Viral vector-containing inhalation powder and method for producing the same

Abstract

This invention provides a viral vector-containing inhalation powder and a method for producing the same. [Solution] An inhalation powder formulation prepared by the SFD method, containing a viral vector and dileucine. This provides an inhalation powder formulation that is excellent in lung reach, as well as in biological activity efficacy and stability.

Description

[Technical Field]

[0001] This specification relates to a viral vector-containing inhalation powder and a method for producing the same. [Background technology]

[0002] Inhaled medications can be advantageous in the treatment of lung diseases. These include liquid, powder, and aerosol formulations. Liquid formulations are atomized by a nebulizer and inhaled by the patient. However, these inhaled medications have problems such as significant residue in the device and circuit, drug loss due to exhalation, and long administration times.

[0003] Inhaled powder medications, being in powder form, can deliver drugs directly and rapidly to lung tissue, making them a potentially highly effective treatment option.

[0004] For example, it is known that powder formulations can be obtained from raw material liquids containing common drugs using the freeze-drying (FD) method. The FD method allows for formulations that have been processed at low temperatures and have reduced moisture content (for example, Patent Document 1). [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2016-199591 [Overview of the project]

[0006] Since inhalation powder formulations require excellent lung reach, formulation using the spray-freezed-drying (SFD) method, which produces powders composed of hollow, porous microparticles, is expected to be effective.

[0007] In the development of inhalation powder formulations, there are also expectations for the application of viral vectors, such as those used for gene therapy, that are involved in increasing or suppressing gene expression within cells.

[0008] However, the SFD method had a problem in that the drug's effectiveness was easily reduced due to the load incurred when spraying the drug-containing solution. Therefore, especially when using a viral vector as the drug, there was a risk that the drug's efficacy, i.e., its biological activity such as increased or suppressed gene expression, would decrease after formulation. Furthermore, from the perspective of distribution and storage of the formulation, it was also important that the biological activity of the viral vector be stably maintained within the formulation. In addition, the inhaled formulation needed to have good airborne dispersibility to ensure lung reach. To date, it has not been possible to obtain an inhaled powder formulation that satisfies the requirements of viral vector efficacy, stability, and lung reach using the SFD method.

[0009] This specification provides an inhalation powder formulation prepared by the SFD method, comprising a viral vector as an active ingredient, and exhibiting excellent lung reach, biological activity efficacy, and stability, as well as a method for producing the same. [Means for solving the problem]

[0010] The inventors investigated various components for the production of inhalation powders using the SFD method, with the intention of considering the biological activity, specifically the ability of the viral vector to reach the lungs and its gene therapy effect within the lungs, as active ingredients. As a result, it was found that dileucine contributes to the effectiveness and stability of biological activity in inhalation powders produced by the SFD method, in addition to excellent airborne dispersibility and lung reach. Furthermore, it was found that using cellulose derivatives in addition to dileucine can also contribute to the effectiveness of biological activity.

[0011] [1] An inhalation powder comprising a spray freeze-dried powder containing a viral vector and dileucine. [2] The inhalation powder according to [1], further comprising a cellulose derivative. [3] The inhalation powder according to [2], wherein the cellulose derivative is one or more selected from the group consisting of methylcellulose, hydroxypropylcellulose, and carboxymethylcellulose. [4] The inhalation powder according to [2] or [3], wherein the cellulose derivative is hydroxypropylcellulose. [5] The inhalation powder according to any one of [2] to [4], wherein the hydroxypropyl cellulose has a weight-average molecular weight of 10,000 or more and 150,000 or less. [6] The inhalation powder according to [5], wherein the hydroxypropyl cellulose has a weight-average molecular weight of 20,000 or more and 60,000 or less. [7] The inhalation powder according to [5], wherein the hydroxypropyl cellulose has a weight-average molecular weight of 80,000 or more and 120,000 or less. [8] An inhalation powder according to any one of [2] to [7], containing the dileucine and the cellulose derivative in a mass ratio of 4:6 to 6:4. [9] The inhalation powder according to any one of [2] to [8], wherein the viral vector comprises an adeno-associated virus vector.

[10] The viral vector is provided in a quantity of 1.0 × 10 per 1 mg of the total amount of the dileucine and the cellulose derivative. 9 GC or above 25×10 9 An inhalation powder containing GC or less, as described in any of [2] to [9].

[11] The cellulose derivative is hydroxypropyl cellulose, having a weight-average molecular weight of 80,000 or more and 120,000 or less, and the amount of the viral vector is 1.0 × 10¹⁶ mg per 1 mg of the total amount of the dileucine and the cellulose derivative. 9 GC or above 25×10 9 An inhalation powder containing GC or less, as described in any of [2] to

[10] .

[12] The inhalation powder according to any one of [1] to

[11] , wherein the average particle size of the spray freeze-dried powder is 5 μm or more and 30 μm or less.

[13] An inhalation powder described in any of [1] to

[12] for storage at 1°C or above and 30°C or below.

[14] A method for producing an inhalation powder containing spray freeze-dried powder, A step of obtaining a spray-frozen product by spray-freezing a sample solution containing a viral vector, a cellulose derivative, and dileucine, A step of obtaining spray freeze-dried powder by sublimating the water in the spray-frozen material, A manufacturing method that includes the following features.

[15] The method for producing the sample solution according to

[14] , wherein the sample solution contains the cellulose derivative and dileucine in a total amount of 10 mg / ml or more and 50 mg / ml or less. [Brief explanation of the drawing]

[0012] [Figure 1] This figure shows an overview of the composition of spray-freeze-dried adeno-associated virus vector (AAVV)-containing and non-containing inhalation powders in the examples. [Figure 2] This figure shows an electron microscope image of particles contained in an AAVV-free inhalation powder. [Figure 3] This figure shows the evaluation results of the particle size distribution (airborne dispersibility) and aerosol performance (lung reach) of an AAVV-free inhalation powder. [Figure 4] This figure shows the evaluation results of gene expression activity and cytotoxicity of an AAVV-containing inhalation powder. [Figure 5] This figure shows the results of the evaluation of the storage stability of an AAVV-containing inhalation powder. [Figure 6] This figure shows the results of evaluating the gene expression effect of intrapulmonary administration (low content) of an AAVV-containing inhalation powder. [Figure 7] This figure shows the results of evaluating the gene expression effect of intrapulmonary administration (high content) of an AAVV-containing inhalation powder. [Figure 8] This figure shows the results of evaluating the gene expression effect of intrapulmonary administration of an AAVV-containing inhalation powder. [Modes for carrying out the invention]

[0013] This specification describes the inhalation powder (hereinafter also simply referred to as "the Product") and its manufacturing method.

[0014] This product contains a viral vector as its active ingredient and at least dileucine. Dileucine contributes to airborne dispersibility and lung reach, as well as to the effectiveness and stability of the viral vector's biological activity, thus providing an excellent inhalation powder formulation of the viral vector.

[0015] Furthermore, this product may contain a cellulose derivative in addition to dileucine. The cellulose derivative may contribute to the effectiveness of the biological activity of the viral vector, and can provide an excellent viral vector inhalation powder.

[0016] Hereinafter, representative and non-limiting specific examples of the present invention will be described in detail with reference to the drawings as appropriate. This detailed description is simply intended to show those skilled in the art details for carrying out preferred examples of the present invention and is not intended to limit the scope of the present invention. Furthermore, additional features and inventions disclosed below may be used separately from or in conjunction with other features and inventions to provide a further improved "viral vector-containing inhalation powder and method for producing the same".

[0017] Furthermore, the combinations of features and processes disclosed in the following detailed description are not essential for carrying out the present invention in the broadest sense, and are described solely to illustrate representative examples of the present invention. Moreover, the various features of the representative examples described above and below, as well as the various features described in the independent and dependent claims, do not necessarily have to be combined in the same way as the examples described herein or in the order listed, in order to provide additional and useful embodiments of the present invention.

[0018] All features described herein and / or in the claims are intended to be disclosed individually and independently of each other, as limitations to the original disclosure and claimed features, separate from the configurations of features described in the examples and / or claims. Furthermore, all descriptions of numerical ranges and groups or clusters are intended to disclose intermediate configurations as limitations to the original disclosure and claimed features.

[0019] (Inhalation powder containing a viral vector) This product contains a viral vector as its active ingredient. In this specification, a viral vector is a nucleic acid that can be used to treat diseases, prevent diseases, maintain or improve health in animals such as humans. Examples of nucleic acids in viral vectors include single-stranded DNA, double-stranded DNA, single-stranded RNA, and single-stranded or double-stranded DNA / RNA hybrids. Generally, viral vectors contain not only such nucleic acids but also virus-derived proteins and lipids, constituting nanoparticles.

[0020] The viral vector is not particularly limited, but can be appropriately selected depending on the intended biological activity of the drug. For example, DNA vectors such as adeno-associated virus vectors (AAVV) can be used. There are many serotypes and genotypes of such DNA vectors, but AAVV1, 6, 6.2, and 9 are useful, for example.

[0021] This drug, containing such viral vectors as its active ingredient, is used, for example, for the prevention or treatment of various diseases in animals, including humans. While not limited to these, this drug is effective for respiratory diseases, including lung diseases, due to its excellent ability to reach the lungs. Examples of such diseases include lung cancer and other cancers, interstitial pneumonia, lung diseases such as asthma, infectious diseases, and congenital disorders.

[0022] When this drug is intended to induce immunity, it can specifically target both systemic and local immunity. Therefore, it is effective against common infections.

[0023] The content of the viral vector in this agent is not particularly limited. However, due to the characteristics of the viral vector, for example, it is 0.1 mg / mg or less, 0.05 mg / mg or less, 0.01 mg / mg or less.

[0024] For example, when the viral vector can be detected by copy number, it is not particularly limited. However, for example, 16×10 10 GC (genomic copy) / mg or less, 14×10 10 GC / mg or less, 12×10 10 GC / mg or less, 10×10 10 GC / mg or less, 8×10 10 GC / mg or less, 6×10 10 GC / mg or less, 4×10 10 GC / mg or less, 2×10 10 GC / mg or less, 1×10 10 GC / mg or less, 8×10 9 GC / mg or less, 6×10 9 GC / mg or less, 4×10 9 GC / mg or less, 2×10 9 GC / mg or less, 1.5×10 9 GC / mg or less, 1×10 9 GC / mg or less, etc. can be used.

[0025] , Also, it is not particularly limited. However, for example, 4×10 8 GC / mg or more and 10×10 9 GC / mg or less, 8×10 8 GC / mg or more and 6×10 9 GC / mg or less, 1×10 9 GC / mg or more and 4×10 9 GC / mg or less, 2.5×10 9 GC / mg or more and 20×10 9 GC / mg or less, etc. can be used.

[0026] A suitable range can be set by appropriately selecting these upper and lower limits. Also, it is not particularly limited. However, for example, 4×10 8GC / mg or more 10×10 9 GC / mg or less, 8×10 8 GC / mg or more 6×10 9 GC / mg or less, 1×10 9 GC / mg or more 4×10 9 GC / mg or less, 2.5×10 9 GC / mg or more 20×10 9 It can be set to GC / mg or less, etc.

[0027] The viral vector content in this drug may be appropriately set in relation to the total amount of the functional excipients, dileucine and cellulose derivatives. For example, 16 × 10 10 GC / mg or less, 14×10 10 GC / mg or less, 12×10 10 GC / mg or less, 10×10 10 GC / mg or less, 8×10 10 GC / mg or less, 6×10 10 GC / mg or less, 4×10 10 GC / mg or less, 2×10 10 GC / mg or less, 1×10 10 GC / mg or less, 8×10 9 GC / mg or less, 6×10 9 GC / mg or less, 4×10 9 GC / mg or less, 2×10 9 GC / mg or less, 2.5×10 9 GC / mg or less, 1×10 9 It can be set to GC / mg or less, etc. Also, although not particularly limited, for example, 4 × 10 8 GC / mg or more, 6×10 8 GC / mg or more, 8×10 8 GC / mg or more, 10×10 8 It can be set to GC / mg or higher, etc. Also, although not particularly limited, for example, 4 × 10 8 GC / mg or more 10×10 9 C / mg or less, 8×10 8 GC / mg or more 6×10 9 GC / mg or less, 1×10 9 GC / mg or more 4×10 9 GC / mg or less, 2.5×109 GC / mg or more 20×10 9 It can be set to GC / mg or less, etc.

[0028] The viral vector content (GC) in this drug is not particularly limited, but can be measured by PCR methods such as RT-PCR or antigen quantification methods such as ELISA.

[0029] (Dileucine) This drug contains dileucine. In this specification, dileucine is a leucine dimer in which two leucine molecules are linked by a peptide bond. The leucine contained in dileucine is either L-leucine or D-leucine. Examples of dileucine include L-leucine dimers, D-leucine dimers, and L-leucine and D-leucine dimers. Typically, it is an L-leucine dimer. As previously described, dileucine contributes well to the lung reach of this drug, as well as to the effectiveness and stability of the biological activity of the viral vector.

[0030] (Cellulose derivatives) This drug may contain a cellulose derivative in addition to dileucine. The cellulose derivative can contribute to the effectiveness of the biological activity of the viral vector. For example, it may suppress the decrease in biological activity during the manufacturing of this drug by the SFD method and allow the biological activity in the lungs to be continuously exerted after transpulmonary administration.

[0031] Cellulose is a polymer in which D-glucose is linked by β-1,4 glycosidic bonds. Cellulose derivatives only need to have this basic cellulose skeleton; for example, the hydrogen atoms of the hydroxyl groups of glucose in the cellulose skeleton may be substituted.

[0032] For example, the cellulose derivative may have a lower alkoxyl group in which the hydrogen atoms of the hydroxyl group of glucose are substituted with lower alkyl groups such as methyl, ethyl, or propyl groups. In this specification, a lower alkyl group means a linear or branched alkyl group having 1 to 4 carbon atoms. Such a cellulose derivative is not particularly limited, but examples include methylcellulose.

[0033] Furthermore, the cellulose derivative may have a hydroxy lower alkoxyl group in which the hydrogen atom of the hydroxyl group is substituted with a hydroxy lower alkyl group. Examples of such cellulose derivatives include hydroxypropyl cellulose.

[0034] Alternatively, the hydrogen atom may be substituted with a carboxyl lower alkyl group in a cellulose derivative. For example, carboxymethylcellulose is one such derivative.

[0035] The ratio of hydroxyl groups in a cellulose derivative in which a hydrogen atom is substituted (substitution ratio) is not particularly limited and can be used as appropriate in a range of several percent to several tens of percent.

[0036] The molecular weight of the cellulose derivative is not particularly limited. For example, any cellulose derivative with any molecular weight can be used. For instance, cellulose derivatives containing hydroxy-lower alkoxyl groups, such as hydroxypropylcellulose, can have a weight-average molecular weight of 10,000 to 150,000. Using one or more cellulose derivatives with a weight-average molecular weight within this range can make a favorable contribution to the biological activity of this drug.

[0037] Cellulose derivatives may also have a weight-average molecular weight of 20,000 to 60,000. Using cellulose derivatives with a weight-average molecular weight in this range can suppress the decrease in the biological activity of the viral vector during formulation by the SFD method. When the viral vector is included in a low content, its retention in the lungs and biological activity can be maintained during transpulmonary administration. For example, values ​​of 25,000 to 55,000, 30,000 to 50,000, and 35,000 to 45,000 are possible.

[0038] Cellulose derivatives may also have a weight-average molecular weight of 80,000 to 120,000. Using cellulose derivatives with a weight-average molecular weight in this range can suppress the decrease in the biological activity of the viral vector during formulation by the SFD method, and, although not particularly limited, can maintain the retention and biological activity of the viral vector in the lungs during transpulmonary administration, even when the viral vector is included in a low content. For example, the ranges are 85,000 to 115,000, 90,000 to 110,000, and 95,000 to 105,000.

[0039] In this specification, the weight-average molecular weight can be obtained as Mw by methods such as GPC using polystyrene as an indicator.

[0040] This drug may contain only dileucine as a functional component against viral vectors, or it may contain dileucine in addition to a cellulose derivative. The ratio (mass ratio, hereinafter the same) of dileucine to cellulose derivative is not particularly limited, but can be set appropriately depending on the type of viral vector and the intended use of this drug. For example, the dileucine:cellulose derivative ratio in this drug can be 10:0 to 1:9. That is, it can be 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, etc. The range of this ratio is not particularly limited, but for example, it can be 8:2 to 4:6, 7:3 to 4:6, 6:4 to 4:6, etc.

[0041] The total amount of dileucine and cellulose derivatives in this drug is not particularly limited, but it may contain an amount necessary to ensure a certain level of lung reach, effectiveness, and stability of the biological activity of the viral vector, which is the active ingredient. Therefore, this total amount can be appropriately determined by a person skilled in the art. For example, the content of dileucine and cellulose derivatives in this drug may be 0.45 mg / mg or more, 0.50 mg / mg or more, 0.60 mg / mg or more, 0.70 mg / mg or more, 0.80 mg / mg or more, 0.90 mg / mg or more, 0.95 mg / mg or more, etc., depending on the content of the cellulose derivative described later. Alternatively, it may be 0.99 mg / mg or less, 0.95 mg / mg or less, 0.90 mg / mg or less, 0.85 mg / mg or less, 0.80 mg / mg or less, 0.75 mg / mg or less, 0.60 mg / mg or less, etc. Furthermore, the content range of dileucine and cellulose derivatives can be, for example, 0.45 mg / mg or more and 0.99 mg / mg or less, 0.50 mg / mg or more and 0.99 mg / mg or less, 0.55 mg / mg or more and 0.95 mg / mg or less, 0.60 mg / mg or more and 0.99 mg / mg or less, 0.65 mg / mg or more and 0.95 mg / mg or less, 0.70 mg / mg or more and 0.95 mg / mg or less, 0.75 mg / mg or more and 0.99 mg / mg or less, 0.80 mg / mg or more and 0.95 mg / mg or less, 0.85 mg / mg or more and 0.99 mg / mg or less, 0.85 mg / mg or more and 0.95 mg / mg or less, etc.

[0042] The total amount of dileucine and cellulose derivatives relative to the viral vector in this drug can also be set within the range of the viral vector content described above.

[0043] (Other ingredients) This drug may consist only of a viral vector, a cellulose derivative, and dileucine, but may also contain other components such as carbohydrates (including monosaccharides, oligosaccharides, polysaccharides, and sugar alcohols) and sugar derivatives. These may also be effective against the biological activity of the viral vector. Examples of carbohydrates include trehalose, mannitol, and dextran. Examples of sugar derivatives include compounds in which an organic group such as an alkyl group is introduced in place of a hydrogen atom in the hydroxyl group of a carbohydrate. The content of these components in this drug is not particularly limited and can be set as appropriate.

[0044] Furthermore, this drug may also contain amino acids such as leucine, and dipeptides such as glycylleucine. These may also be effective against the biological activity of viral vectors. These concentrations in this drug are not particularly limited and can be set as appropriate.

[0045] (spray freeze-dried powder) This product contains a viral vector, dileucine, and optionally a cellulose derivative as a spray-freeze powder. The spray-freeze powder can be obtained by the SFD method. The spray-freeze powder generally consists of particles containing the viral vector and dileucine. The particles of the spray-freeze powder in this product are typically porous spherical particles.

[0046] The 50% particle diameter (D50, hereinafter simply referred to as the average particle diameter) in the volume-based particle diameter distribution measured by laser diffraction and scattering is, for example, 5.0 μm to 30.0 μm, 10.0 μm to 30.0 μm, or 10.0 μm to 20.0 μm. Furthermore, the span, expressed by the following formula using D50, the 10% particle diameter (D10), and the 90% particle diameter (D90), is, for example, 5.0 or less, 4.0 or less, 3.0 or less, and 2.0 or less.

[0047] Span=(D90-D10) / D50

[0048] (Air dispersibility) Dispersibility in air is evaluated by the average particle size. In this specification, the average particle size of the spray freeze-dried powder of this agent can be determined from the volume-based particle size distribution by laser diffraction-scattering. Typically, the spray freeze-dried powder is dispersed in air, for example, with compressed air at 0.2 MPa, and the particle size distribution is measured using a laser diffraction-scattering particle size distribution analyzer LMS-2000e (product name, Seishin Corporation) or an apparatus capable of measuring particle size distribution by laser diffraction-scattering with equivalent or higher accuracy and precision.

[0049] (Aerosol performance or lung reach) This drug reaches the lungs via inhalation (gas flow during suction from the oral cavity to the bronchi). The aerosol performance, i.e., the ability of this drug to reach the lungs, can be evaluated using the multi-stage liquid impinger (MSLI) method.

[0050] (Evaluation method using the Multi-Stage Liquid Impinger (MSLI) method) In this specification, the MSLI method refers to the measuring apparatus described in 6.15 Aerodynamic particle size determination method for inhalants, 5.1 Multistage liquid impinger method (apparatus 1), of the 18th revised Japanese Pharmacopoeia, First Supplement. A pre-separator may be used as appropriate.

[0051] The overview of the measuring device and the measurement method can be based on the general test method described above. The evaluation of this drug by the MSLI method can be based on the measurement procedure for inhaled powders in 5.1.2 of the general test method 5.1 Multistage Liquid Impinger Method (Device 1) described above.

[0052] (lung reachability) At the time of use, i.e., to evaluate lung reach by inhaler, the particle release rate (EF) and the recovery rate of particles equivalent to 5 μm or less (FPF) are based on the above general test method 6.15 Aerodynamic particle size measurement method for inhalants 6. Calculation, and are calculated using the following formulas (1) and (2). EF(%)=M emitted / M total×100 (1) FPF(%)=M ≦5μm * / M total ×100 (2) (However, M total This is the total deposition amount, M emitted This is the amount of deposition after the induction port, and M ≦5μm This represents the deposition amount corresponding to particles smaller than 5 μm.

[0053] Note that * indicates the amount of constituent particles calculated by interpolation, applying it to the calculation in Table 6.15-6 Apparatus 1 of the general test method 6.15 Aerodynamic particle size measurement method for inhalants 6. Calculation.

[0054] EF (Emission Factor) is an indicator of the amount released from the inhaler, while FPF (Frequency Factor) is an indicator of how well the medication reaches the lungs.

[0055] In aerosol performance evaluation using the MSLI method, this drug can achieve, for example, an EF of 80% or higher. This is because an EF of 80% or higher indicates good release properties. The OE can also be, for example, 85% or higher, 90% or higher, or 95% or higher.

[0056] Furthermore, in aerosol performance evaluation by MSLI, this drug can achieve, for example, an FPF of 20% or higher, 25% or higher, 30% or higher, 35% or higher, 40% or higher, or 45% or higher. A higher value generally indicates better lung penetration. However, depending on the active ingredient and intended use of the inhaled powder, an FPF of 20% or higher may be sufficient.

[0057] (Biological activity of viral vectors after formulation) According to the SFD method, there is a concern that the biological activity of the viral vector may decrease during formulation. This drug can retain, for example, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more of the biological activity of the viral vector before formulation, after formulation by the SFD method (for example, after SFD and return to room temperature). Cellulose derivatives containing dileucine and hydroxyalkylcellulose such as hydroxypropylcellulose contribute to maintaining the biological activity of the viral vector after formulation. Cellulose derivatives are effective when the viral vector content is low (for example, when the genome copy number is 20 × 10⁶). 9 Less than GC / mg, 1 x 10 9 ~15×10 9 GC / mg, 1 x 10 9 ~10×10 9 GC / mg, 1 x 10 9 ~5×10 9 GC / mg, 1 x 10 9 ~3×10 9 GC / mg may make a significant contribution to maintaining biological activity.

[0058] (Biological activity of viral vectors after storage at room temperature) This drug exhibits excellent stability at room temperature (1-30°C). When stored at room temperature after formulation, it may retain over 60%, 70%, 80%, or even 90% of its biological activity immediately after formulation for up to one month. In particular, dileucine may contribute significantly to this room-temperature storage stability.

[0059] This drug may exhibit superior expression and persistence of the biological activity of the viral vector at the local lung site upon reaching the lungs. Therefore, it is an effective drug for the treatment or prevention of lung-borne diseases.

[0060] This drug can be administered using any known method of administration applicable to inhaled powder formulations.

[0061] As described above, this drug is an inhalation powder formulation that excels in airborne dispersibility, lung reach, stability of the biological activity of the viral vector after formulation and subsequent storage, and the expression and continuity of biological activity at the local level in the lungs.

[0062] (Method of manufacturing inhalation powder) A method for producing this drug may include the steps of: spray-freezing a sample solution containing a viral vector and dileucine to obtain a spray-frozen product; and sublimating the water in the spray-frozen product to obtain the spray-freeze-dried powder. This method can produce an inhalation powder that is excellent in air dispersibility, lung reach, stability of the biological activity of the viral vector after formulation and during subsequent storage, and expression and persistence of biological activity at the local level in the lungs.

[0063] (spray freezing process) In the spray freezing process, a sample solution containing a viral vector and dileucine is spray-freezed to obtain a spray-freezed product. The content of the viral vector, and the total amounts of dileucine and cellulose derivatives in the sample solution are set so that they are effective in expressing the biological activity of the viral vector in the final spray-freeze-dried powder.

[0064] While not particularly limited, the sample solution may contain the total amount of cellulose derivative and dileucine at concentrations of, for example, 5 mg / ml to 50 mg / ml, 5 mg / ml to 40 mg / ml, 5 mg / ml to 30 mg / ml, 8 mg / ml to 30 mg / ml, or 10 mg / ml to 20 mg / ml. Furthermore, the sample solution may contain the viral vector at a concentration of, for example, 1.0 × 10⁻⁶ of the total amount of cellulose derivative and dileucine. 9 GC / mg or more 30×10 9 GC / mg or less, 2.0×10 9 GC / mg or more 25×10 9 GC / mg or less, 2.5×10 9 GC / mg or more 20×10 9 It can be included in amounts of GC / mg or less.

[0065] The primary solvent in the sample solution is water. Other components mentioned above can be added as appropriate.

[0066] The spray freezing process is not particularly limited, but known spray freezing apparatuses can be used. For example, the sample solution is dropletized using a known spraying method, and the droplets of the sample solution are frozen by directly or indirectly cooling them with a cooling medium. For example, the liquid cooling medium is not particularly limited, and in addition to liquid nitrogen, liquid helium, liquid argon, etc. can be used. As a gaseous cooling medium, nitrogen gas obtained by evaporating liquid nitrogen, argon gas obtained by evaporating liquid argon, dry ice, carbon dioxide gas, etc. can be used.

[0067] (drying process) The drying process involves sublimating the water in the spray-frozen material to obtain spray-freeze-dried powder. While not particularly limited, any known freeze-drying apparatus can be used in the drying process. For example, it can be performed at -40°C under reduced pressure (5 Pa or less), followed by drying at approximately 10-25°C for 10-40 hours.

[0068] By following the spray freezing and drying processes described above, a spray freeze-dried powder can be obtained. This manufacturing method, although employing the SFD method, suppresses the decrease in the biological activity of the viral vector associated with SFD, improves storage stability after manufacturing, has excellent lung reach, and provides an inhalation powder that is excellent in expressing and maintaining high biological activity at the local level in the lungs. [Examples]

[0069] The following examples embody the disclosures of this specification, but these examples are not intended to limit the disclosures of this specification. Furthermore, unless otherwise specified in the following description, % represents mass %. [Examples]

[0070] (Manufacturing of AAVV-containing powder formulations by spray freeze-drying method) In this example, the present agent, which is a spray-freeze-dried powder, was prepared using the following AAVV, dileucine, and cellulose derivatives. An overview is shown in Figure 1.

[0071] (AAVV) A serotype 6 adeno-associated virus vector (AAVV) encoding the CAG promoter (cytomegalovirus enhancer / chicken β-actin promoter) and the firefly luciferase (FLuc) gene was used. Hydroxypropylcellulose (HPC), leucine (Leu, L-leucine), and dileucine (diLeu, L-leucine dimer) were used as excipients, and sodium fluorocein (FlNa) was used as a labeling agent for quantification. The number after HPC represents the weight-average molecular weight (K).

[0072] (Manufacturing of spray freeze-dried AAVV formulations (hereinafter simply referred to as SFD formulations)) Each additive was dissolved in ultrapure water and adjusted to the predetermined concentration shown in Figure 1. Then, the solutions of each additive were filtered through a membrane filter with a pore size of 0.45 μm, and each AAVV stock solution (~1 × 10⁻¹) was added to the resulting solution to achieve the predetermined content shown in Figure 1. 13 Various sample solutions were prepared by adding GC (genome copy) / ml. When combining two types of excipients, the sample solution was prepared so that the total volume reached the predetermined concentration shown in Figure 1, with a mass ratio of 1:1.

[0073] Using a spray nozzle (B1 / 4JCO-SS+SU1A-SS, Spraying Systems Japan LLC), each sample solution was sprayed into liquid nitrogen with compressed air at 150 kPa and rapidly frozen. The solutions were then transferred to shelves in a freeze-dryer (FDU-2100 and DRC-1000, Tokyo Rikakikai Co., Ltd.) that had been pre-cooled to -40°C, where the water was sublimated to obtain various SFD formulations with different AAVV content and additive compositions.

[0074] Note that in Figure 1, the numbers after the hyphens correspond to the AAVV content (2.5 × 10⁻⁶). 9 GC / mg: low content SFD formulation, 20×10 9GC / mg: High-content SFD formulation). Note that the mass ratio of AAVV and its components in the stock solution to the SFD formulation is extremely low, so in the following experiments, their masses were ignored (mass of SFD formulation = mass of additives) to determine the amount of AAVV added (GC / well) and the dose administered (GC / mouse). For the same reason, it is considered that the effect of the presence of AAVV and its components in the stock solution on the powder properties of the SFD formulation is small, and further considering that AAVV is a substance that needs to be prevented from diffusion, in Examples 2 and 3 below, evaluations were performed using SFD formulations that did not contain AAVV. [Examples]

[0075] (Evaluation of particle structure) In this example, each SFD formulation was dispersed onto conductive carbon double-sided tape attached to a sample stage using a powder dispersion device (created by connecting a syringe, a three-way stopcock, and a pipette tip). After the SFD formulations on the tape were platinum-coated (JEC-3000FC, JEOL Ltd.), the particle structure was observed using a scanning electron microscope (JSM-IT100LA, JEOL Ltd.). The results are shown in Figure 2.

[0076] As shown in Figure 2, electron microscopy observations of all SFD formulations revealed a large number of particles with a diameter of 10-20 μm and rich in the hollow porosity characteristic of the SFD method. No clear differences in particle structure were observed based on the additive composition, however, SFD formulations without leucine and dileucine (HPC40-0 and HPC100-0) tended to have a greater proportion of particle aggregates. [Examples]

[0077] (Evaluation of airborne dispersibility and lung reach) [1] Measurement of particle size distribution Each SFD formulation was dispersed in air using compressed air at 0.2 MPa, and the particle size distribution was measured using a laser diffraction / scattering particle size distribution analyzer (LMS-2000e, Seishin Enterprise Co., Ltd.). From the obtained cumulative distribution, the 10% particle size (D10), 50% particle size (D50: median diameter), and 90% particle size (D90) were determined respectively. Also, as an index value indicating the variation in particle size, Span was calculated from the following formula. The results are shown in Figure 3.

[0078] Span = (D90 - D10) / D50

[0079] [2] Evaluation of aerosol performance A No. 2 hydroxypropyl methylcellulose capsule (Qualicaps Co., Ltd.) filled with 1 mg of each SFD formulation was installed in an inhalation device (Jethaler (registered trademark) Standard, Tokiko Systems Solutions Co., Ltd.) and connected to a multi-stage liquid impinger (Copley Scientific). After suction at a flow rate of 80 L / min for 3 seconds using a vacuum pump, the SFD formulation deposited on each site was dissolved and recovered with phosphate buffer solution (PBS), and the deposition rate of the SFD formulation at each site was determined by measuring the fluorescence intensity derived from FlNa using a multimode plate reader (EnSight, PerkinElmer). From the obtained deposition pattern, the emission fraction (EF) and fine particle fraction (FPF) were calculated using the following formulas (1) and (2) respectively. In the measurement of EF and FPF, it is carried out according to the measurement procedure of inhaled powder in 5.1.2 of the multi-stage liquid impinger method (Apparatus 1) in General Test Method 5.1 and the aerodynamic particle size measurement method of inhalants in General Test Method 6.15 of the 18th Revision of the Japanese Pharmacopoeia First Supplement. The results are shown in Figure 3.

[0080] EF (%) = M emitted / M total ×100 (1) FPF (%) = M ≦5μm / M total ×100 (2)

[0081] As shown in Figure 3, the SFD formulations without leucine and dileucine (HPC40-0 and HPC100-0) showed high aggregation properties, with D50 values ​​of 90 μm or more and Span values ​​of 3 or more. On the other hand, the SFD formulations containing leucine and dileucine showed low values, with D50 values ​​of 10-20 μm and Span values ​​of 2 or less, suggesting high air dispersibility.

[0082] Furthermore, as shown in Figure 3, SFD formulations without leucine and dileucine (HPC40-0 and HPC100-0) showed relatively high EF (Ejection Factor) of over 70%, but poor lung reach with a fast-flow factor (FPF) of less than 4%. On the other hand, SFD formulations containing leucine and dileucine showed aerosol performance suitable for inhalation applications, with an EF of over 85% and an FPF of over 25%. The inclusion of HPC tended to further increase the molecular weight of HPC, resulting in a decrease in FPF, but this was similar to the FPF of commercially available inhalation powder formulations (20-50%).

[0083] Based on these results, it was demonstrated that using dileucine as an excipient significantly improves the airborne dispersibility and lung reach of SFD formulations, similar to leucine. [Examples]

[0084] (Evaluation of gene expression activity (after formulation and after storage) and cytotoxicity of SFD formulations) Each SFD formulation is dissolved in Opti-MEM(registered trademark), and 1 × 10 9 Treatment solutions were prepared to match the AAVV addition rate per GC / well. Human lung adenocarcinoma (NCI-H441) cells seeded in a light-shielded 96-well microplate were treated with each solution, and after 4 hours, the culture medium was replaced and the cells were incubated for a further 44 hours. Subsequently, alamarBlue® reagent and luciferin were added, and cytotoxicity and gene expression activity were evaluated by detecting and analyzing the fluorescence (due to reaction with alamarBlue reagent) and luminescence (corresponding to FLuc expression) using a real-time in vivo imaging system (IVIS, Revvity). The results are shown in Figure 4.

[0085] Furthermore, to examine storage stability, the day the sample was added for the initial evaluation of gene expression activity (the day each SFD preparation was manufactured) was designated as day 0. Thereafter, each SFD preparation and AAVV solution (AAVV stock solution diluted with PBS) were stored at room temperature (approximately 25°C), and after a predetermined number of days had elapsed, the sample was added to cells in the same manner and gene expression activity was evaluated. The results are shown in Figure 5.

[0086] As shown in Figure 4, regarding the gene expression activity of various SFD preparations with different AAVV content and additive compositions, the SFD preparation using leucine alone (Leu-2.5) showed a significant decrease in gene expression activity to less than 2% of the AAVV solution (Original AAVV). On the other hand, SFD preparations containing HPC and dileucine were able to maintain gene expression activity at more than 70% of the Original AAVV level. Gene expression activity was similarly maintained in both low-content and high-content SFD preparations, and no effect of AAVV content was observed. Furthermore, cell viability was observed at more than 95% in all treatments, and no cytotoxicity was detected.

[0087] Furthermore, as shown in Figure 5, when the daily changes in gene expression activity under room temperature storage were evaluated and compared, the gene expression activity of the AAVV solution decreased significantly over time, and after one month, the gene expression activity was approximately 15% of that of Original AAVV. On the other hand, the SFD formulation was able to maintain gene expression activity for a longer period, with the gene expression activity remaining at more than 40% of that of Original AAVV even after three months.

[0088] Based on these results, we have clarified the protective effect of dileucine on the SFD formulation of AAVV and its subsequent storage. [Examples]

[0089] (Evaluation of gene expression effects of intrapulmonary administration of SFD preparations) In this example, 50 μL of AAVV solution (AAVV stock solution diluted with PBS) and 0.5 mg of each SFD formulation were administered via the lungs through a cannula inserted into the trachea from the mouse's oral cavity. For the administration of the AAVV solution and the SFD formulation, a microsyringe and a powder dispersion device were used, respectively. The AAVV solution was prepared to have the same AAVV dosage as the low-content SFD formulation and the high-content SFD formulation.

[0090] After administration, luciferin solution was intraperitoneally administered on each measurement day, and 10 minutes later, a luminescence image of the whole mouse body was taken using IVIS to analyze the amount of FLuc expression in the lungs (corresponding to the luminescence intensity). From the time course of FLuc expression amount in each individual mouse, the maximum FLuc expression amount (FLuc max ), FLuc max reach time (T max ), and the area under the curve (AUC) of the FLuc expression amount-time curve were calculated, respectively. The results are shown in FIGS. 6 to 8.

[0091] As shown in FIGS. 6 to 8, even in the administration of the SFD formulation similar to the AAVV solution, a local gene expression effect in the lungs could be confirmed over 2 months after administration. The gene expression effect was higher in the high-content SFD formulation than in the low-content SFD formulation, and the dosage dependence of AAVV was confirmed.

[0092] In addition, both the low-content SFD formulation and the high-content SFD formulation showed a gene expression effect equal to or higher than that of the AAVV solution. In particular, among the low-content SFD formulations, HPC100 / diLeu-2.5 showed a 3.6-fold higher value in FLuc max and a 3.2-fold higher value in AUC compared to the AAVV solution. Among the high-content SFD formulations, diLeu-20 showed a 1.8-fold higher value in FLuc max and a 2.2-fold higher value in AUC compared to the AAVV solution. The T max of the SFD formulation was generally 7 to 21 days regardless of the AAVV content and the additive composition, and there was no clear difference from the AAVV solution.

[0093] From the above results, it was found that an AAVV-containing SFD formulation showing an excellent gene expression effect in the lungs can be obtained by using dilucine as an excipient.

Claims

1. An inhalation powder containing a spray-freeze-dried powder containing a viral vector and dileucine.

2. Furthermore, the inhalation powder according to claim 1, further containing a cellulose derivative.

3. The inhalation powder according to claim 2, wherein the cellulose derivative is one or more selected from the group consisting of methylcellulose, hydroxypropylcellulose, and carboxymethylcellulose.

4. The inhalation powder according to claim 2 or 3, wherein the cellulose derivative is hydroxypropylcellulose.

5. The inhalation powder according to any one of claims 2 to 4, wherein the hydroxypropyl cellulose has a weight-average molecular weight of 10,000 or more and 150,000 or less.

6. The inhalation powder according to claim 5, wherein the hydroxypropyl cellulose has a weight-average molecular weight of 20,000 or more and 60,000 or less.

7. The inhalation powder according to claim 5, wherein the hydroxypropyl cellulose has a weight-average molecular weight of 80,000 or more and 120,000 or less.

8. The inhalation powder according to claim 2, comprising the dileucine and the cellulose derivative in a mass ratio of 4:6 to 6:

4.

9. The inhalation powder according to claim 2, wherein the viral vector includes an adeno-associated virus vector.

10. The aforementioned viral vector comprises a viral vector, wherein the viral vector is present in a quantity of 1.0 × 10⁻¹⁶ per 1 mg of the total amount of the dileucine and the cellulose derivative. ９ GC or higher 25×10 ９ The inhalation powder according to claim 2, containing GC or less.

11. The cellulose derivative is hydroxypropyl cellulose, with a weight-average molecular weight of 80,000 or more and 120,000 or less, and the viral vector is contained in a quantity of 1.0 × 10⁻¹⁶ mg per 1 mg of the total amount of dileucine and the cellulose derivative. ９ GC or higher 25×10 ９ The inhalation powder according to claim 10, containing GC or less.

12. The inhalation powder according to claim 1, wherein the average particle size of the spray freeze-dried powder is 5 μm or more and 30 μm or less.

13. An inhalation powder according to any one of claims 1 to 12, for storage at a temperature of 1°C or higher and 30°C or lower.

14. A method for producing an inhalation powder containing spray freeze-dried powder, A step of obtaining a spray-frozen product by spray-freezing a sample solution containing a viral vector, a cellulose derivative, and dileucine, A step of obtaining spray freeze-dried powder by sublimating the water in the spray-frozen material, A manufacturing method that includes the following features.

15. The method for producing the product according to claim 14, wherein the sample solution contains the cellulose derivative and dileucine in a total amount of 10 mg / ml or more and 50 mg / ml or less.

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