JP2020193160A - Drug carrier for pulmonary delivery and pulmonary disease therapeutic drug containing the same - Google Patents

Abstract

To provide a drug carrier for pulmonary delivery that can deliver a gene-nucleic acid medicine to lungs safely and efficiently by systemic administration, and can sustainedly express the effect of a target gene-nucleic acid medicine in the lungs for a long time.SOLUTION: A drug carrier for pulmonary delivery includes a complex composed of a nucleic acid and dendrigraft poly-L-lysine coated with γ-polyglutamic acid, and has a negative surface charge, and has a diameter of 50 nm-250 nm.SELECTED DRAWING: Figure 1

Description

The present invention relates to a drug carrier for lung delivery and a therapeutic agent for lung disease including the same.

In recent years, gene / nucleic acid drugs have been attracting attention as new therapeutic agents for intractable respiratory diseases (lung diseases) such as pulmonary fibrosis and chronic obstructive pulmonary disease (COPD). Since it is not easy to secure stability and membrane permeability of gene / nucleic acid drugs and it is difficult to efficiently deliver them into cells, for example, fine particles, polymers, multifunctional carriers, etc. were used. The Drug Delivery System (hereinafter referred to as DDS) is under development.

DDS has the advantage that it not only enhances the therapeutic effect of drugs, but also reduces side effects. Therefore, it is desirable that the biocompatible polymer that serves as a carrier of DDS has low irritation and toxicity to the living body, is biocompatible, and is biodegradable and metabolized after administration, and is a drug contained therein. It is said that it is preferable that the particles are sustained and gradually released.

The present inventor has proposed a drug delivery complex that is less injurious to the living body and can selectively deliver a drug to cells at a target site (Patent Documents 1 and 2).

Specifically, the drug delivery complex of Patent Document 1 contains a complex of a drug such as a nucleic acid and a cationic molecule and an anionic molecule containing the complex, and is substantially uncharged or has a negative surface. It is a charged drug delivery complex characterized in that the anionic molecule is selected from the group consisting of γ-polyglutamic acid, chondroitin sulfate, alginic acid and salts thereof. Further, the drug delivery complex of Patent Document 2 is characterized in that it contains an antigen or a complex of a drug and a cationic molecule, and an anionic molecule containing the antigen or drug, and has directivity toward cells or organs. .. The drug delivery complexes of Patent Documents 1 and 2 are highly safe and have been confirmed to have remarkable efficacy for drug delivery.

On the other hand, the present inventors prepared a novel complex consisting of siRNA, dendrigraft poly-L-lysine, and γ-PGA, and the effect / toxicity of this complex at the cellular level and direct effect on mouse tumors. We have reported on the silencing effect of administration (Non-Patent Document 1).

JP-A-2010-59064 International Publication WO 2011/105520

Application of biodegradable dendrigraft poly-llysine to a small interfering RNA delivery system JOURNAL OF DRUG TARGETING, 2017 VOL. 25, NO. 1, 49-57

However, conventional drug delivery complexes such as Patent Documents 1 and 2 do not enable selective delivery to the lungs, and it is considered difficult to use them as therapeutic agents for lung diseases. On the other hand, transpulmonary administration of gene / nucleic acid drugs is also attracting attention as an administration method for enabling selective delivery to the lungs, but it may not be applicable to patients with impaired respiratory function, and sometimes side effects. Is also a problem. For this reason, no preparation has been developed at present that can safely and efficiently deliver gene / nucleic acid drugs to the lungs, especially by systemic administration.

In addition, Non-Patent Document 1 mentions the effect of direct administration of the novel complex to mouse tumors, but has not studied the selective delivery of the drug to the lung.

The present invention has been made in view of the above circumstances, and by systemic administration, a gene / nucleic acid drug can be safely and efficiently delivered to the lung, and a target gene / nucleic acid drug can be delivered to the lung for a long period of time. An object of the present invention is to provide a drug carrier for lung delivery capable of continuously exhibiting an effect, and a therapeutic drug for lung disease containing the same.

In order to solve the above problems, in the drug carrier for lung delivery of the present invention, the complex consisting of nucleic acid and dendrigraft poly-L-lysine is coated with γ-polyglutamic acid and has a negative surface charge. It is characterized by a diameter of 50 nm to 250 nm.

In this drug carrier for lung delivery, the charge ratio of nucleic acid, dendrigraft poly-L-lysine and γ-polyglutamic acid is preferably 1: 2 to 8: 4 to 16.

The therapeutic agent for lung disease of the present invention is characterized by containing the drug carrier for lung delivery.

In this lung disease drug, lung diseases include acute respiratory distress syndrome, asthma, cystic fibrosis, α-1-antitryptosine deficiency, primary and metastatic lung cancer, chronic obstructive pulmonary disease (COPD), and lung fiber. It is preferably one or more of illness, respiratory infection, and severe pneumonia.

The method for producing a drug carrier for lung delivery of the present invention is:
The first step of mixing nucleic acid and dendrigraft poly-L-lysine to prepare a complex, and
The second step of mixing the complex with γ-polyglutamic acid and coating the complex with γ-polyglutamic acid, and
It is characterized by including.

In this method for producing a drug carrier for lung delivery, in the first step, the nucleic acid and dendrigraft poly-L-lysine are mixed so as to have a charge ratio of 1: 2 to 1: 8, and in the second step, It is preferable to mix the nucleic acids, dendrigraft poly-L-lysine and γ-polyglutamic acid so that the charge ratio is 1: 2 to 8: 4 to 16.

The drug carrier for lung delivery of the present invention can safely and efficiently deliver a gene / nucleic acid drug to the lung by systemic administration, and continuously exerts the effect of the target gene / nucleic acid drug in the lung for a long period of time. Can be done. The therapeutic agent for lung disease of the present invention can treat various lung diseases by means of a gene / nucleic acid drug delivered to the lung.

The drug delivery complex (drug carrier for lung delivery) prepared in Example 1 was intravenously administered to mice, and 24 hours after the administration, each organ (liver, kidney, spleen, heart, etc.) was subjected to an in vivo imaging system (IVIS). It is a figure which observed the luciferase activity in lung). The drug delivery complex (drug carrier for lung delivery) prepared in Example 1 was intravenously administered to mice 6 hours, 24 hours, 72 hours, 1 week, 2 weeks, 4 weeks, and 6 weeks after the administration. , It is a figure which shows the result of having determined the luciferase activity in each organ (liver, kidney, spleen, heart, lung).

An embodiment of a drug carrier for lung delivery and a therapeutic agent for lung disease of the present invention will be described.

The drug carrier for lung delivery of the present invention has a complex consisting of nucleic acid and dendrigraft poly-L-lysine coated with γ-polyglutamic acid, has a negative surface charge, and has a diameter of 50 nm to 200 nm. ..

The nucleic acid is not particularly limited, and may be any DNA, RNA, a chimeric nucleic acid of DNA and RNA, a hybrid of DNA / RNA, and the like. The nucleic acid can be any 1 to 3 strands, but is preferably single strand or double strand. Nucleic acids are other types of nucleotides that are N-glycosides of purine or pyrimidine bases, or other oligomers with a non-nucleotide skeleton (eg, commercially available peptide nucleic acid (PNA)) or other oligomers containing special bonds. (However, the oligomer contains a nucleotide having a arrangement that allows base pairing and base attachment as found in DNA or RNA). Furthermore, those with known modifications, such as those with a label known in the art, those with a cap, those with a methylation, those in which one or more natural nucleotides are replaced with an analog, Intramolecular nucleotide modifications, such as those with uncharged bonds (eg, methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.), charged or sulfur-containing bonds (eg, phosphorothioate, phosphoro, etc.) Those having dithioates (such as dithioates), such as those having side chain groups such as proteins (nucleases, nuclease inhibitors, toxins, antibodies, signal peptides, etc.) and sugars (eg, monosaccharides), intercurrent compounds (such as intercurrent compounds) For example, those with aclysine, psolarene, etc., those containing chelate compounds (eg, metals, radioactive metals, boron, oxidizing metals, etc.), those containing alkylating agents, modified bonds. It may be a compound (for example, α-anomeric type nucleic acid).

For example, the type of DNA can be appropriately selected depending on the purpose of use, and is not particularly limited, and examples thereof include plasmid DNA, cDNA, antisense DNA, chromosomal DNA, PAC, BAC, and the like, and plasmid DNA is preferable. , CDNA, antisense DNA. Circular DNA such as plasmid DNA can be appropriately digested with a restriction enzyme or the like and used as linear DNA.

The type of RNA can be appropriately selected according to the purpose of use and is not particularly limited, but is not particularly limited, for example, siRNA, miRNA, shRNA, antisense RNA, messenger RNA, single-stranded RNA genome, double-stranded RNA genome. , RNA replicon, transfer RNA, ribosomal RNA and the like, preferably siRNA, miRNA, shRNA, mRNA, antisense RNA, RNA replicon and the like.

The size of the nucleic acid is not particularly limited, from the chromosome (artificial chromosomes, etc.) giant nucleic acid molecules (e.g., a size of about 10 ⁷ kbp), such as, the introduction of a low-molecular nucleic acid (e.g., size of about 5 bp) However, considering the efficiency of nucleic acid introduction into cells, it is preferably 15 kbp or less. For example, the size of a high molecular nucleic acid such as plasmid DNA is 2 to 15 kbp, preferably 2 to 10 kbp, and more preferably 4 to 10 kbp. Moreover, the size of a relatively small molecule nucleic acid such as siRNA is 5 to 1000 bp, preferably 10 to 500 bp, and more preferably 15 to 200 bp.

The nucleic acid may be either naturally occurring or synthesized, but if the size is about 100 bp or less, a nucleic acid automatic synthesizer usually used by the phosphotriethyl method, the phosphodiester method, etc. is used. Can be synthesized.

dendrigraft poly-L-lysine (DGL) is a dendrimer (a dendrimer having a structure that branches regularly from the center) that has a positive charge and is composed of lysine. For example, dendrigraft poly manufactured by COLCOM. -Commercial products such as L-lysine can be used. The dendrigraft poly-L-lysine (DGL) used in the drug carrier for lung delivery of the present invention can exemplify, for example, a molecular weight in the range of 50,000 to 200,000, preferably 150,000 to 200,000. The diameter can be exemplified in the range of 10 nm to 20 nm, preferably 15 nm to 20 nm. The number of lysines can be exemplified in the range of 300 to 1000, preferably 800 to 1000.

γ-Polyglutamic acid (γ-PGA) has a negative charge. γ-Polyglutamic acid (γ-PGA) may be a salt thereof. Examples of γ-polyglutamic acid (γ-PGA) or a salt thereof include a compound represented by the following formula (1).

(In the formula, R is a hydrogen atom, an alkali metal atom such as sodium, potassium and lithium, and a tertiary such as trimethylamine, triethylamine, dimethylamine, diethylamine, triethanolamine, trimethanolamine, diethanolamine, dimethanolamine and ethanolamine. It is an amine or a quaternary amine such as tetramethylamine or tetraethylamine, and the R present in the molecule may be the same or different, and n is an integer of 40 or more.)
Charge ratios of nucleic acids, dendrigraft poly-L-lysine and γ-polyglutamic acid in the drug carrier for lung delivery of the present invention (phosphate group of nucleic acid, amino group of dendrigraft poly-L-lysine, carboxyl group of γ-polyglutamic acid The molar ratio) is 1: 2 to 8: 4 to 16, and particularly preferably 1: 6: 10.

The drug carrier for lung delivery of the present invention has a negative surface charge. The diameter of the drug carrier for lung delivery of the present invention is 50 nm to 250 nm, preferably 50 nm to 100 nm.

The drug carrier for lung delivery of the present invention can safely and efficiently deliver a gene / nucleic acid drug to the lung by systemic administration, and continuously exerts the effect of the target gene / nucleic acid drug in the lung for a long period of time. Can be done. Therefore, the drug carrier for lung delivery of the present invention can be used as a therapeutic drug for lung diseases in the form of a gene therapy drug, a nucleic acid drug, a DNA vaccine, etc. by selecting a nucleic acid according to the purpose.

The therapeutic agent for lung disease of the present invention includes the above-mentioned drug carrier for lung delivery. The lung disease to be treated by the drug for treating lung disease is not particularly limited, and for example, acute respiratory distress syndrome, asthma, cystic fibrosis, α-1-antitryptocin deficiency, primary and metastatic lung cancer, and chronic obstructive disorder. One or more of lung disease (COPD), pulmonary fibrosis, respiratory infections, severe pneumonia and the like can be exemplified. In addition to primary lung cancers such as small cell lung cancer, squamous cell lung cancer, and adenocarcinoma of the lung, lung cancer also includes metastatic lung cancer.

Here, the "gene therapy drug" is a gene expression of a defect in a cell that is dysfunctional due to having an abnormal gene from a specific DNA gene to produce a protein having a desired function. It is a drug that repairs and corrects and treats diseases. The protein produced in a gene therapy drug can exemplify a normal protein originally encoded by a gene to be treated in gene therapy.

For example, cystic fibrosis, α-1-antitryptocin deficiency, and lung cancer are expected to have therapeutic effects by introducing a single gene. Specifically, cystic fibrosis is known to be a disease caused by a mutation in a gene encoding a protein called cystic fibrosis transmembrane regulator (CFTR), which is normal in the form of a gene therapy drug. It is expected that a therapeutic effect can be obtained by introducing a nucleic acid containing the CFTR gene (for example, plasmid DNA). In addition, α-1-antitryptocin deficiency is known to be a disease caused by the absence or low secretion of the protein inhibitor α-1-antitryptosine (AAT) from the liver. In the form of a gene therapy drug, it is expected that a therapeutic effect can be obtained by introducing a nucleic acid containing a normal AAT gene (for example, plasmid DNA).

Furthermore, as therapeutic gene candidates for lung cancer, for example, tumor suppressor gene p53, interleukin (IL) 12, interferon (IFN) -β, γ and the like can be exemplified.

Further, for pulmonary fibrosis, COPD, etc., a plasmid DNA encoding HGF, which is expected to have a therapeutic effect thereof, can be exemplified as a nucleic acid in the drug carrier for lung delivery of the present invention.

Further, the "nucleic acid drug" is a drug having a natural nucleotide or a chemically modified nucleotide as a basic skeleton, and acts directly on a living body without mediated gene expression, and is produced by chemical synthesis.

When the therapeutic agent for lung disease of the present invention is in the form of a nucleic acid drug, examples of the nucleic acid include antisense oligonucleotides (ASO), siRNA, microRNA (miRNA) and the like.

A "DNA vaccine" is a vaccine that uses DNA, which is a design drawing of the components that make up a pathogen, and synthesizes proteins that are part of the pathogen according to the instructions of the DNA, creating immunity to the proteins and creating a disease. It contributes to the treatment of.

Examples of proteins produced in DNA vaccines include proteins that are part of pathogens that cause diseases to be treated / prevented. Specifically, for example, DNA vaccines encoding malaria antigens. , A DNA vaccine encoding GP63, which is a candidate vaccine antigen for Leishmania disease and Shagas disease, a pneumonia vaccine using PNAG, a DNA vaccine encoding a melanoma-related antigen, and the like can be exemplified.

Mammals including humans (monkeys, mice, rats, cows, etc.) can be exemplified as subjects to which the drug carrier for lung delivery and the therapeutic agent for lung diseases of the present invention are administered.

The administration form of the therapeutic agent for lung disease of the present invention is not particularly limited, and may be oral administration or parenteral administration. Examples of parenteral administration include injection administration such as intramuscular injection, intravenous injection, and subcutaneous injection, transdermal administration, nasal administration, oral cavity administration, and pulmonary administration.

The therapeutic agent for lung disease of the present invention may be formulated by adding additives such as pharmaceutically acceptable carriers, excipients, vehicles, preservatives, stabilizers and binders. Dosage forms include, for example, liquids (for example, injections), dispersants, suspensions, tablets, pills, powders, suppositories, powders, fine granules, granules, capsules, syrups, lozenges, Examples thereof include inhalants, ointments, eye drops, nasal drops, ear drops, and poultices.

Formulations include, for example, excipients, binders, disintegrants, lubricants, solubilizers, solubilizers, colorants, flavoring agents, stabilizers, emulsifiers, absorption promoters, surfactants, pH adjusters. It can be carried out by a conventional method by appropriately using an agent, a preservative, an antioxidant and the like.

Examples of ingredients used in formulation include purified water, saline solution, phosphate buffer, dextrose, glycerol, ethanol and other pharmaceutically acceptable organic solvents, animal and vegetable oils, lactose, mannitol, glucose, sorbitol, crystalline cellulose. , Hydroxypropyl cellulose, starch, corn starch, silicic anhydride, aluminum magnesium silicate, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethyl cellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch , Pectin, methyl cellulose, ethyl cellulose, xanthan gum, gum arabic, tragant, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, vaseline, paraffin, octyldodecyl myristate, isopropyl myristate, higher alcohol, stearyl alcohol, stear Examples thereof include acids and human serum glucose.

When the therapeutic agent for lung disease of the present invention is administered to mammals (for example, humans, mice, rats, guinea pigs, rabbits, dogs, horses, monkeys, pigs, etc.), in particular, the dose to be administered is determined by symptoms, patient age, and gender. , Body weight, sensitivity difference, administration method, administration interval, type of active ingredient, type of preparation, and is not particularly limited, but for example, about 40 μg to 200 mg can be administered once or in several divided doses. In the case of injection administration, about 2000 μg / kg to 4000 μg / kg may be administered once or in several divided doses depending on the body weight of the patient. Next, an embodiment of the method for producing a drug delivery complex of the present invention will be described. The description of the parts common to the above-mentioned description of the lung-directed drug delivery complex will be omitted.

The method for producing a lung-directed drug delivery complex of the present invention is:
The first step of mixing nucleic acid and dendrigraft poly-L-lysine to prepare a complex, and
The second step of mixing the complex and γ-polyglutamic acid and coating the complex with γ-polyglutamic acid, and
including.

In the first step, the charge ratio of nucleic acid to dendrigraft poly-L-lysine (molar ratio of nucleic acid phosphate group, dendrigraft poly-L-lysine amino group) is 1: 2 to 1: 8, preferably 1: 1. Mix to 4 to 1: 6 and incubate at 15 ° C to 25 ° C for 0.5 to 300 minutes, preferably 10 to 180 minutes to make the complex. The concentration of the nucleic acid in the mixture can be appropriately set in consideration of the use, size (molecular weight), etc., but when the nucleic acid is DNA, for example, the range of 0.01 to 1000 ng / μL can be exemplified.

In the second step, the complex prepared in the first step and γ-polyglutamic acid are mixed with the charge ratio of nucleic acid, dendrigraft poly-L-lysine and γ-polyglutamic acid (phosphate group of nucleic acid, dendrigraft poly-L-lysine). The amino group and the molar ratio of the carboxyl group of γ-polyglutamic acid) are 1: 2 to 8: 4 to 16, particularly preferably 1: 6: 10, and 0.5 at 15 ° C to 25 ° C. The complex is coated with γ-polyglutamic acid by incubating for ~ 300 minutes, preferably 10 ~ 180 minutes. This makes it possible to obtain a drug carrier for lung delivery of the present invention having a negative surface charge. By systemic administration, this drug delivery complex can safely and efficiently deliver a gene / nucleic acid drug to the lungs, and can continuously express the effects of the target gene / nucleic acid drug in the lungs for a long period of time.

The drug carrier for lung delivery and the therapeutic agent for lung disease of the present invention are not limited to the above embodiments. For example, in the drug carrier for lung delivery of the present invention, when the drug delivery into a cell or organ is intended for diagnosis or imaging of a cell or organ, the drug carrier for lung delivery is a modification suitable for a diagnostic device or an imaging device. (For example, a radiation label, a label with a fluorescent dye, etc.) may be attached. In addition, the method for producing a drug delivery complex of the present invention may include known operations / steps other than the above-mentioned steps.

Hereinafter, the drug carrier for lung delivery of the present invention will be described together with Examples.

<Example 1> Preparation of drug delivery complex (1) Material For plasmid DNA, luciferase cDNA obtained from a pGL3-control vector manufactured by Promega Co., Ltd. using Hind III and Xba I was multicloned into a pcDNA3 vector manufactured by Invitrogen Co., Ltd. By inserting into the site, a plasmid DNA (pCMV-Luc) encoding luciferase was prepared.

For dendrigraft poly-L-lysine (DGL), COLCOM's dendrigraft poly-L-lysine (G5) was used. As γ-polyglutamic acid (γ-PGA), γ-polyglutamic acid (γ-PGA: molecular weight 10,000 to 100,000) manufactured by Yakult Co., Ltd. was used.

(2) Preparation method The above-mentioned pCMV-Luc and DGL are mixed at a charge ratio of 1: 6 (phosphate group of pCMV-Luc: amino group in DGL), allowed to stand at room temperature for 30 minutes or more, and pCMV-Luc. And a complex consisting of DGL was constructed. Then, γ-PGA is mixed with this complex so as to have a charge ratio of 1: 6: 10 (phosphate group of pCMV-Luc: amino group in DGL: carboxyl group in γ-PGA), and 30 at room temperature. By leaving it for more than a minute, a drug delivery complex having a negative surface charge was constructed in which the complex consisting of pCMV-Luc and DGL was coated with γ-PGA.

<Example 2> Administration of drug delivery complex (1) Pulmonary orientation of drug delivery complex The drug delivery complex prepared in Example 1 was intravenously administered to mice in the tail vein, and an in vivo imaging system was administered 24 hours after administration. In (IVIS), the luciferase activity expressed from pCMV-Luc in each organ (liver, kidney, spleen, heart, lung) was determined and observed.

The results are shown in FIG. High gene expression (luciferase activity) was observed only in the lungs of this drug delivery complex, and no gene expression was observed in other organs. Therefore, it was confirmed that this drug delivery complex (drug carrier for lung delivery) has remarkable lung directivity and can be used as a therapeutic drug for lung diseases.

(2) Persistence of long-term action of drug delivery complex The drug delivery complex (drug carrier for lung delivery) prepared in Example 1 was intravenously administered to mice in the tail vein, and 6 hours, 24 hours, 72 hours, 1 after administration. After weeks, 2, 4, and 6 weeks, the liver, kidneys, spleen, heart, and lungs were removed. Each organ was homogenized, a substrate luciferase assay buffer (Picagene) was added, and the luminescence generated was measured to determine the luciferase activity.

The results are shown in FIG. Gene expression in the lungs of this drug delivery complex was observed even 6 weeks after administration, confirming that the action was sustainable for a long period of time.

Claims (6)

A drug carrier for lung delivery characterized in that the complex consisting of nucleic acid and dendrigraft poly-L-lysine is coated with γ-polyglutamic acid, has a negative surface charge, and has a diameter of 50 nm to 250 nm. .. The drug carrier for lung delivery according to claim 1, wherein the charge ratio of nucleic acid, dendrigraft poly-L-lysine and γ-polyglutamic acid is 1: 2 to 8: 4 to 16. A drug for treating lung disease, which comprises the drug carrier for lung delivery according to claim 1 or 2. Lung diseases include acute respiratory distress syndrome, asthma, cystic fibrosis, α-1-antitryptosine deficiency, primary and metastatic lung cancer, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, respiratory infections, The drug for treating lung disease according to claim 3, wherein the drug is one or more of severe pneumonia. The first step of mixing nucleic acid and dendrigraft poly-L-lysine to prepare a complex, and
The second step of mixing the complex with γ-polyglutamic acid and coating the complex with γ-polyglutamic acid, and
A method for producing a drug carrier for lung delivery, which comprises. In the first step, the nucleic acid and dendrigraft poly-L-lysine are mixed so that the charge ratio is 1: 2 to 1: 8, and in the second step, the nucleic acid, dendrigraft poly-L-lysine and γ- The method for producing a drug carrier for lung delivery according to claim 5, wherein the polyglutamic acid is mixed so as to have a charge ratio of 1: 2 to 8: 4 to 16.