis for the nasal glucagon powder formulation is for up to approximately 24 months. Surprisingly, the nasal glucagon powder has a total impurity level of about 20% (a / a) or less. For powdered preparations, the current USP monograph (USP41-NF36) states that the concentration is 31% (a / a) or less. The current market limit specifies that the total impurities and related compounds present must be less than 100%. It has total impurity levels significantly lower than those recommended for glucagon emergency kits. [Table 3]

[0071] Clinical efficacy of nasal glucagon powder formulation The results are shown in Table 1. The clinical efficacy of nasal glucagon powder formulation was evaluated in the NCT03339453 clinical trial study (Sui Co et al., EASD-2008; Abstract 150). To evaluate the efficacy and safety of glucagon powder (NG) in the treatment of insulin-induced hypoxia. During glucose administration, intramuscular glucagon (IMG) was compared with intranasal glucagon in adults with type 1 diabetes. The glucagon powder formulation is loaded into a device for delivery to one nostril at a dose of 3.0 mg. will be done.

[0072] The results, shown in Table 4, showed that 100% of patients were successfully treated with either NG or IMG. and that NG activity is comparable to IMG activity in this study. [Table 4]

[0073] array (SEQ ID NO:1) His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr The present invention may include the following aspects. (1) 1. A method for preparing a peptide powder formulation, comprising: a. forming a first mixture of an acid, a phospholipid surfactant, and a cyclodextrin in an aqueous carrier; b. subjecting the first mixture to a first filtration step, the filter comprising a membrane having a pore size of about 0.4 μm to about 0.5 μm; c. adding a peptide to the first filtered product to form a second mixture, and subjecting the second mixture to a second filtration step. wherein the filter comprises a membrane having a pore size of about 0.4 μm to about 0.5 μm; d. drying the second filtered product to form a solid formulation and processing the solid formulation to produce a final powder formulation. (2) 3. The method of claim 1, wherein the peptide is glucagon or a glucagon analog. The method of claim 2, wherein the peptide is glucagon. (4) 4. The method of claim 1, wherein the surfactant, the cyclodextrin and the peptide together comprise about 1.5% to about 3% by weight of the second mixture. (5) 5. The method of claim 4, wherein the surfactant, the cyclodextrin and the peptide together comprise about 2% by weight of the second mixture. (6) 5. The method of claim 4, wherein the surfactant, the cyclodextrin and the peptide together comprise about 2.5% by weight of the second mixture. (7) 7. The method of claim 1, wherein the membrane in both the first and second filtration steps comprises a polyvinylidene fluoride (PVDF) membrane. (8) 8. The method of claim 1, wherein the membrane in both the first and second filtration steps comprises a pore size of about 0.45 μm. (9) The method according to any one of claims 1 to 8, wherein the acid is citric acid or acetic acid. 10. The method of claim 9, wherein the acid is acetic acid. (11) 11. The method of claim 10, wherein the acetic acid is at a concentration of 1M. (12) The method according to any one of claims 1 to 11, wherein the surfactant is dodecylphosphocholine, didecylphosphatidylcholine, lysolauroylphosphatidylcholine, dioctanoylphosphatidylcholine, or dilauroylphosphatidylglycerol. The method of claim 12, wherein the surfactant is dodecylphosphocholine. (14) 14. The method according to claim 1, wherein the cyclodextrin is α-cyclodextrin, β-cyclodextrin, hydroxypropyl β-cyclodextrin, or γ-cyclodextrin. (15) 15. The method of claim 14, wherein the cyclodextrin is a β-cyclodextrin. (16) 1. A method for preparing a peptide powder formulation, comprising: a. forming a first mixture of a phospholipid surfactant and a cyclodextrin in an aqueous carrier; b. subjecting the first mixture to a first filtration step, the filter comprising a membrane having a pore size of about 0.4 μm to about 0.5 μm; c. adding a peptide to the first filtration product to form a second mixture and subjecting the second mixture to a second filtration step, wherein the filter comprises a membrane having a pore size of about 0.4 μm to about 0.5 μm; d. drying the second filtered product to form a solid formulation and processing the solid formulation to produce a final powder formulation. (17) 17. The method of any one of claims 1-16, wherein greater than 98% of the peptide in the final powder formulation is non-aggregated peptide as measured by reverse phase HPLC. (18) 18. A powder formulation prepared by the method according to any one of claims 1 to 17. (19) 1. A method for preparing a glucagon powder formulation comprising: a. forming a first mixture of acetic acid, dodecylphosphocholine, and β-cyclodextrin in an aqueous carrier; b. subjecting the first mixture to a first filtration step, the filter comprising a membrane having a pore size of about 0.4 μm to about 0.5 μm; c. adding glucagon to the first filtration product to form a second mixture, and subjecting the second mixture to a second filtration step, wherein the filter comprises a membrane having a pore size of about 0.4 μm to about 0.5 μm; d. drying the second filtered product to form a solid formulation and processing the solid formulation to produce a final powder formulation. (20) 20. The method of claim 19, wherein the surfactant, the cyclodextrin and the peptide together comprise about 2.5% by weight of the second mixture. (21) 21. The method of claim 19 or claim 20, wherein the membrane in both the first and second filtration steps comprises a PVDF membrane. (22) 22. The method of any one of claims 19 to 21, wherein the membrane in both the first and second filtration steps comprises a pore size of about 0.45 μm. (23) 23. The method according to claim 19, wherein the acetic acid is at a concentration of 1M. (24) 24. The method of any one of claims 19 to 23, wherein greater than 98% of the glucagon in the final powder formulation is non-aggregated glucagon as measured by reverse phase HPLC. (25) 25. A powder formulation prepared by the method according to any one of claims 19 to 24. (26) 26. The powder formulation of claim 25, wherein greater than 98% of the glucagon in the final powder formulation is non-aggregated glucagon as measured by reverse phase HPLC.

Claims

1. 1. A method for preparing a peptide powder formulation, comprising: a. forming a first mixture of an acid, a phospholipid surfactant, and a cyclodextrin in an aqueous carrier; b. subjecting the first mixture to a first filtration step, the filter comprising a membrane having a pore size of about 0.4 μm to about 0.5 μm; c. adding a peptide to the first filtration product to form a second mixture and subjecting the second mixture to a second filtration step, wherein the filter comprises a membrane having a pore size of about 0.4 μm to about 0.5 μm; and d. drying the second filtered product to form a solid formulation and processing the solid formulation to produce a final powder formulation having a residual moisture content of less than 5%, wherein the peptide is selected from the group consisting of amylin, amylin analogs, recombinant human factor VIII (rfVII), calcitonin gene-related peptide (CGRP), calcitonin, GLP-1 analogs, GLP-1-GLP dual agonists, GIP agonists, recombinant human growth hormone (rhGH), octapeptide CCR5 inhibitor D-Ala-peptide T-amylin, recombinant human insulin, insulin analogs, PTH 1-31 cyclic peptide analogs, interferon-beta, interferon beta-1a, interferon beta-1b, interleukin-2 (IL-2), erythropoietin (EPO), pramlintide acetate, glucagon, or a glucagon analog.

2. 10. The method of claim 1, wherein greater than 98% of the peptide in the final powder formulation is non-aggregated peptide as measured by reverse phase HPLC.

3. The method according to claim 1 or 2, wherein the pH of the solution before drying is maintained at 2-3.

4. The method of any one of claims 1 to 3, wherein the phospholipid surfactant comprises a mixture of phospholipid surfactants or a single type of phospholipid surfactant.

5. 5. The method of any one of claims 1 to 4, wherein step d comprises freeze-drying or spray-drying the second filtered product to form a solid formulation and processing the solid formulation to produce a final powder formulation.

6. 6. The method of any one of claims 1 to 5, wherein the membrane in both the first and second filtration steps comprises a polyvinylidene fluoride (PVDF) membrane, a cellulose acetate membrane, a cellulose nitrate membrane, a polytetrafluoroethylene (PTFE, Teflon) membrane, a polyvinyl chloride membrane, or a polyethersulfone membrane.

7. The method of any one of claims 1 to 6, wherein the peptide is glucagon or a glucagon analogue.

8. The method of claim 7, wherein the peptide is glucagon.

9. The method according to any one of claims 1 to 8, wherein the acid is citric acid or acetic acid.

10. 10. The method of claim 1, wherein the phospholipid surfactant is dodecylphosphocholine, didecylphosphatidylcholine, lysolauroylphosphatidylcholine, dioctanoylphosphatidylcholine, or dilauroylphosphatidylglycerol.

11. The method according to any one of claims 1 to 10, wherein the cyclodextrin is α-cyclodextrin, β-cyclodextrin, hydroxypropyl β-cyclodextrin, or γ-cyclodextrin.

12. the peptide is glucagon, and the method comprises: a. forming a first mixture of acetic acid, dodecylphosphocholine, and β-cyclodextrin in an aqueous carrier; b. subjecting the first mixture to a first filtration step, the filter comprising a membrane having a pore size of about 0.4 μm to about 0.5 μm; c. adding glucagon to the first filtration product to form a second mixture and subjecting the second mixture to a second filtration step, wherein the filter comprises a membrane having a pore size of about 0.4 μm to about 0.5 μm; d. drying the second filtered product to form a solid formulation and processing the solid formulation to produce a final powder formulation.

13. 13. The method of claim 12, wherein greater than 98% of the glucagon in the final powder formulation is non-aggregated glucagon as measured by reverse phase HPLC.

14. 14. The method of claim 12 or 13, wherein the acetic acid is at a concentration of 1 M.

15. The method of any one of claims 12 to 14, wherein the membrane in both the first and second filtration steps comprises a PVDF membrane.

16. 16. The method of any one of claims 12 to 15, wherein the membrane in both the first and second filtration steps comprises a pore size of about 0.45 μm.

17. The method of any one of claims 12 to 16, wherein the drying comprises freeze drying, spray drying, or tray drying.

18. 18. The method of any one of claims 12 to 17, wherein the final powder formulation comprises glucagon, dodecylphosphocholine and β-cyclodextrin in a weight ratio of 10:10:80 (glucagon:dodecylphosphocholine:β-cyclodextrin).

19. The method of any one of claims 12 to 18, wherein the final powder formulation is formulated for intranasal administration.

20. The method of any one of claims 12 to 19, further comprising the step of loading the final powder formulation into a nasal delivery device.

21. A method according to any one of claims 1 to 20, wherein the amount of acid is less than 10% w / w.

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