Nano-embedded micron (NIM) encapsulated therapeutic nucleic acid dry powder and pharmaceutical comprising same

Abstract

The present invention relates to nano-embedded micron (NIM) encapsulated biologically active therapeutic nucleic acid dry powder comprising lipid nanoparticles comprising at least ionizable cationic lipids, helper lipids, stealth lipids and a therapeutic nucleic acid wherein the lipid nanoparticles are encapsulated in a pharmaceutically acceptable excipient, the excipient includes at least one substance selected from the group consisting of monosaccharides, polysaccharides, sugar alcohols, (poly) peptides, proteins, esters, polyurethanes, phosphate esters, phosphazenes, amino acids, surfactants, polymers. The invention also relates to a mass ratio of the therapeutic nucleic acid to the excipient of greater than 0.10%, preferably between 0.90% and 1.30%.

Description

[0001] describe This invention relates to a therapeutic nucleic acid dry powder encapsulated in nano-in-micro (NIM) layers according to claim 1. The invention also relates to a pharmaceutical product according to claim 11, particularly a pharmaceutical product for use with a dry powder inhalation device, said pharmaceutical product comprising the dry powder located within at least one sealed chamber of packaging.

[0002] Directly applying drugs to their site of action is the best way to reduce dosage and side effects. Lung delivery is therefore favored for lung diseases such as asthma. Substances susceptible to enzymes are ideal candidates for this route of administration due to their relatively low enzyme activity and slow surface clearance. Furthermore, dry powder inhalers enable the delivery of drugs with long shelf lives and provide patients with a rapid and easy-to-use tool, while offering high adherence. Although various treatments are available for lung diseases, nucleic acid therapy is a promising new tool for addressing uncontrolled disease variants (such as severe, uncontrolled asthma), and particularly for viruses for which no antiviral compounds are currently available. Knowing the viral genome is sufficient to develop nucleic acid-based therapeutics that can inhibit viral replication. Small interfering RNA (siRNA) can silence messenger RNA, pathologically upregulating protein translation and alleviating disease symptoms. Besides siRNA, other therapeutic nucleic acids (such as miRNA, shRNA, dsRNA, mRNA, circRNA, saRNA, tracrRNA, and lncRNA) are also promising candidates for nucleic acid-based therapeutics. However, siRNA-based and other therapeutic nucleic acid-based therapeutics face several challenges related to intracellular delivery and enzyme stability. To address these issues, nanoparticles are preferred for protecting and encapsulating therapeutic nucleic acids such as siRNA.

[0003] Existing technology WO2022 / 079105A1 discloses a method for producing nano-embedded, micron-encapsulated siRNA dry powder by spray drying from an aqueous suspension, which preserves the siRNA intact in the produced dry powder, i.e., largely retains its biological activity. This represents a significant advance in providing such gene therapy and corresponding clinical dosages of the drug. However, in the prior art, the amount (mass ratio) of siRNA to sugar, and this amount in the produced dry powder, remains relatively low. The prior art specifies that the mass ratio of siRNA to sugar and / or sugar alcohol is preferably between 0.001% and 0.02 to exhibit a high yield or high proportion of intact / biologically active siRNA in the dry powder. At this amount or concentration of siRNA, treating patients by inhaling the dry powder would require numerous repeated doses. Even if patients could inhale such large amounts of dry powder, such treatment or therapy would significantly reduce patient adherence.

[0004] However, increasing the quantity or mass ratio of therapeutic nucleic acids (especially siRNA) in the corresponding dry powder presents several challenges. First, if the mass ratio of therapeutic nucleic acids increases, the shielding effect of excipients or lipid nanoparticles that protect the therapeutic nucleic acids from environmental influences (especially heat) may decrease. This makes the therapeutic nucleic acids more susceptible to degradation during the production of the dry powder. Consequently, the amount of biologically active therapeutic nucleic acids may decrease, which may in turn prevent a sufficient net increase in the amount of biologically active therapeutic nucleic acids in the dry powder. Second, an increase in therapeutic nucleic acids also requires a corresponding increase in the mass percentage of lipid nanoparticles. Both changes can lead to increased hygroscopicity of the dry powder. This will adversely affect the shelf life of the dry powder and its delivery to the site of action, especially if this requires passage through a moist and warm environment (such as the patient's oropharynx) before reaching the site of action (i.e., the lungs). Furthermore, strong hygroscopic properties make it increasingly difficult to dry the powder to acceptable levels. This may necessitate higher temperatures during spray drying, which in turn may impair or destroy the function of the therapeutic nucleic acids. In addition, the high mass ratio of lipid nanoparticles to therapeutic nucleic acids provided in aqueous suspensions for spray drying makes it more difficult for lipid nanoparticles to chemically / physically bind with water-soluble excipients (such as sugars and / or sugar alcohols), thus leading to problems in the microencapsulation of lipid nanoparticles loaded with therapeutic nucleic acids.

[0005] The scientific article by Jingya Wu et al., "The Influence of Thermal and Shear Stress on the Stability of siRNA Dry Powder in Spray Drying" (International Journal of Pharmaceutics; 566(2019) 32-39), discloses that high dose or mass ratios (1 / 100) of siRNA to mannitol can be spray-dried without significantly affecting the bioactivity of the spray-dried siRNA. However, this article only covers one system in which siRNA and mannitol are dissolved in RNase-free water at a weight ratio of 1 / 100 to a concentration of 30 mg / mL. This spray-dried powder or its redispersible dispersion in the solvent cannot be transfected into cells and therefore cannot be internalized due to the lack of a suitable carrier system.

[0006] In other words, this method does not teach or imply the effect of the increased mass ratio of lipid nanoparticles on the system during or after spray drying, and generally does not result in powder that could potentially be used in pharmaceutical applications, as the siRNA would not have the opportunity to transfect into cells. Similarly, WO2022 / 079105A1 cited above teaches a system that can be transfected into cells due to lipid nanoparticle carriers, but because the amount of siRNA used is very small, it only teaches the use of very small amounts of lipid nanoparticles.

[0007] Therefore, the object of the present invention is to provide a therapeutic nucleic acid dry powder with nano-embedded micron-encapsulated structure, which can be used clinically or as a pharmaceutical preparation. Summary of the Invention

[0008] This problem is solved by the bioactive therapeutic nucleic acid dry powder encapsulated in nano-embedded micrometers (NIM) as described in claim 1.

[0009] Advantageous embodiments of the invention are presented in the following description, drawings, and dependent claims. Features described below or claims that are in the form of apparatus or product features should also be considered as disclosed and claimed as method features, and vice versa.

[0010] The bioactive therapeutic nucleic acid dry powder of the present invention, which is nano-embedded in micrometers (NIM) and encapsulated, comprises lipid nanoparticles, said lipid nanoparticles comprising at least ionizable cationic lipids, auxiliary lipids, occult lipids, and therapeutic nucleic acids, wherein said lipid nanoparticles are encapsulated in a pharmaceutically acceptable excipient, said excipient comprising at least one substance selected from the group consisting of: monosaccharides, polysaccharides, sugar alcohols, (poly)peptides, proteins, esters, urethanes, phosphate esters, phosphazenes, amino acids, surfactants, and polymers, wherein the mass ratio of said therapeutic nucleic acid to said excipient is greater than 0.10%, particularly between 0.90% and 1.30%.

[0011] Surprisingly, it has been found that nano-embedded micron-encapsulated therapeutic nucleic acids (preferably siRNA) can be produced, especially by the spray drying method disclosed in WO2022 / 079105A1, wherein the mass ratio of siRNA or therapeutic nucleic acid to excipient is high enough to enable clinical or pharmaceutical applications in humans.

[0012] The contents of WO2022 / 079105A1 concerning the process / method of spray drying and the bioactivity analysis of therapeutic nucleic acids in dry powder are incorporated herein by reference.

[0013] It has been demonstrated that this result can be achieved without losing the shielding effect on the therapeutic nucleic acid, thereby reproducing high yields of bioactive / intact spray-dried therapeutic nucleic acids. Furthermore, it has been demonstrated that the dry powder can be produced without excessive or overly hygroscopic properties. Also surprisingly, the significant increase in the amount of lipid nanoparticles did not adversely affect the spray-dried powder and / or the encapsulated therapeutic nucleic acid (preferably siRNA), thus not hindering the use of the dry powder in pharmaceutical and commercial applications (especially for inhalation). The amount of lipid nanoparticles may need to reach approximately 10% of the mass of the dry powder (see below). Unexpectedly, this significant change in the overall formulation did not have a significant impact on the spray-drying process, the state of the spray-dried powder and / or the therapeutic nucleic acid (preferably siRNA) in the dried state. In particular, with a proportionally reduced amount of stabilizing excipients, the thermal energy input per unit of therapeutic nucleic acid during spray drying is much higher, and therefore, surprisingly, the therapeutic nucleic acid is not damaged during the spray-drying process.

[0014] One difference compared to the spray drying process disclosed in WO2022 / 079105A1 is that the concentration of lipid nanoparticles is increased before adding the lipid nanoparticles with encapsulated siRNA or therapeutic nucleic acid to the aqueous excipient (preferably a 10% lactose solution in purified water). This concentration is determined by standard procedures and is necessary because lipid nanoparticles can only be produced at limited concentrations. In a preferred variant of this known method, this concentration is increased from approximately 16 mM of the direct preparation to 60 mM to 70 mM.

[0015] Surprisingly, the concentrated lipid nanoparticles can be combined with water-soluble excipients without significant dispersion problems, and can be spray-dried without significantly negatively affecting the state or activity of the spray-dried siRNA or therapeutic nucleic acid.

[0016] The ionizable cationic lipids used may include or be composed of lipids that can carry a positive charge under specific pH conditions. Their functions include facilitating nucleic acid encapsulation in lipid nanoparticles (LNPs), mediating endosome membrane rupture to release nucleic acids, and potentially supporting endosome uptake. Examples include, for instance, 1,2-dioleoyl-3-dimethylammonium propane (DODAP), DLin-MC3-DMA (a lipid that is a key component in LNP formulations used in Pfizer-BioNTech and Moderna COVID-19 vaccines), and other similar ionizable lipids.

[0017] Stealth lipids (typically PEGylated lipids or alternatives thereof) are added to LNPs to improve their stability, prolong their circulation time in vivo, and help them evade the immune system. Examples of stealth lipids include polyethylene glycol-lipids (e.g., DSPE-PEG2000), polyvinylpyrrolidone (PVP) lipids, polyvinyl alcohol (PVA) lipids, poloxamer lipids, and polysarcosine lipids. All of these examples are used to provide stealth properties to LNPs, which enhances their stability and improves their biodistribution.

[0018] Supporting lipids support the stability of LNPs during storage and circulation. They can include a range of lipids such as sterols, phospholipids, and glycerides. Examples include cholesterol (a common sterol), DOPE (dioleoylphosphatidylethanolamine, a phospholipid), and DOPC (dioleoylphosphatidylcholine, another phospholipid). These lipids are typically noncationic and contribute to improving the fluidity and flexibility of LNPs, as well as their overall stability. In a preferred embodiment of the invention, two supporting lipids are used. When using two supporting lipids, it is particularly advantageous that one of them is cholesterol.

[0019] The selection of each lipid group depends largely on the specific formulation and desired properties of the LNP. Different applications may require adjustments to the type and proportion of these components.

[0020] As suggested in WO2022 / 079105A1, the initial spray drying process is carried out using sugars and / or sugar alcohols as pharmaceutically acceptable excipients. However, excipients may also be selected from at least one or a combination of at least one of the following substances: monosaccharides, polysaccharides, sugar alcohols, (poly)peptides, proteins, esters, polyurethanes, phosphate esters, phosphazenes, amino acids, surfactants, and polymers. Excipients generally have a relatively small impact on the spray drying process and can therefore be adapted to the specific needs of the corresponding dry powder.

[0021] On the other hand, the excipient composition has a significant impact on the LNP stability properties of dry powder formulations. The excipient composition is adjusted based on the overall composition of the LNP (lipids and cargo molecules) and the total LNP concentration in the dry powder to optimally preserve the LNP and ensure long-term storage stability.

[0022] In addition, the composition of excipients affects the physicochemical properties of dry powder, such as particle size and particle size distribution, moisture content, surface properties and morphology, density, flow properties, disintegration time and solubility, crystallinity and polymorphism, drug distribution and uniformity, electrostatic properties and thermal properties.

[0023] The combination of excipients significantly determines the solubility of the dry powder and subsequently ensures its uptake into cells.

[0024] In addition, they determine the inhalable properties of dry powder, such as delivery dose, median aerodynamic diameter, and fine particle fraction.

[0025] According to a preferred embodiment of the dry powder, the therapeutic nucleic acid comprises at least one substance selected from the group consisting of siRNA, miRNA, shRNA, dsRNA, mRNA, circRNA, saRNA, tracrRNA, and lncRNA. The therapeutic nucleic acid can achieve a very wide range of applications, especially the pharmaceutical use of the corresponding dry powder. The therapeutic nucleic acid is structurally similar to siRNA sufficiently that the established principle of nano-intercalation and micro-encapsulation via spray drying can be transformed into different types of therapeutic nucleic acids having the high mass ratio of the therapeutic nucleic acid to excipient of the present invention.

[0026] For example, antisense oligonucleotides (ASOs), a subgroup of therapeutic nucleic acids, are single-stranded DNA or RNA molecules engineered to complement specific target mRNA sequences. Their characteristics, such as a smaller number of nucleotides, need to be optimized for nanoparticle formulations. While small interfering RNAs (siRNAs) do not necessarily require chemical modification of nucleotides, modification of ASOs is often necessary to ensure intracellular stability.

[0027] Generally, siRNA and ASO can be used for similar purposes (such as gene silencing), but they have different mechanisms of action. ASO binds directly to a complementary mRNA sequence, preventing translation or promoting degradation through an RNase H-mediated mechanism. In contrast, siRNA is integrated into an RNA-induced silencing complex (RISC), which then binds to and degrades the target mRNA. Therefore, the degradation kinetics differ between the two approaches.

[0028] According to a preferred embodiment of the dry powder, at least 50%, preferably at least 75%, more preferably at least 85% of the therapeutic nucleic acid is bioactive in the dry powder state and / or after redispersibility. The bioactivity of the spray-dried therapeutic nucleic acid can be evaluated as disclosed in WO2022 / 079105A1. In a preferred embodiment, the therapeutic nucleic acid in the dry powder is structurally intact. In other words, the therapeutic nucleic acid in the dry powder is free of degradation products. A high percentage of bioactivity of the therapeutic nucleic acid is particularly important for the quality ratio of the therapeutic nucleic acid in the dry powder. This is because even if a powder with a high initial amount or proportion of therapeutic nucleic acid can be spray-dried, the bioactivity after spray drying determines whether the powder can be practically or conveniently used, for example, as a pharmaceutical agent for dry powder inhalers (DPIs).

[0029] According to another advantageous embodiment of the invention, the residual moisture content of the dry powder is less than 7.5%, preferably less than 5%, and especially less than 3%. This results in a longer shelf life for the obtained dry powder. Preferably, this residual moisture content can be achieved through an additional drying process following the initial spray drying.

[0030] According to an advantageous variation of the invention, the lipid mass ratio of the dry powder is 8% or more, preferably 10% or more. This is advantageous because a high lipid mass ratio supports a high quality ratio of the therapeutic nucleic acid. Surprisingly, even with such a high amount or mass ratio of lipids in the dry powder, the hygroscopic properties of the dry powder remain within acceptable ranges for use as a dry powder inhaler.

[0031] According to another preferred embodiment, the median diameter (MMD) of the dry powder is between 1 μm and 6 μm. This preferred MMD optimizes the use of the dry powder for pulmonary delivery via inhalation.

[0032] In another preferred embodiment, the dry powder (particularly in its redispersed state in an aqueous medium) exhibits cell transfectivity into living cells. As expressed above, for the therapeutic effect of bioactive therapeutic nucleic acids encapsulated in nano-embedded micrometers (NIMs), it is not only important to apply a substance with a sufficiently high ratio or amount of therapeutically active ingredient (such as therapeutic nucleic acid) near the target cells. Furthermore, the uptake or internalization of the therapeutic nucleic acid is a key factor enabling it to exert its therapeutic effect. Therefore, endowing the dry powder or its redispersed form in an aqueous medium with cell transfectivity into living cells is a very important aspect for using the dry powder in therapeutic applications.

[0033] According to another embodiment of the invention, the excipient comprises or is composed of sugars and / or sugar alcohols, preferably lactose, mannitol, trehalose, and / or leucine. It is believed that the excipients endorsed above have a particularly advantageous effect on the resulting dry powder. However, the corresponding mechanism has not been fully understood or proven. The remaining excipients of claim 1 are generally equally suitable for the production of the dry powder.

[0034] In another embodiment of the invention, the lipid nanoparticles conform to an Onpattro® formulation or a patisiran formulation. The use of Onpattro® is a clinically approved substance, and the corresponding Onpattro® formulations are readily available, making the use of Onpattro® and its derivatives a good starting point for LNPs, as they are expected to facilitate easier clinical approval. The corresponding lipid nanoparticles are disclosed in US 8,158,601B2, which is incorporated herein by reference. The corresponding formulation is recorded by the EMA under the product name EU / 3 / 11 / 857 and is given the non-proprietary name “PATISIRAN”.

[0035] In an alternative preferred embodiment of the present invention, the ionizable cationic lipid is selected from the group consisting of: C12-200, DOTAP (1,2-dioleoyl-3-trimethylammonium propane), DODAP (1,2-dioleoyl-3-dimethylammonium propane), DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane), DLinDMA (N,N-dilinoleoyl-N,N-dimethylammonium chloride), DLin-KC2-DMA (6Z,9Z,28Z,31Z-heptadec-6,9,28,31-tetraen-19-yloxy(N,N-dimethylcarbamoylmethyl)amine), HGT4003 (chemical name not specified), cKK-E12 ((C2-dimethylamino) Ethyl methyl thiosulfonate, ICE (4A3-SC8), ALC-0315 ((6Z,9Z,28Z,31Z)-heptadec-6,9,28,31-tetraen-19-yloxy(N,N-dimethylcarbamoylmethyl)amine), SM-102 (9-heptadecyl 8-((2-hydroxyethyl)(6-oxo-6-((pentylamino)methyl)hexyl)amino)octanoate), and combinations thereof. These ionizable cationic lipids enable highly efficient intracellular transfection.

[0036] In another preferred embodiment, the dry powder is used as a pharmaceutical dosage form, particularly for pulmonary delivery. In this regard, it is particularly advantageous that when produced by a spray drying process (such as the spray drying process disclosed in WO2022 / 079105A1), the dry powder produces a particle size and particle size distribution suitable for pulmonary delivery. This allows the powder to be used directly without further processing; that is, the dry powder can be directly packaged and used. This is achieved, in particular, by the fact that the particles of the dry powder have a therapeutic nucleic acid to excipient mass ratio greater than 0.10% (particularly between 0.90% and 1.30%). Surprisingly, the amount of lipid nanoparticles required to achieve the corresponding therapeutic nucleic acid mass ratio (which can account for 10% or more of the mass of the dry powder) does not affect the spray drying process, the resulting dry powder, or the state and function of the therapeutic nucleic acid in the dried and / or redispersed state.

[0037] According to another aspect of the invention, the therapeutic nucleic acid in the dry powder is active in silencing the translation of messenger RNA and / or silencing the replication of viral RNA leading to lung disease into proteins. This enables very potent application opportunities because the dry powder is, in some cases (directly after spray drying), in a condition suitable for use with DPIs. Therefore, the treatment of lung diseases and the use of corresponding therapeutic nucleic acids are promising applications of the dry powder of the present invention.

[0038] In another preferred embodiment of the dry powder, the therapeutic nucleic acid is active in promoting the expression of natural proteins, especially autologous proteins. This enables the dry powder to be used in protein replacement therapy applications where the body is unable to produce a certain protein. By promoting the expression of natural proteins, especially autologous proteins, the deficiency of natural proteins, especially autologous proteins, can be overcome. In these applications, clinical manifestations are triggered by the absence of endogenous or autologous proteins. The body incorrectly produces the protein, produces a non-functional form of the protein, or does not produce the protein at all. One possible application is for protein replacement therapy in cystic fibrosis, where the therapeutic nucleic acid promotes the expression of the membrane protein cystic fibrosis transmembrane conduction regulator (CFTR), which is incorrectly produced by the cells.

[0039] In another preferred embodiment of the invention, the therapeutic nucleic acid is active in promoting the expression of proteins characteristic of the specific virus and recognizable by the immune system. In this embodiment, the dry powder can be used in vaccine applications similar to SARS-CoV-2 vaccination, in which mRNA is used as a therapeutic nucleic acid to promote the expression of the SARS-CoV-2 spike protein. The immune system recognizes these characteristic spike proteins as foreign substances and responds to them. Among other things, it forms antibodies against the virus-specific spike protein.

[0040] The aforementioned problems are also solved by a medicine, particularly for use with a dry powder inhaler, comprising a package that seals at least one sealed chamber (preferably multiple sealed chambers), wherein the dry powder of any of the previously described embodiments is sealed within the at least one sealed chamber. This allows for very convenient use of the dry powder by the patient. The package can be constructed and shaped to be used with one or more different types of dry powder inhalers. In a preferred embodiment, the package can be implemented as a blister pack. Preferably, the package comprises multiple sealed chambers. According to a preferred embodiment, the package is constructed and shaped to be used with a dry powder inhaler in a magazine-like function, thereby allowing the sealed chambers to be “loaded” into the administration position and / or mechanically opened when the preparation mechanism (such as a loading lever) of the dry powder inhaler is activated. Preferably, the package is constructed to release the dry powder into an airflow (preferably an air stream) when the release mechanism of the dry powder inhaler is activated. In an alternative preferred embodiment, the package can have the form and function of a capsule, within which the dry powder is sealed. Attached Figure Description

[0041] Various aspects of the invention have been described with reference to the accompanying drawings, which illustrate examples and advantageous embodiments of the invention.

[0042] These figures show: Figure 1 Scanning electron microscope image of an embodiment of the dry powder of the present invention; Figure 2 The results of the siRNA integrity test of the dry powder according to the present invention; Figure 3 The particle size distribution of the dry powder according to the invention after its release from a capsule-based dry powder inhaler is shown. Figure 4 This demonstrates in vitro gene silencing of EGFP in the H1299-EGFP cell line—a human epithelial-like lung cancer cell line carrying a plasmid for enhanced green fluorescent protein (EGFP). Figure 5 This study presents an in vivo mouse model of asthma, demonstrating the downregulation of type 2 asthma-related cytokines and % eosinophils in bronchoalveolar lavage (BAL) fluid following asthma NIM treatment. Figure 6 The residual moisture levels of different dry powder formulations according to the present invention, as evaluated by Karl Fischer (KF) titration, are shown.

[0043] Figure 7 The figure shows the fine particle fraction (FPF) of the dry powder according to the invention—the percentage of powder composed of MMD between 1 μm and 5 μm.

[0044] Figure 1 A scanning electron microscope (SEM) image of the dry powder (01) according to the invention is shown. The particles (02) of the dry powder show a spherical shape. As can be seen from the scale bar (03) representing a length / distance of 10 μm, the particles (02) have a diameter of less than 10 μm.

[0045] Figure 2 The results of a therapeutic nucleic acid integrity test (bioanalyzer) are shown. Columns two and three represent a comparison between fresh and spray-dried lipid nanoparticles with the corresponding therapeutic nucleic acids. The test bands / lines at positions 4 and 20 of the step ladder represent structurally and functionally intact therapeutic nucleic acids. Figure 2 As shown, for the dry powder, there are no internal lines between 4 and 20. This means that the degradation of the therapeutic nucleic acid has not occurred, and the therapeutic nucleic acid is intact in the dry powder.

[0046] Figure 3 The particle size distribution of the dry powder according to the invention after release from a capsule-based dry powder inhaler is shown. The median diameter (MMD) was evaluated by laser diffraction (Sympatec) with an inhalation module. It is shown that the MMD is between 1 μm and 6 μm, which is required for inhaled dry powder lung delivery.

[0047] Figure 4 In vitro gene silencing of EGFP in the H1299-EGFP cell line—a human epithelial-like lung cancer cell line carrying a plasmid for enhanced green fluorescent protein (EGFP)—is illustrated. The gene silencing efficiency of the different dry powder formulations according to the invention is not significantly different compared to the fresh nanoparticle formulation prior to spray drying, thus preserving the silencing efficiency.

[0048] Figure 5An in vivo asthma model demonstrated downregulation of type 2 asthma-related cytokines and % eosinophils in bronchoalveolar lavage (BAL) fluid following asthma NIM treatment. An OVA-induced acute asthma mouse model was used. Over two weeks, animals in the treatment groups—one group treated with active oligonucleotides and the other with non-coding oligonucleotides—were sensitized with OVA and adjuvants, followed by three intranasal OVA challenges to induce asthma symptoms. Prior to these challenges, three doses of 2 mg anti-GATA3 NIM dry powder and a non-coding control NIM were administered intratracheally to assess whether NIM could reduce anaphylactic reactions. Animals were sacrificed 24 hours after the last treatment. BAL fluid was collected from the animals.

[0049] Figure 6 The residual moisture levels of different dry powder formulations according to the invention, as evaluated by Karl Fischer (KF) titration, are shown. All values ​​maintain a water content below 3.5% in the final formulation.

[0050] Figure 7 The fine particle fraction (FPF) of the dry powder according to the invention after release from a capsule-based dry powder inhaler is shown—the percentage of powder consisting of MMD (micron-to-diameter ratio) between 1 μm and 5 μm. The FPF was evaluated by laser diffraction with an inhalation module (Sympatec). A high FPF is shown.

Claims

1. A bioactive therapeutic nucleic acid dry powder encapsulated in nano-embedded micrometers (NIM), the dry powder comprising lipid nanoparticles, wherein the lipid nanoparticles comprise at least ionizable cationic lipids, accessory lipids, occult lipids, and therapeutic nucleic acids, wherein, The lipid nanoparticles are encapsulated in a pharmaceutically acceptable excipient, which comprises at least one substance selected from the group consisting of: monosaccharides, polysaccharides, sugar alcohols, (poly)peptides, proteins, esters, polyurethanes, phosphate esters, phosphazenes, amino acids, surfactants, and polymers. Its features are, The mass ratio of the therapeutic nucleic acid to the excipient is greater than 0.10%, preferably between 0.90% and 1.30%.

2. The dry powder according to claim 1, Its features are, The therapeutic nucleic acid includes at least one substance selected from the following: siRNA, miRNA, shRNA, dsRNA, mRNA, circRNA, saRNA, tracrRNA, and lncRNA.

3. The dry powder according to claim 1 or 2, Its features are, At least 50%, preferably 75%, and most preferably at least 85% of the therapeutic nucleic acid is biologically active in the dry powder state and / or after redispersibility.

4. The dry powder according to any one of the preceding claims, Its features are, The residual moisture content of the dry powder is less than 7.5%, particularly less than 5%, and preferably less than 3%.

5. The dry powder according to any one of the preceding claims, Its features are, The lipid content of the dry powder is 8% or more, preferably 10% or more.

6. The dry powder according to any one of the preceding claims, Its features are, The mass median diameter (MMD) of the dry powder is between 1 μm and 6 μm.

7. The dry powder according to any one of the preceding claims, Its features are, The dry powder, especially in its redispersed state in an aqueous medium, exhibits cell transfection capability into living cells.

8. The dry powder according to any one of the preceding claims, Its features are, The excipients include sugars and / or sugar alcohols, or are composed of sugars and / or sugar alcohols, preferably lactose, mannitol, trehalose and / or leucine.

9. The dry powder according to any one of the preceding claims, Its features are, The ionizable cationic lipids are selected from the group consisting of: C12-200, DOTAP (1,2-dioleoyl-3-trimethylammonium propane), DODAP (1,2-dioleoyl-3-dimethylammonium propane), DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane), DLinDMA (N,N-dilinoleyl-N,N-dimethylammonium chloride), DLin-KC2-DMA (6Z,9Z,28Z,31Z-heptadec-6,9,28,31-tetraen-19-yloxy(N,N-dimethyl)- (N,N-dimethylcarbamoylmethyl)amine), HGT4003 (chemical name not specified), cKK-E12 ((C2-dimethylamino)ethyl methylthiosulfonate), ICE (4A3-SC8), ALC-0315 ((6Z,9Z,28Z,31Z)-heptadec-6,9,28,31-tetraen-19-yloxy(N,N-dimethylcarbamoylmethyl)amine), SM-102 (9-heptadecyl-8-((2-hydroxyethyl)(6-oxo-6-((pentylamino)methyl)hexyl)amino)octanoate), and combinations thereof.

10. The dry powder according to claims 1 to 9, which is used as a pharmaceutical dosage form, especially for pulmonary delivery.

11. The dry powder according to claims 1 to 10, in, The therapeutic nucleic acid is active in silencing the translation of messenger RNA into proteins and / or silencing viral RNA replication that leads to lung disease.

12. The dry powder according to any one of the preceding claims, Its features are, The therapeutic nucleic acid is active in promoting the expression of natural proteins, especially autologous proteins.

13. The dry powder according to any one of the preceding claims, Its features are, The therapeutic nucleic acid is active in promoting the expression of proteins that are characteristic of the specific virus and can be recognized by the immune system.

14. A medicine, particularly for use with a dry powder inhaler, said medicine comprising a package enclosing at least one sealed chamber, preferably multiple sealed chambers, wherein, The dry powder is sealed within the sealed chamber. Its features are, Dry powder according to any one of the preceding claims.

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