Liquid formulation of GM-CSF for inhalation

Abstract

SOLUTION: Provided herein is a liquid formulation comprising recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF) for inhalation by nebulization.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a liquid formulation for inhalation containing recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF).

Background Art

[0002] Granulocyte macrophage colony-stimulating factor (GM-CSF) was originally identified as a hematopoietic growth factor. Human GM-CSF stimulates the growth of myeloid and erythroid progenitor cells in vitro and activates monocytes, macrophages, and granulocytes in several immune and inflammatory processes (Gasson JC et al. Prog Clin Biol Res. 1990;352:375-384.; Gasson JC et al. Prog Clin Biol Res. 1990;338:27-41; Hart SP et al. Biochem Soc Trans. 1998;26(4):650-652; Rapoport AP et al. Blood Rev. 1992;6(1):43-57.). It is produced by many cell types, including lymphocytes, monocytes, endothelial cells, fibroblasts, and some malignant cells (Metcalf D. Blood. 1986;67(2):257-267; Clark SC, Kamen R. Science. 1987;236(4806):1229-1237; Hart PH et al. J Immunol. 1988;141(5):1516-1521; Metcalf D et al. Blood. 1986;67(1):37-45). In addition to having growth-stimulating and differentiating functions on hematopoietic progenitor cells, GM-CSF has also been found to have various effects on cells of the immune system that express the GM-CSF receptor (for reviews, see Hamilton JA. Trends Immunol. 2002;23(8):403-408; de Groot RP et al. Cell Signal. 1998;10(9):619-628).

[0003] Granulocyte macrophage colony-stimulating factor inhalation therapy has been disclosed for treating patients suffering from autoimmune alveolar proteinosis (aPAP) (Tazawa R et al. N Engl J Med. 2019;381(10):923-932), nontuberculous mycobacterial (NTM) infection (Scott JP et al. Eur Respir J. 2018;51(4):1702127), acute radiation syndrome (Singh VK et al. Cytokine. 2015;71(1):22-37), acute respiratory distress syndrome (Herold S et al. Am J Respir Crit Care Med. 2014;189(5):609-611), and cystic fibrosis (Heslet L et al. J Inflamm Res. 2012;5:19-27.) by nebulization.

[0004] Inhalation therapy reduces systemic side effects and offers the best possibility for optimal delivery to the lungs. Inhalation procedures target the airways by reducing the amount of drug required for optimal efficacy. Furthermore, the incidence of systemic side effects is usually low, along with the rapid onset of action of the drug. Nebulizers are a very suitable treatment method for fungal or bacterial lung infections because they fill the air with a very fine mist of respirable droplets that can be deposited at all sites of the lungs.

[0005] However, protein formulations as liquids for inhalation therapy are a very complex process because products that can be added to nebulizers and maintain a stable state during aerosol generation are required. The formulation should maintain stability during long-term storage, minimize the effect of nebulization on the integrity of the protein, and have an appropriate tonicity for transpulmonary delivery. Therefore, the delicate balance between stability, tolerance in the lungs including potential toxicity, and isotonicity is the key to achieving an effective protein formulation for inhalation therapy.

[0006] When the protein is formulated as a lyophilized product for resuspension, the same problem does not occur because long-term stability is ensured such that isotonicity can be achieved upon resuspension. Thus, the same excipient composition is not necessarily applicable to two different formulations.

[0007] A liquid formulation of GM-CSF (LEUKINE®) was previously disclosed for intravenous administration (US2002141970A1). US2002141970A1 suggests the addition of a chelating agent as an excipient to increase the stability of the compound.

[0008] The formulation of the medicament for atomization aims to optimize the solubility and stability of the drug, and minor changes in the formulation may also affect the inhalation amount, particle size distribution, and treatment time.

[0009] Atomization inherently has the risk of destabilizing the protein. The main cause of protein instability during atomization is unfolding and aggregation at the gas-liquid interface. Therefore, the use of GM-CSF for atomization requires a stable liquid formulation that is ready for use for inhalation and does not cause significant changes during atomization. SUMMARY OF THE INVENTION

[0010] The present invention discloses a stable liquid formulation of rhGM-CSF that is ready for use for inhalation and does not exhibit undue instability during atomization.

[0011] In one embodiment of the present invention, a combination of a sugar alcohol or sugar, albumin, and polyethylene glycol (PEG) with water provides a highly stable liquid formulation of rhGM-CSF.

[0012] In one embodiment, the liquid formulation described above is used as a medicament for atomization.

[0013] In one embodiment, the liquid formulation of rhGM-CSF is for use in the treatment of aPAP or NTM infection.

[0014] In one embodiment, a method for the pulmonary delivery of recombinant rhGM-CSF is disclosed, which includes the step of atomizing rhGM-CSF, a sugar alcohol, albumin, and PEG.

[0015] In a further embodiment, an atomizer containing a liquid formulation of rhGM-CSF is disclosed for use in the treatment of aPAP or NTM infection.

Brief Description of the Drawings

[0016]

Figure 1

Figure 2

Figure 3

Figure 4A

Figure 4B

Figure 5

Mode for Carrying Out the Invention

[0017] The present invention relates to a liquid formulation of recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) for use in administration by inhalation, wherein the liquid formulation comprises rhGM-CSF, a sugar alcohol or sugar, albumin, and water.

[0018] A specific combination of excipients ensures the high stability of rhGM-CSF in a liquid formulation that is ready for use in inhalation therapy.

[0019] In one embodiment, the rhGM-CSF is molgramostim (SEQ ID NO: 1), sargramostim (SEQ ID NO: 2), or Regramostim.

[0020] The amino acid sequence of molgramostim is identical to the amino acid sequence of endogenous human GM-CSF. The amino acid sequence of sargramostim differs from natural GM-CSF by a leucine substitution at position 23. In a preferred embodiment, the rhGM-CSF is molgramostim (SEQ ID NO: 1). Molgramostim is a non-glycosylated rhGM-CSF expressed in Escherichia coli.

[0021] In one embodiment, the liquid formulation of rhGM-CSF contains a sugar, wherein the sugar is selected from the group consisting of maltose, trehalose, sucrose, mannose, lactose, or galactose.

[0022] In another embodiment of the present invention, the liquid formulation of rhGM-CSF contains a sugar alcohol selected from the group consisting of mannitol or sorbitol. The addition of a polyol sugar to the liquid formulation of rhGM-CSF increases stability. Furthermore, the polyol sugar is used as an isotonic agent to ensure that the formulation is isotonic with human plasma. In a preferred embodiment, the sugar alcohol is mannitol.

[0023] Albumin is a family of globular proteins, and the most common one is serum albumin. Human serum albumin (HSA) is the most common protein contained in human plasma. The addition of HSA contributes to the stability of the rhGM-CSF liquid formulation. Also, due to its abundance and natural self-tolerance to albumin, the possibility of HSA inducing an immune response in a certain patient population can be minimized. Therefore, in one embodiment of the present invention, the liquid formulation of rhGM-CSF contains albumin. In a preferred embodiment, the albumin is HSA. In another embodiment of the present invention, the albumin is recombinant human albumin (rHA).

[0024] In one embodiment of the present invention, the liquid formulation of rhGM-CSF further contains PEG. Such PEGs include, but are not limited to, PEG1000, PEG1550, PEG2000, PEG3000, PEG3350, PEG4000, or PEG8000. Therefore, in one embodiment, the liquid formulation of rhGM-CSF further contains a PEG selected from the group consisting of PEG1000, PEG1550, PEG2000, PEG3000, PEG3350, PEG4000 or PEG8000. In a preferred embodiment, the PEG is PEG-4000.

[0025] The protein concentration is crucial to ensure a stable and ready-to-use formulation. Increasing the concentration of rhGM-CSF can increase the stability of the rhGM-CSF liquid formulation. In one embodiment of the present invention, the concentration of rhGM-CSF is at least 150 μg / mL. In a further embodiment of the present invention, the concentration of rhGM-CSF in the disclosed liquid formulation is in the range of at least 150 - 500 μg / mL. In another embodiment of the present invention, the concentration of rhGM-CSF in the disclosed liquid formulation is in the range of 200 - 300 μg / mL. The present disclosure shows that increasing the concentration of rhGM-CSF stabilizes the liquid formulation in the presence of the sugar alcohol mannitol (Figure 2).

[0026] To ensure the balance between isotonicity and stability of the liquid formulation of inhaled rhGM-CSF, embodiments of the present invention disclose a liquid formulation of rhGM-CSF, where the concentration of the sugar alcohol is at least 25 mg / mL. In a further embodiment of the present invention, the concentration of the sugar alcohol is in the range of 45-100 mg / mL. In another embodiment of the present invention, the concentration of the sugar alcohol is in the range of 45-55 mg / mL. As can be seen in Figure 1 of the present disclosure, increasing the concentration of the sugar alcohol mannitol improves the stability of rhGM-CSF. The stabilizing effect of mannitol is confirmed at a concentration of 25 mg / mL of the sugar alcohol. This effect increases significantly at a concentration of 50 mg / mL.

[0027] In one embodiment of the present invention, the liquid formulation of rhGM-CSF contains albumin at a concentration of at least 0.2 mg / mL. In a further embodiment, the liquid formulation of rhGM-CSF contains albumin in the concentration range of 0.2-10 mg / mL, for example, 0.3-5 mg / mL, for example, 0.5-1.5 mg / mL. In Figure 3 of the present disclosure, it is shown that in the rhGM-CSF liquid formulation (composite formulation) containing rHA and PEG4000 as stabilizers under both cooling and accelerated conditions, almost no impurities containing rhGM-CSF are formed. Furthermore, Figure 5 shows that the stabilizing effect of albumin can already be confirmed at a concentration of 0.2 mg / mL, and the stabilizing effect increases by increasing the concentration.

[0028] In one embodiment of the present invention, the liquid formulation of rhGM-CSF contains PEG. For example, the concentration of PEG is at most 50, for example, at most 40, for example, at most 30, for example, at most 20, for example, at most 10, for example, at most 1 mg / mL. In another embodiment of the present invention, the concentration of PEG is in the concentration range of, for example, 0.05-0.5 mg / mL, for example, 0.05-0.3 mg / mL, for example, 0.05-0.2 mg / mL. In a further embodiment of the present invention, the liquid formulation of rhGM-CSF contains PEG in the concentration range of 0.05-0.15 mg / mL.

[0029] The selection of the optimal combination and concentration of excipients and biotherapeutics is a very specific and complex process that is unique to individual protein formulations. The present invention discloses one embodiment of a liquid formulation of rhGM-CSF, where rhGM-CSF is molgramostim (SEQ ID NO: 1), the sugar alcohol is mannitol, the albumin is recombinant human albumin, and the PEG is PEG-4000.

[0030] In one embodiment, the concentration of rhGM-CSF is at least 150 μg / mL, the concentration of mannitol is at least 25 mg / mL, the concentration of recombinant human albumin is at least 0.2 mg / mL, and the concentration of PEG-4000 is at most 1 mg / mL.

[0031] In another embodiment of the present invention, the concentration of rhGM-CSF is 150 - 500 μg / mL, the concentration of mannitol is 45 - 100 mg / mL, the concentration of recombinant human albumin is 0.5 - 1.5 mg / mL, and the concentration of PEG-4000 is 0.05 - 0.15 mg / mL.

[0032] In a preferred embodiment, the concentration of rhGM-CSF is in the range of 200 - 300 μg / mL, the concentration of mannitol is 50 mg / mL, the concentration of recombinant human albumin is 1 mg / mL, and the concentration of PEG-4000 is 0.1 mg / mL.

[0033] Buffers were added to the formulation to adjust the pH, stabilize, and optimize drug solubility and stability. For drugs for inhalation, it is desirable that the pH of the product is close to physiological pH. In one embodiment of the present invention, the liquid formulation of rhGM-CSF further comprises a buffer. In a preferred embodiment, the buffer comprises citric acid monohydrate or Na2HPO4.

[0034] The molality osmotic concentration of the liquid formulation is an important parameter for the suitability of the liquid formulation for inhalation. Preferably, the liquid formulation is essentially isotonic. Thus, in one embodiment of the present invention, the molality osmotic concentration of the formulation is 250 to 375 mOsm / L, preferably 325 to 335 mOsm / L.

[0035] In one embodiment of the present invention, a liquid formulation of rhGM-CSF is disclosed in which the % of rhGM-CSF remaining after storage at 40 °C for 3 months exceeds 60%. In a further embodiment of the present invention, a liquid formulation of rhGM-CSF is disclosed in which the level of impurity formation after 6 months at 25 °C is less than 5%.

[0036] In one embodiment, the present invention further discloses a method for delivering rhGM-CSF to the lungs, which includes atomization of rhGM-CSF, sugar alcohol, albumin and PEG.

[0037] The liquid formulation of rhGM-CSF presented herein is particularly well-suited for atomization. The data of the present disclosure demonstrate that the efficacy of molgramostim for a liquid formulation containing rhGM-CSF, sugar alcohol or sugar, albumin, PEG and water retains 99.4% of the biological efficacy of rhGM-CSF upon atomization. Furthermore, it has been found that after atomization of this liquid formulation of rhGM-CSF, most of the rhGM-CSF remains in its original state after the atomization process. Finally, even when the liquid formulation presented herein is atomized at up to 18 months, little or no change in performance stability is observed.

[0038] In one embodiment of the present invention, at least 90%, such as at least 95%, such as at least 98%, such as at least 99% of the biological efficacy of rhGM-CSF is retained after atomization.

[0039] In a further embodiment of the present invention, the formulation has storage stability at 25 °C for at least 18 months.

[0040] In another embodiment of the present invention, the liquid formulation is for use in pulmonary administration. To facilitate pulmonary administration, the formulation must be aerosolized into small respirable droplets, most of which are less than 5 μm in diameter. In the case of biological preparations, the formulation must maintain stability during the aerosolization process, which can cause protein denaturation and / or aggregation due to high levels of mechanical energy and an increase in the area of the gas-liquid interface. The formulation should also contain excipients that are safe for administration to the lungs and have an appropriate range of pH and isotonicity.

[0041] In another embodiment of the present invention, the liquid formulation of rhGM-CSF is for use in the treatment of lung infections.

[0042] In a further embodiment, the liquid formulation of rhGM-CSF is for use in the treatment of bacterial, viral, or fungal infections in the lung. In one embodiment, the bacterial infection is selected from the group consisting of Streptococcus pneumoniae, Haemophilus species, Staphylococcus aureus, and Mycobacterium tuberculosis. In another embodiment, the viral infection is selected from the group consisting of bronchitis, pneumonia, and bronchiolitis. In yet another embodiment, the fungal infection is selected from the group consisting of Aspergillus, Cryptococcus, Pneumocystis, and endemic fungi.

[0043] In a preferred embodiment, the liquid formulation of rhGM-CSF is for use in the treatment of autoimmune pulmonary alveolar proteinosis (aPAP) or nontuberculous mycobacterial (NTM) infections, tuberculosis, ARDS, influenza infections, coronavirus infections, acute and chronic radiation syndromes, bronchial asthma, COPD, pulmonary fibrosis, lung cancer, and inflammatory disorders of the respiratory system. The formulation may also be used prophylactically to stimulate an immune response and protect against lung infections.

[0044] In an embodiment of the present invention, a liquid formulation of rhGM-CSF is disclosed for use as a drug for atomization. In another embodiment, a liquid formulation of rhGM-CSF is disclosed for use in the treatment of aPAP or NTM infections.

[0045] In one embodiment, a method of treating a medical condition is provided, the method comprising administering to a subject a liquid formulation of rhGM-CSF by atomization. In another embodiment, a method of treating aPAP or NTM infections is provided, the method comprising administering to a subject a liquid formulation of rhGM-CSF.

[0046] APAP is a rare autoimmune lung disease. It is the most common form (90% of cases) of pulmonary alveolar proteinosis (PAP). Most cases occur in adults between 20 and 50 years of age. Some people show no symptoms, while others experience progressive shortness of breath and breathlessness during exertion. Other signs and symptoms may include dry chronic cough, fatigue, weight loss, chest pain, and vague complaints. In rare cases, hemoptysis, clubbing and swelling of the fingertips, and cyanosis may be present. Autoimmune PAP is caused by immune system dysfunction due to IgG antibodies that block the effects of GM-CSF. GM-CSF regulates the removal of surfactant (a mixture of proteins and fats) by alveolar macrophages. Surfactant accumulates in the air sacs of the lungs (alveoli) and ultimately makes breathing impossible. The standard treatment is a procedure called lung lavage.

[0047] NTM is a mycobacterium that does not cause tuberculosis or leprosy. NTM causes lung diseases similar to tuberculosis. The most common clinical symptom of NTM disease is lung disease, but lymphatic, skin / soft tissue, and disseminated diseases are also important. Lung diseases caused by NTM are most commonly seen in postmenopausal women and patients with underlying lung diseases such as cystic fibrosis, bronchiectasis, and previous tuberculosis. It is not uncommon for patients with alpha1-antitrypsin deficiency, Marfan syndrome, and primary ciliary dyskinesia to develop pulmonary NTM colonization and / or infections. Pulmonary NTM can also be found in individuals with AIDS and malignant diseases.

[0048] The range and intensity of clinical symptoms vary but generally include a chronic cough with purulent sputum. Hemoptysis may also be seen. Systemic symptoms include malaise, fatigue, and weight loss in advanced disease. The diagnosis of Mycobacterium abscessus (M. abscessus) lung infection requires the presence of symptoms, radiological abnormalities, and microbial culture.

[0049] An inhaler containing a liquid formulation of rhGM-CSF according to the present disclosure. In a preferred embodiment, the inhaler is a nebulizer. Types of nebulizers include jet nebulizers, ultrasonic, and vibrating mesh. Other embodiments include soft mist inhalers, surface acoustic wave atomization, and capillary aerosol generators.

[0050] In one embodiment, the present invention discloses a method for delivering rhGM-CSF to the lungs, including atomization of a liquid formulation of rhGM-CSF, where the liquid formulation includes rhGM-CSF, sugar alcohol or sugar, albumin, and water, and the rhGM-CSF is delivered to the lungs by this method.

[0051] In another embodiment, the present invention discloses a nebulizer containing a liquid formulation of rhGM-CSF for use in the treatment of aPAP or NTM infections. This includes jet nebulizers, ultrasonic, and vibrating mesh nebulizers.

Examples

[0052] The effects of different formulations on the stability of rhGM-CSF were studied using HPLC. The content and purity of each formulation were evaluated by reverse-phase high-performance liquid chromatography (RP-HPLC). The determination of the higher-order structure (aggregates) and degradation products of rhGM-CSF was evaluated by size-exclusion high-performance liquid chromatography (SE-HPLC).

[0053] Example 1 (Figure 1) - Effect of mannitol concentration Three formulation parameters of molgramostim were varied according to the following table (Table 1). 1. Mannitol level (0 - 50 mg / mL): The buffer concentration was adjusted simultaneously so that the formulation maintained isotonicity. 2. Stabilizer (one of four options): rHA and PEG, rHA alone, no stabilizer, and Tween 80 alone 3. Molgramostim level (0.1 - 0.4 mg / ml)

[0054]

Table 1

[0055] Data were collected by the RP-HPLC method. The vials were stored at 40 °C for 3 months. There were two preparations for each of the 15 formulations in the above table. The main findings of this study were those shown in Figure 1. The level of mannitol had a significant effect on the stability under accelerated conditions over 3 months.

[0056] Example 2 (Figure 2) - Effect of rhGM-CSF concentration From the data collected as described in Example 1, it was further observed that as the rhGM-CSF concentration increased, a significant effect was also seen on the stability of the rhGM-CSF formulation at a specific concentration of mannitol.

[0057] Example 3 - Effect of rHA and PEG-4000 (Figure 3) The effects of rHA and PEG-4000 on the stability of molgramostim formulations for nebulization were studied under the following conditions.

[0058]

Table 2

[0059] Formulation preparation As described below, stability tests were carried out using versions (complex and simple) of the molgramostim formulation.

[0060] Molggramostim composite formulation The complex formulation was prepared on a weight basis according to the following description. Sterilization was carried out only on the finally formulated product. The content of the finally formulated product is as shown in the table below.

[0061]

Table 3

[0062] To produce the complex rhGM-CSF formulation, a buffer solution was first prepared according to the following procedure.

[0063] Preparation of citric acid / phosphoric acid / HSA buffer (e.g., batch size = 2000 g)

[0064]

Table 4

[0065] rHSA was taken out from a refrigerator at 2 - 8°C and equilibrated to room temperature. The required amounts of mannitol, PEG4000, citric acid monohydrate, disodium hydrogen phosphate (anhydrous) and rHSA were pre-weighed into a suitable container. A clean beaker / bottle and a stir bar were placed on a top-loading balance, and the weight of the beaker and stir bar was recorded to tare. Approximately 800 g of Water for Injection (WFI) was added to the beaker. The required amounts of citric acid monohydrate, disodium hydrogen phosphate (anhydrous), mannitol and PEG4000 were added to the WFI, the beaker was transferred to a magnetic stirrer plate, and the mixture was stirred until all the solids were dissolved. The beaker was returned to the top-loading balance, and the required amount of rHSA was added to the beaker. Then, the beaker was transferred to the magnetic stirrer plate and gently stirred for about 5 minutes or until the solution was homogeneous. Foaming should be avoided. Finally, WFI was added up to the target volume of 2000 g, and the solution was gently stirred again for about 10 minutes while avoiding foaming. The buffer was labeled and stored at 2 - 8°C until further processing. The buffer can be stored at 2 - 8°C for up to 100 hours.

[0066] Preparation of formulated product of rhGM-CSF (batch size in the example = 1000 g) After the preparation of the buffer, the rhGM-CSF complex formulation was prepared according to the following procedure. Weight (g) of rhGM-CSF solution = A Weight (g) of the formulated product = A+(10.1507×A)

[0067]

Table 5

[0068] rhGM-CSF was thawed at 2 - 8°C for 24 hours ± 4 hours. If the bulk material is not thawed within this time, gently rotate the bottle at room temperature to continue thawing. The bottle should not be shaken, and foaming should be avoided. The required amount of rhGM-CSF was pre-weighed into a container of appropriate size. A clean bottle and stir bar were placed on the top-loading balance. The weight of the beaker and stir bar was recorded to tare the balance. Approximately 800 g of citric acid / phosphate / rHA buffer was added to the beaker, and then the pre-weighed amount of rhGM-CSF was also added to the beaker. The container of rhGM-CSF was rinsed with citric acid / phosphate / HSA buffer, and the rinse was transferred to the beaker. Then, the beaker was placed on a magnetic stirrer and gently stirred for about 5 minutes while avoiding foaming. It was confirmed that the mixture was completely homogeneous. Citric acid / phosphate / HSA buffer was added to bring the final weight to 1000 g, and then gently stirred for about 10 minutes while avoiding foaming. The formulated product was filtered using a Millipore Stericup filter unit (PVDF membrane (Durapore), very low protein binding, pore size 0.22 μm), protected from light, and stored at 2 - 8°C.

[0069] Molggramostim simple formulation The simple formulation of rhGM-CSF is prepared on a volume basis because the density of the buffer for the simple formulation is unknown. The component concentrations and compounding amounts per vial of the final formulated product are shown in the following table.

[0070] [Table 6]

[0071] Preparation of the formulated product of rhGM-CSF (Example batch size = 1000 mL)

[0072] [Table 7]

[0073] The required amounts of mannitol, citric acid monohydrate, and disodium hydrogen phosphate (anhydrous) were pre-weighed in a suitable container. Approximately 800 mL of water for injection (WFI) was added to a beaker equipped with a stir bar. Citric acid monohydrate, disodium hydrogen phosphate (anhydrous), and mannitol were added to the WFI. The mixture was stirred until all the solids were dissolved. The required amount of rhGM-CSF was added to the beaker, the container of rhGM-CSF was rinsed with WFI, and the rinse solution was transferred to the beaker. The beaker was placed on a stirring plate, and the mixture was gently stirred for about 5 minutes while avoiding foaming. Check that it is completely homogeneous. The contents of the beaker were transferred to a 1 L volumetric flask. The beaker was rinsed with WFI, and the rinse solution was transferred to the volumetric flask. Care should be taken to avoid foaming during the transfer. WFI was added to the volumetric flask up to 1 L QS. Also, the contents were mixed by inverting the flask gently. The formulated product was filtered using a Millipore Stericup filter unit (PVDF membrane (Durapore), very low protein binding, pore size 0.22 μm). The final formulated product was protected from light and stored at 2 - 8°C.

[0074] The main findings of this study are as shown in Figure 2. In the formulation containing rHA and PEG4000 as a stabilizer (complex formulation), the formation of impurities of rhGM-CSF was less under both refrigeration and accelerated conditions. Furthermore, the following table shows that the complex formulation has significantly higher purity when stored at 40°C for 12 days compared to the simple formulation.

[0075]

Table 8

[0076]

Table 9

[0077] Example 4 - Stability after Atomization The stability of the rhGM-CSF complex formulation after atomization was evaluated by comparing the non-atomized formulation and the concentrated atomized formulation, and comparing the detected impurities and protein aggregates. The aerosol generated by the vibrating mesh was collected immediately after atomization by collecting it into a polypropylene tube placed against the mesh. Impurities and protein aggregates were detected by high performance liquid chromatography (HPLC) and size exclusion chromatography (SEC) respectively. The formulations evaluated in this study were identical to the above rhGM-CSF complex formulations except that the rhGM-CSF concentration was 300 μg / mL instead of 250 μg / mL. High concentrations of molgramostim are likely to show instability and represent the worst-case scenario.

[0078] The detection of unknown impurities determined by HPLC is shown in Table 2 below. The relative retention times (RRT) of these peaks relative to the main peak were 0.87, 0.96, and 1.07. After atomization using the Head type30 device, a slight increase (0.26%) in RRT 0.87 and residual in the reservoir (0.32%) were detected in the atomized sample. These results demonstrate that most of the rhGM-CSF remains in its original state after the atomization process.

[0079]

Table 10

[0080] The generation of impurities and aggregates due to atomization was initially examined by collecting and analyzing the aerosol produced by a vibrating mesh nebulizer (PARI eFlow). In the evaluation of aggregation by SEC, observable differences were found in albumin-related aggregates designated as HMW1 and HMW2. From this data, it is speculated that some aggregation of albumin occurs during the atomization process, but most of the proteins in the formulation remain non-aggregated.

[0081]

Table 11

[0082]

Table 12

[0083] The data collected in Table 4 are the rhGM-CSF concentrations of the complex formulation before and after atomization by the vibrating mesh nebulizer. The content was measured by RP-HPLC.

[0084] Finally, the efficacy of molgramostim was also evaluated before and after atomization. In the case of the complex formulation, it was confirmed that 99.4% of the biological efficacy of GM-CSF was retained during atomization. The biological efficacy was measured by TF-1 cell line assay.

[0085] Example 5 - Performance stability of rhGM-CSF complex formulation. The performance stability of the rhGM-CSF complex formulation during atomization was evaluated by storage at 5°C and 25°C for up to 18 months. The following three parameters were evaluated: DD = dose under simulated inspiration (<1601> by USP, adult), FPF = fine particle fraction (droplets less than 5 μm), and MMAD = mass median aerodynamic diameter. At the 18-month time point, there was little or no change in the performance stability due to atomization. The performance data were generated by pharmaceutical cascade impactor in accordance with USP <601>.

[0086] Example 6 - Stability of rHA low-concentration formulation A version of the complex rhGM-CSF containing low-concentration (0.2 mg / mL) rHA was evaluated and shown to have improved stability compared to a simple formulation without rHA or PEG4000. The content of rhGM-CSF was measured by RP-HPLC.

Claims

1. A liquid formulation of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) for use in administration by inhalation, said liquid formulation comprising rhGM-CSF, a sugar alcohol or sugar, albumin and water.

2. The liquid formulation of claim 1, wherein the rhGM-CSF is molgramostim (SEQ ID NO: 1), sargramostim (SEQ ID NO: 2), or regramostim.

3. 3. The liquid formulation of claim 1 or 2, wherein the sugar is maltose, trehalose, sucrose, mannose, lactose, or galactose, or the sugar alcohol is mannitol or sorbitol.

4. The liquid formulation of any one of claims 1 to 3, wherein the albumin is recombinant human albumin.

5. 5. The liquid formulation of any one of claims 1 to 4, further comprising polyethylene glycol (PEG), wherein the PEG is selected from the group consisting of PEG 1000, PEG 1550, PEG 2000, PEG 3000, PEG 3350, PEG 4000, or PEG 8000, preferably PEG-4000.

6. The liquid formulation according to any one of claims 1 to 5, wherein the concentration of rhGM-CSF is at least 150 μg / mL.

7. The liquid formulation according to claim 6, wherein the concentration of rhGM-CSF is in the range of 150 to 500 μg / mL, preferably 200 to 300 μg / mL.

8. The liquid formulation of any one of claims 1 to 7, wherein the concentration of the sugar alcohol is at least 25 mg / mL.

9. The liquid formulation according to claim 8, wherein the concentration of the sugar alcohol is in the range of 45 to 100 mg / mL, preferably 45 to 55 mg / mL.

10. 10. The liquid formulation of any one of claims 1 to 9, wherein the concentration of albumin is at least 0.2 mg / mL.

11. The liquid formulation according to any one of claims 1 to 10, wherein the concentration of albumin is in the range of 0.5 to 1.5 mg / mL.

12. 12. The liquid formulation according to any one of claims 1 to 11, wherein the concentration of PEG is up to 50, such as up to 40, such as up to 30, such as up to 20, such as up to 10, such as up to 1 mg / mL.

13. The liquid formulation of any one of claims 1 to 12, wherein the concentration of the PEG is in the range of 0.05 to 0.15 mg / mL.

14. The liquid formulation according to any one of claims 1 to 13, wherein the rhGM-CSF is molgramostim (SEQ ID NO: 1), the sugar alcohol is mannitol, the albumin is recombinant human albumin, and the PEG is PEG-4000.

15. 2. The liquid formulation of claim 1, wherein the concentration of rhGM-CSF is at least 150 μg / mL, the concentration of mannitol is at least 25 mg / mL, the concentration of recombinant human albumin is at least 0.2 mg / mL, and the concentration of PEG-4000 is at most 1 mg / mL.

16. 16. The liquid formulation of claim 15, wherein the concentration of rhGM-CSF is 200-300 μg / mL, the concentration of mannitol is 45-100 mg / mL, the concentration of recombinant human albumin is 0.5-1.50 mg / mL, and the concentration of PEG-4000 is 0.05-0.15 mg / mL.

17. Citric acid monohydrate or sodium ２ H.P.O. ４ The liquid formulation of any one of claims 1 to 16, further comprising a buffer such as

18. The liquid formulation according to any one of claims 1 to 17, wherein the liquid formulation has an osmolality of 250 to 375 mOsm / L, preferably 325 to 335 mOsm / L.

19. The liquid formulation according to any one of claims 1 to 18, wherein the % of rhGM-CSF remaining after 3 months storage at 40°C is greater than 60% and / or the level of impurity formation after 6 months storage at 25°C is less than 5%.

20. The liquid formulation according to any one of claims 1 to 19, wherein at least 90%, such as at least 95%, such as at least 98%, such as at least 99% of the biological potency of rhGM-CSF is retained after nebulization.

21. 21. The liquid formulation of any one of claims 1 to 20, wherein the liquid formulation is stable upon storage at 25°C for at least 18 months.

22. The liquid formulation of any one of claims 1 to 21, wherein the liquid formulation for inhalation is nebulized.

23. The liquid formulation of any one of claims 1 to 22, wherein the liquid formulation is for use in pulmonary administration.

24. The liquid formulation according to any one of claims 1 to 23, wherein the use is for the treatment of a pulmonary infection.

25. The liquid formulation according to any one of claims 1 to 24, wherein said use is for the treatment of a bacterial, viral or fungal infection in the lungs.

26. The liquid formulation according to any one of claims 1 to 25, wherein the use is for the treatment of autoimmune pulmonary alveolar proteinosis (aPAP) or nontuberculous mycobacterial (NTM) infections.

27. An inhaler comprising a liquid formulation according to any one of claims 1 to 26.

28. 28. The inhaler of claim 27, wherein the inhaler is a nebulizer.

29. A pulmonary formulation of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) comprising rhGM-CSF, a sugar alcohol or sugar, albumin and water.

30. A method for delivering rhGM-CSF to the lungs comprising nebulizing a liquid formulation of rhGM-CSF, said liquid formulation comprising rhGM-CSF, a sugar alcohol or sugar, albumin and water, and wherein said method delivers said rhGM-CSF.

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