JP7787381B2 - Pharmaceutical compositions and particles - Google Patents

Description

The present invention relates to pharmaceutical compositions, particles, etc.

When using small molecular weight compounds as pharmaceuticals, their toxicity must be evaluated. If the concentration at which a candidate compound exhibits toxicity is close to its effective concentration, it is difficult to use the compound as a pharmaceutical. For this reason, only a very limited number of small molecular weight compounds are used as pharmaceuticals.

In recent years, cell preparations have been used as pharmaceuticals. For example, Patent Document 1 discloses a preparation containing statin-encapsulated nanoparticles in which a statin is encapsulated in nanoparticles containing a bioabsorbable polymer, the nanoparticles having a number-average particle size of less than 1000 nm, which is used to enhance the function of stem cells, as well as stem cells that have taken up and contained the statin-encapsulated nanoparticles.

International Publication No. 2016/076227

If it were possible to make drugs such as low-molecular-weight compounds exert their effects at lower concentrations than usual, it might be possible to use them as pharmaceuticals, even if the concentration at which toxicity manifests is close to the effective concentration. For this reason, there is a demand for drug administration methods that can lower the effective concentration of drugs.

However, the administration method for such drugs and the pharmaceutical compositions to be used in such administration methods have not yet been established.

Accordingly, an object of the present invention is to provide a pharmaceutical composition that allows for a lower dosage per dosage unit of a drug than when the drug acts alone, as well as particles and the like used in the pharmaceutical composition.

As a result of extensive research into resolving the above-mentioned problems, the inventors discovered that the above-mentioned problems could be solved by using a population of cells in which cells that accumulate at the site of inflammation have been introduced with particles containing a bioabsorbable polymer and a drug, leading to the completion of the present invention.

The present invention includes the following embodiments.
[1]
comprising a population of cells,
the cells are cells that accumulate at an inflammatory site and into which particles containing a bioabsorbable polymer and a drug have been introduced;
The drug is cyclosporine or minoxidil.
Pharmaceutical compositions.
[2]
For use in a method comprising administering cyclosporine or minoxidil in a dose less than the dose per dosage unit of cyclosporine or minoxidil,
The pharmaceutical composition described in [1].
[3]
The cells include at least one of immune cells and mesenchymal stem cells.
The pharmaceutical composition according to [1] or [2].
[4]
A pharmaceutical composition for suppressing an immune response, comprising:
the cells comprise peripheral blood mononuclear cells;
the drug is cyclosporine;
The pharmaceutical composition according to [1] or [2].
[5]
A pharmaceutical composition for promoting angiogenesis, comprising:
the cells comprise mesenchymal stem cells;
The drug is minoxidil.
The pharmaceutical composition according to [1] or [2].
[6]
A pharmaceutical composition for promoting angiogenesis, comprising:
A pharmaceutical composition comprising a population of mesenchymal stem cells and minoxidil.
[7]
comprising a population of cells,
the cells are cells that accumulate at an inflammatory site and into which particles containing a bioabsorbable polymer and a drug have been introduced;
A pharmaceutical composition for use in a method comprising administering said agent in a dosage amount less than the dosage amount per dosage unit of said agent.
[8]
1. A particle for use in a method comprising administering a drug at a dose less than the per-dosage unit dose of said drug,
The particles are
a bioabsorbable polymer and the drug;
It is introduced into cells that accumulate at the site of inflammation,
particle.
[9]
A method of administering a drug, comprising:
encapsulating a drug in a bioabsorbable polymer to form drug-encapsulated particles;
introducing the drug-encapsulated particles into cells that accumulate at the site of inflammation;
administering the cells.
method.
[10]
The method according to [9], comprising administering the drug at a dose less than the dose per dosage unit of the drug.
[11]
The method according to [9] or [10], wherein the cells accumulate at an inflammatory site.
[12]
[12] The method according to any one of [9] to [11], wherein the drug is cyclosporine or minoxidil.
[13]
[13] The method according to any one of [9] to [12], wherein the cells include at least one of immune cells and mesenchymal stem cells.
[14]
the cells comprise peripheral blood mononuclear cells;
the drug is cyclosporine;
The method according to any one of [9] to [13].
[15]
the cells comprise mesenchymal stem cells;
The drug is minoxidil.
The method according to any one of [9] to [13].

The present invention provides a pharmaceutical composition that allows for a lower dosage per dosage unit of a drug than when the drug acts alone, as well as particles and the like used in the pharmaceutical composition.

Results of immune responses in Example 1. Fluorescence microscopy results under various conditions in Example 2. Arrows indicate blood vessel-like structures. Note that in each figure, mcM means μM, and hAP means that mesenchymal stem cells into which minoxidil-encapsulated PLGA particles were introduced were used.

An embodiment of the present invention (hereinafter referred to as "the present embodiment") will be described in detail below. However, the present invention is not limited to the following embodiment, and various modifications are possible without departing from the spirit of the present invention.

[Pharmaceutical composition]
In one aspect of this embodiment, the pharmaceutical composition comprises a population of cells, the cells being cells that accumulate at the site of inflammation into which particles comprising a bioabsorbable polymer and a drug have been introduced, and the drug is cyclosporine or minoxidil.
In another aspect of this embodiment, the pharmaceutical composition comprises a population of cells, the cells being cells that accumulate at an inflammatory site into which particles comprising a bioabsorbable polymer and a drug have been introduced, and the pharmaceutical composition is for use in a method comprising administering the drug at a dose less than the dose per dosage unit of the drug.

As will be shown in the examples below, in this embodiment, a drug is encapsulated in particles containing a bioabsorbable polymer, the particles are introduced into cells that accumulate at the site of inflammation, and the drug is administered using the cells. This allows the drug to be effective at a lower dose than when the drug is administered alone.

This is thought to be because, with such pharmaceutical compositions, the accumulation ability of cells that accumulate at the site of inflammation causes cells to accumulate at the affected area, and as the bioabsorbable polymer in the particles contained in the accumulated cells decomposes, the drug is slowly released, resulting in the simultaneous exertion of the effects of the cells and the drug. However, this speculation is not intended to limit the scope of the present invention.

(cell)
The pharmaceutical composition of this embodiment includes cells that accumulate at inflammatory sites and into which particles containing a bioabsorbable polymer and a drug have been introduced (hereinafter also referred to as "cells of this embodiment"). Note that the pharmaceutical composition of this embodiment may also include cells other than the cells of this embodiment. Examples of such cells include cells that have not been introduced with particles containing a bioabsorbable polymer and a drug and that accumulate at inflammatory sites, and cells that have been introduced with particles containing a bioabsorbable polymer and a drug and that do not accumulate at inflammatory sites.

Cells that accumulate at inflammatory sites are not particularly limited as long as they have the ability to accumulate at inflammatory sites, but examples include immune cells and mesenchymal stem cells.

Immune cells are not particularly limited as long as they are cells involved in the immune response, but may include, for example, T cells, B cells, natural killer (NK) cells, macrophages, monocytes, dendritic cells, and granulocytes (eosinophils, neutrophils, basophils, mast cells, etc.), as well as combinations of one or more of these. Immune cells may also be a combination of multiple types of cells, such as mononuclear cells, particularly peripheral blood mononuclear cells.

Mesenchymal stem cells may be derived from any tissue, as long as they are somatic stem cells derived from mesenchyme and have the ability to self-renew and differentiate. Examples of mesenchymal stem cells include those derived from adipose tissue, bone marrow, dental pulp, blood (peripheral blood, umbilical cord blood, etc.), placenta, umbilical cord, synovium, periosteum, perichondrium, muscle, ligament, tendon, meniscus, or skin. Among these, adipose-derived mesenchymal stem cells are preferably used. Adipose-derived mesenchymal stem cells can be obtained from adipose tissue, which can be easily obtained from subcutaneous fat using minimally invasive techniques such as liposuction. Abundant adipose-derived mesenchymal stem cells can be extracted and separated from the adipose tissue obtained as described above using a Celution System (Cytori Pharmaceuticals, Inc.) or similar.

The cells that accumulate at the site of inflammation may be cells derived from a non-human animal or from a human, but are preferably human-derived cells. Non-human animals are not particularly limited, and examples include mice, rats, guinea pigs, hamsters, rabbits, monkeys, cows, minipigs, pigs, sheep, goats, dogs, and cats.

Furthermore, the cells that accumulate at the site of inflammation may be derived from cells of the individual to whom the pharmaceutical composition of this embodiment or the cells of this embodiment is to be administered (autologous cells), or may be derived from cells other than that individual (allogeneic cells). Furthermore, the cells that accumulate at the site of inflammation may be xenogeneic or allogeneic cells.

The cells that accumulate at the site of inflammation may be cells collected from a living body or cells obtained by culturing such cells, or may be cells obtained by inducing differentiation from pluripotent stem cells. Examples of pluripotent stem cells include embryonic stem cells (ES cells) and iPS cells.

The method for introducing particles containing a bioabsorbable polymer and a drug (hereinafter also referred to as "particles of this embodiment" in this specification) into cells is not particularly limited, but includes co-culturing the particles of this embodiment with cells. By co-culturing the particles of this embodiment with cells that accumulate at the site of inflammation, the cells take up the particles of this embodiment into the cells through endocytosis or membrane permeation, making it possible to easily obtain cells of this embodiment.

The conditions for co-culturing the particles of this embodiment with cells that accumulate at inflammatory sites are not particularly limited, and may be, for example, normal culture conditions for culturing cells that accumulate at inflammatory sites or culture in an appropriate buffer. The culture time is also not particularly limited, but from the perspective of ensuring that the cells take up the particles of this embodiment, it is preferably 5 minutes to 2 hours, 10 minutes to 1 hour, or 15 to 45 minutes. After co-culturing the particles of this embodiment with the cells, the cell population may be washed as appropriate to remove any particles of this embodiment that have not been taken up by the cells.

The pharmaceutical composition of this embodiment may be in the form of a cell suspension containing the cells of this embodiment, or in the form of a cell sheet or sheet-like cell culture obtained by culturing the cells of this embodiment to form a sheet-like tissue. The cell sheet may be a composite cell sheet having a layer structure of the cells of this embodiment and feeder cells.

(particle)
The particles of this embodiment are particles containing a bioabsorbable polymer and a drug, and can also be described as drug-encapsulated particles in which a drug is encapsulated in a particle containing a bioabsorbable polymer.

Bioabsorbable polymers are not particularly limited as long as they are bioabsorbable or biodegradable polymeric materials, and examples include polymers and copolymers of hydroxy acids such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and poly(lactic-co-glycolic acid) (PLGA); polymers and copolymers such as PEG, polyanhydrides, poly(ortho)esters, polyesters, polyurethanes, poly(butyric acid), poly(valeric acid), poly(caprolactone), poly(hydroxyalkanoic acid), and poly(lactide-co-caprolactone); and natural polymers such as proteins such as albumin, collagen, gelatin, and prolamins, alginates, cellulose derivatives, and polyhydroxyalkanoates. Among these, preferred bioabsorbable polymers are hydroxy acid polymers and copolymers such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and poly(lactic-co-glycolic acid) (PLGA), with poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and poly(lactic-co-glycolic acid) (PLGA) being more preferred. The bioabsorbable polymer preferably includes at least one selected from the group consisting of poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and poly(lactic-co-glycolic acid) (PLGA). Note that bioabsorbable polymers may also be referred to as biodegradable polymers.

The particles of this embodiment may contain components other than the bioabsorbable polymer and drug. Examples of such components include water, distilled water for injection, saline, glucose solution, isotonic solutions (e.g., solutions of sodium chloride, potassium chloride, glycerin, mannitol, sorbitol, boric acid, borax, propylene glycol, etc.), other aqueous solvents, and aqueous or oily bases. The drug contained in the particles of this embodiment will be described later.

The particle size of the particles of this embodiment is not particularly limited as long as it is within a range that can be taken up by cells, and may be changed appropriately depending on the type of cell. The particle size of the particles of this embodiment may be, for example, 1000 nm or less, preferably 50 nm to 400 nm, and more preferably 100 nm to 200 nm. Therefore, the particles of this embodiment can also be called nanoparticles. The particle size of the particles of this embodiment can be measured by light scattering using a particle suspension.

The method for forming particles in this embodiment is not particularly limited, but may be, for example, by encapsulating other components, including a drug, in a bioabsorbable polymer. Specifically, the particles may be formed by, for example, spherical crystallization. The spherical crystallization method is a method that allows for the design of spherical crystal particles by controlling the crystal formation and growth process in the final process of compound synthesis, and for the direct control and processing of their physical properties. Examples of spherical crystallization methods include the emulsion solvent diffusion method (ESD method).

The emulsion solvent diffusion method can be carried out, for example, by the following method. A bioabsorbable polymer, a drug, and optionally other ingredients are dissolved in a good solvent that can dissolve the bioabsorbable polymer to obtain a mixed solution. By adding the mixed solution dropwise to a poor solvent that does not dissolve the bioabsorbable polymer while stirring, spherical crystalline polymer nanoparticles can be formed.

(drugs)
In the pharmaceutical composition of this embodiment, the drug is contained in the particles introduced into the cells. Therefore, in the pharmaceutical composition of this embodiment, the drug is mainly contained in the particles within the cells, but may also be contained in other forms. For example, in the pharmaceutical composition of this embodiment, the drug may be contained in the extracellular region of the intracellular particles, or may be contained in the extracellular region.

The drug to be encapsulated in the particles of this embodiment is not particularly limited and can be selected depending on the intended use of the pharmaceutical composition of this embodiment.
The drug encapsulated in the particles of this embodiment may be, for example, a lipophilic drug. When a lipophilic drug is used, after the drug is released from the particles of this embodiment, the drug can suitably permeate the cell membrane of the cells containing the particles of this embodiment and the cell membrane of other cells in the affected area, so that the effect of this embodiment tends to be more effective and reliable.
In one aspect of this embodiment, in the pharmaceutical composition, the drug is cyclosporine or minoxidil.

The amount of drug encapsulated per particle of the particles of this embodiment is not particularly limited and can be adjusted appropriately depending on the type of drug and the type of cells into which the particles of this embodiment are introduced. The amount of drug encapsulated per particle can be controlled by adjusting the conditions in the above-mentioned method for forming particles of this embodiment. For example, when forming particles of this embodiment using the emulsion solvent diffusion method, the amount of drug encapsulated per particle can be controlled by adjusting the amount of drug added together with the bioabsorbable polymer, adjusting the stirring speed of the poor solvent, or adjusting the time from when the mixed liquid containing the bioabsorbable polymer and drug is dropped into the poor solvent until the particles are removed.

The concentration of the drug encapsulated in the particles of this embodiment may be adjusted appropriately depending on the type of drug, the mixing ratio of the particles of this embodiment to the cells, and other factors. For example, the drug concentration in the suspension of the particles of this embodiment before being introduced into the cells may be, for example, 1 to 500 μg/mL or 10 to 100 μg/mL.

(Other ingredients)
The pharmaceutical composition of this embodiment may contain, in addition to the cell population, a pharmaceutically acceptable carrier and/or additive, the proportion of which may be appropriately determined based on the range commonly used in the pharmaceutical field.

Carriers are not particularly limited, but include, for example, water, distilled water for injection, physiological saline, glucose solution, isotonic solutions (e.g., solutions of sodium chloride, potassium chloride, glycerin, mannitol, sorbitol, boric acid, borax, propylene glycol, etc.), other aqueous solvents, and aqueous or oily bases. These carriers may be used alone or in combination of two or more.

The additives are not particularly limited, but examples thereof include stabilizers, solubilizers, suspending agents, emulsifiers, soothing agents, buffers, preservatives, antiseptics, pH adjusters, colorants, and absorption enhancers.
The stabilizer is not particularly limited, but examples thereof include albumin, globulin, gelatin, mannitol, glucose, dextran, ethylene glycol, propylene glycol, ascorbic acid, sodium bisulfite, sodium thiosulfate, sodium EDTA, sodium citrate, and dibutylhydroxytoluene.
The solubilizing agent is not particularly limited, but examples thereof include alcohols (e.g., ethanol, etc.), polyalcohols (e.g., propylene glycol, polyethylene glycol, etc.), and nonionic surfactants (e.g., Polysorbate 80 (registered trademark), HCO-50, etc.).
The suspending agent is not particularly limited, but examples thereof include glycerin monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, and sodium lauryl sulfate.
The emulsifier is not particularly limited, but examples thereof include gum arabic, sodium alginate, and tragacanth.
The soothing agent is not particularly limited, but examples thereof include benzyl alcohol, chlorobutanol, and sorbitol.
The buffer is not particularly limited, but examples thereof include phosphate buffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer, and Tris buffer.
The preservative is not particularly limited, but examples thereof include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, butyl parahydroxybenzoate, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric acid, and borax.
The preservative is not particularly limited, but examples thereof include benzalkonium chloride, parahydroxybenzoic acid, and chlorobutanol.
The pH adjuster is not particularly limited, but examples thereof include hydrochloric acid, sodium hydroxide, phosphoric acid, and acetic acid.
These additives may be used alone or in combination of two or more.

(Administration)
In this embodiment, a drug is encapsulated in particles containing a bioabsorbable polymer, the particles are introduced into cells that accumulate at the site of inflammation, and the drug is administered using the cells. This allows the drug to exert its effects at a lower dose than when the drug is administered alone. Therefore, the pharmaceutical composition of this embodiment is preferably used in a method comprising administering the drug at a dose lower than the dose per dosage unit of the drug. Here, the drug may be cyclosporine or minoxidil.

The pharmaceutical composition of this embodiment contains a predetermined amount of a drug per dosage unit, which is preferably smaller than the dosage per dosage unit when the drug is administered in a conventional manner, for example, when the drug is administered alone.
The target drug in the pharmaceutical composition of this embodiment is, for example, cyclosporine or minoxidil, which are commercially available drugs. The dosage unit of cyclosporine or minoxidil is described in the package insert or the like in each country. A dosage of the pharmaceutical composition of this embodiment that is less than the dosage per dosage unit of cyclosporine or minoxidil means that the dosage per dosage unit of cyclosporine or minoxidil when administered as the pharmaceutical composition of this embodiment is less than the dosage per dosage unit of cyclosporine or minoxidil when administered as a prescription drug approved in each country.
The predetermined amount may be, for example, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 8% or less, 5% or less, 3% or less, 2% or less, 1% or less, 0.8% or less, 0.5% or less, 0.3% or less, 0.2% or less, or 0.1% or less compared to the dose per dosage unit when the drug is administered in a conventional manner, for example, the drug is administered alone. The predetermined amount may be, for example, 0.1% or more, 0.2% or more, 0.3% or more, 0.5% or more, 1% or more, 2% or more, 3% or more, 5% or more, 8% or more, 10% or more, 20% or more, 30% or more, 40% or more, or 50% or more compared to the dose per dosage unit when the drug is administered in a conventional manner, for example, the drug is administered alone. The predetermined amount may be, for example, within a range of 0.1 to 90% of the dose per dosage unit when the drug is administered in a normal manner, for example, when the drug is administered alone, or within a range in which the upper and/or lower limits of the range are arbitrarily replaced with the above-mentioned values.

The dosage and administration interval of the pharmaceutical composition of this embodiment can be appropriately selected depending on the type of drug, the subject to be administered, the administration route, the disease, the age, weight, and symptoms of the subject.
The pharmaceutical composition of this embodiment may be administered multiple times (e.g., 2 to 10 times), and the intervals between administrations are not particularly limited and may be, for example, twice a day, once a day, twice a week, once a week, or once every two weeks. The pharmaceutical composition of this embodiment may also be administered periodically until treatment of the disease is completed, and the intervals between administrations are not particularly limited and may be as described above.
The number of cells contained in one dosage unit of the pharmaceutical composition of this embodiment is not particularly limited, but may be, for example, in the range of 1 x ¹⁰ to 1 x ¹⁰ cells, or 1 x ¹⁰ to 1 x ¹⁰ cells. The above number of cells may be administered multiple times as a single dose, or this dose may be administered in multiple divided doses. The pharmaceutical composition of this embodiment may also be administered together with one or more other drugs.

The pharmaceutical composition and the cells of this embodiment can be administered by direct application to tissue, but are not particularly limited thereto. Examples of administration routes include intravascular administration, particularly intravenous administration, or intramuscular administration, intrathecal administration, intraperitoneal administration, intestinal administration, rectal administration, vaginal administration, intraocular administration, intracerebral administration, subcutaneous administration, nasal administration, sublingual administration, oral administration, inhalation, transdermal administration, implantation, spraying onto the surface of an organ, and direct (local administration to the affected area) administration by applying a sheet or the like.
The administration may be carried out, for example, as an injection or infusion, or by surgical transplantation of a cell sheet, etc. When the cells of this embodiment are in the form of a sheet, the cell sheet of this embodiment may be fixed to the target tissue with a fastening means such as sutures or staples when applied to the tissue.

(Application)
The pharmaceutical composition of this embodiment can be used for various purposes depending on the type of drug encapsulated in the particles of this embodiment. For example, the pharmaceutical composition of this embodiment can be used for the treatment or prevention of a disease.

In one embodiment, when the drug is cyclosporine, i.e., when the particles of this embodiment contain cyclosporine, the pharmaceutical composition of this embodiment may be used to suppress immune responses. In this case, the pharmaceutical composition of this embodiment may be used to treat or prevent autoimmune diseases, inflammatory diseases, post-transplant rejection, etc. Specifically, the pharmaceutical composition of this embodiment may be used to suppress rejection in organ transplants such as kidney transplants, liver transplants, heart transplants, lung transplants, pancreas transplants, and small intestine transplants, suppress rejection and graft-versus-host disease in bone marrow transplants, suppress immune responses associated with cell transplants, and treat or prevent Behçet's disease and other non-infectious uveitis, psoriasis vulgaris, pustular psoriasis, psoriatic erythroderma, psoriatic arthritis, aplastic anemia, pure red cell aplasia, nephrotic syndrome, generalized myasthenia gravis, atopic dermatitis, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, autoimmune hepatitis, and dermatomyositis. Furthermore, the pharmaceutical composition of this embodiment may be used for the treatment or prevention of diseases known to be applicable to cyclosporine or for which cyclosporine is known to be usable for the treatment or prevention, or may be used for the treatment or prevention of diseases for which the treatment or prevention with cyclosporine has been approved.
In this case, the cells into which the particles of this embodiment are introduced preferably include peripheral blood mononuclear cells.

In one embodiment, when the drug is minoxidil, i.e., when the particles of this embodiment contain minoxidil, the pharmaceutical composition of this embodiment may be used to promote angiogenesis. In this case, the pharmaceutical composition of this embodiment may be used for the treatment or prevention of diseases in which angiogenesis may be involved, alopecia, and wound healing. Specific examples of diseases in which angiogenesis may be involved include ischemic diseases, and examples of ischemic diseases include cardiovascular diseases or ischemic heart diseases (e.g., coronary artery disease, coronary artery thrombosis, myocardial infarction, angina pectoris, acute coronary syndrome, atrial fibrillation, sudden ischemic death, and transient ischemic attack), peripheral ischemic diseases or peripheral arterial diseases (e.g., peripheral occlusive arterial disease, lower limb arterial ischemic disease, distal limb ischemic disease, vascular embolism, venous thrombosis, deep vein thrombosis, thrombophlebitis, arterial embolism, and wound healing in diabetic patients), and ischemic cerebral diseases (e.g., cerebral contusion, Parkinson's disease, multiple sclerosis, and cerebral infarction). Furthermore, the pharmaceutical composition of this embodiment may be used for the treatment or prevention of diseases for which minoxidil is known to be applicable or for which minoxidil is known to be usable for the treatment or prevention, or for the treatment or prevention of diseases for which minoxidil has been approved for treatment or prevention.
In this case, the cells into which the particles of the present embodiment are introduced preferably include mesenchymal stem cells. Examples of mesenchymal stem cells include the mesenchymal stem cells described above in the section (Cells), and among these, it is preferable to use adipose-derived mesenchymal stem cells.

The subject to which the pharmaceutical composition and cells of this embodiment are administered may be a mammal. Mammals are not particularly limited, but include, for example, humans, non-human primates, domestic animals, laboratory animals, and livestock, with humans being particularly preferred.

In the examples described below, it was shown that mesenchymal stem cells have a low angiogenesis-promoting effect when administered in small amounts to endothelial cells, but that angiogenesis is promoted when combined with minoxidil. Therefore, the present specification provides a pharmaceutical composition for promoting angiogenesis, comprising a population of mesenchymal stem cells and minoxidil; the use of minoxidil to enhance the angiogenesis-promoting effect of mesenchymal stem cells; and the use of minoxidil in the manufacture of a pharmaceutical composition for promoting angiogenesis. In this pharmaceutical composition, the concentration of minoxidil may be 10 μM or more, 20 μM or more, 30 μM or more, 50 μM or more, 70 μM or more, 80 μM or more, or 90 μM or more. Examples of mesenchymal stem cells include the mesenchymal stem cells described above in the (Cells) section, and it is particularly preferable to use adipose-derived mesenchymal stem cells.

[cell]
The cells of this embodiment are cells that accumulate at the site of inflammation and into which particles containing a bioabsorbable polymer and a drug have been introduced. The cells of this embodiment can be used as a pharmaceutical composition, optionally in the form of a cell population containing other cells. The cells of this embodiment can be used for the treatment or prevention of the above-mentioned diseases. The cells of this embodiment may be used in various forms, such as a cell suspension, a cell sheet, or a sheet-like cell culture. The cells of this embodiment are preferably used in a method that includes administering a drug at a dose lower than the dose per dosage unit of the drug.

An aspect of this embodiment is a cell of this embodiment for use in a method comprising administering an agent at a dose less than the dose per dosage unit of the agent.
One aspect of this embodiment is the use of the cells of this embodiment in a method comprising administering a drug at a dose that is less than the dose per dosage unit of the drug.
One aspect of this embodiment is the cell of this embodiment for use in treating or preventing the above-mentioned disease.
One aspect of this embodiment is the use of the cells of this embodiment in a method for treating or preventing the above-mentioned diseases.
One aspect of this embodiment is the use of a cell of this embodiment in the manufacture of a pharmaceutical composition of this embodiment.

[particle]
The particles of this embodiment are particles containing a bioabsorbable polymer and a drug, and are drug-encapsulated particles that are introduced into cells that accumulate at an inflammatory site. The particles of this embodiment are suitable for use in a method that includes administering a drug at a dose smaller than the dose per dosage unit of the drug.

One aspect of this embodiment is the particles of this embodiment for use in a method comprising administering a drug at a dose that is less than the dose per dosage unit of the drug.
One aspect of this embodiment is the use of the particles of this embodiment in a method comprising administering a drug at a dose that is less than the dose per dosage unit of the drug.
One aspect of this embodiment is the particle of this embodiment for use in treating or preventing the above diseases.
One aspect of this embodiment is the use of the particles of this embodiment in a method for treating or preventing the above diseases.
One aspect of this embodiment is the use of the particles of this embodiment in the manufacture of the pharmaceutical composition of this embodiment.

[Drug administration method]
One aspect of this embodiment is a method for administering a drug, the method including: encapsulating a drug in a bioabsorbable polymer to prepare drug-encapsulated particles; introducing the drug-encapsulated particles into cells that accumulate at the site of inflammation; and administering the cells. This drug administration method of this embodiment allows for a reduction in the amount of drug administered per dosage unit compared to administering the drug by a conventional method, for example, administering the drug alone.

In the drug administration method of this embodiment, a drug is first encapsulated in a bioabsorbable polymer to produce drug-encapsulated particles. Here, other components besides the drug may also be encapsulated in the drug-encapsulated particles. The bioabsorbable polymer, drug, other components, and method for encapsulating the drug in the bioabsorbable polymer are as described above. After the drug-encapsulated particles are produced, the amount of drug encapsulated per drug-encapsulated particle may be measured.

The drug-encapsulated particles are then introduced into cells that accumulate at the site of inflammation. The cells that accumulate at the site of inflammation and the method of introduction are as described above. In the drug administration method of this embodiment, the cells that accumulate at the site of inflammation are preferably obtained from the subject to whom the drug is to be administered.

Next, the cells into which the drug-encapsulated particles have been introduced are administered to a subject. The administration subject, cell administration method, and dosage are as described above. The dosage per dosage unit of cells is preferably adjusted by measuring the amount and/or concentration of the drug contained in the administration product, and adjusting the dosage to a predetermined amount that is less than the dosage per dosage unit when the drug is administered by a conventional method, for example, when the drug is administered alone. The predetermined amount is as described above. The amount and/or concentration of the drug contained in the administration product containing the cell population can be measured, for example, by a method combining liquid chromatography and mass spectrometry (e.g., LC/MS/MS).

The drug administration method of this embodiment may include selecting a drug depending on the disease of the subject to be administered the drug before preparing the drug-encapsulated particles.

[Therapeutic or preventive methods]
The pharmaceutical composition of this embodiment can be used for the treatment or prevention of the above-mentioned diseases, and is preferably used for the treatment of the above-mentioned diseases.
One aspect of this embodiment is a method of treating or preventing a disease, comprising administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical composition of this embodiment.
One aspect of this embodiment is a method of treating or preventing a disease, comprising administering to a patient in need thereof a therapeutically effective amount of the cells of this embodiment.
One aspect of this embodiment is a method for treating or preventing a disease, comprising administering a drug to a patient in need thereof by the drug administration method of this embodiment.
Here, the disease may be any of the diseases described above, and the drug contained in the pharmaceutical composition of this embodiment or the cell of this embodiment may be selected depending on the disease, and the drug to be administered by the drug administration method of this embodiment may be selected.

The present invention will be explained in more detail below using examples and comparative examples. The present invention is not limited in any way by the following examples.

[Example 1]
(In vitro immunosuppression evaluation test using human peripheral blood mononuclear cells transfected with cyclosporine-encapsulated PLGA particles)
Treatment of mononuclear cells with magnetic beads bearing CD3/CD28 on their surface induces immune stimulation and promotes the proliferation of T cells contained in the mononuclear cells. The stimulated T cells were allowed to incorporate bromodeoxyuridine (BrdU) for a certain period of time, and T cell proliferation was evaluated by Cell ELISA using an anti-BrdU antibody.

50 mg of lactic acid-co-glycolic acid copolymer (PLGA) and 2.5 mg of cyclosporine were dissolved in a mixture of 1 mL of acetone and 0.5 mL of ethanol to prepare a polymer solution. 50 mL of 4 wt % polyvinyl alcohol (PVA) solution was stirred at 400 rpm while the polymer solution was added dropwise at room temperature to obtain a suspension of PLGA particles encapsulating cyclosporine. The acetone and ethanol were evaporated and then centrifuged, after which the precipitate was collected and resuspended in distilled water. This centrifugation and resuspension in distilled water procedure was repeated three times. The final suspension was dispensed and stored at -80°C.

Next, a suspension of human peripheral blood mononuclear cells was prepared. A frozen tube of normal human peripheral blood mononuclear cells (Fujifilm Wako Pure Chemical Industries, Ltd./551-37651) was removed and thawed by immersing it in an incubator preheated to 37°C. The thawed cell suspension was added to 10 mL of RPMI 1640 medium, mixed, and then centrifuged at 500 g for 10 minutes, and the supernatant was removed. The pelleted cells were washed again with RPMI 1640 medium, and then suspended in RPMI 1640 medium to achieve an appropriate cell concentration.

Cryopreserved cyclosporine-encapsulated PLGA particles (cyclosporine concentration: 25 μg/mL) were thawed immediately before the test, mixed with a mononuclear cell suspension (concentration: 4 × ¹⁰ cells/mL) at a volume ratio of 1:10, and treated at 37°C in the presence of ₅ % CO for 30 minutes to allow the PLGA particles to be taken up by the cells. PLGA particles not taken up by the cells were then removed by washing twice with PBS.

Four types of cell populations were prepared by mixing mononuclear cells transfected with cyclosporine-encapsulated PLGA particles with mononuclear cells not treated with PLGA particles (untreated mononuclear cells) at ratios of 0:100, 25:75, 50:50, and 100:0, respectively. Using a portion of the mononuclear cells that had incorporated cyclosporine-encapsulated PLGA nanoparticles, the cyclosporine concentration in the cells was measured by LC/MS/MS, and the amount of cyclosporine contained per cell was calculated. Based on the calculation results, the cyclosporine concentrations in each well were calculated to be 0 ng/mL, 10 ng/mL, 20 ng/mL, and 40 ng/mL, respectively.

These cell populations were seeded into individual wells of a 96-well plate, treated with CD3/CD28 magnetic beads, and cultured for 24 hours. The total number of cells in each well was adjusted to be the same.

As controls, wells were prepared in which untreated mononuclear cells were added with 1000 ng/mL, 100 ng/mL, 10 ng/mL, or 0 ng/mL of cyclosporine, and wells in which untreated mononuclear cells were not treated with CD3/CD28.

After 24 hours of culture, BrdU was added and the cells were cultured for an additional 6 hours. The cells were then fixed and lysed using the reagents in the BrdU Cell Proliferation Assay Kit (Merck/2750), and the DNA was single-stranded and immobilized before being subjected to Cell ELISA using an anti-BrdU antibody. Two wells were tested for each treatment condition, and the average values were used for analysis. The above test was performed independently three times, and the average values and standard deviations were calculated to analyze the results. The results are shown in Figure 1.

Figure 1 shows that in the control with added cyclosporine, the immunosuppressive effect of cyclosporine increased as the cyclosporine concentration increased to 10 ng/mL, 100 ng/mL, and 1000 ng/mL. Furthermore, it was found that peripheral blood mononuclear cells transfected with cyclosporine-encapsulated PLGA particles exhibited immunosuppressive effects even at lower cyclosporine concentrations compared to the control with added cyclosporine alone. In particular, a high immunosuppressive effect was observed even at a low cyclosporine concentration of 40 ng/mL.

Furthermore, under conditions containing 50% untreated mononuclear cells and 50% mononuclear cells transfected with cyclosporine-encapsulated PLGA particles, the immune activation value was predicted to be the average of the immune activation value obtained under conditions containing only untreated mononuclear cells and the immune activation value obtained under conditions containing only mononuclear cells transfected with cyclosporine-encapsulated PLGA particles; however, the actual value was lower than the predicted value. The percentage difference between this predicted value and the actual value was calculated to be 40%. A similar calculation was performed under conditions containing 75% untreated mononuclear cells and 25% mononuclear cells transfected with cyclosporine-encapsulated PLGA particles, and the percentage difference between the theoretical value and the actual value was 30%.

In this way, when peripheral blood mononuclear cells transfected with cyclosporine-encapsulated PLGA particles were present in the presence of untreated mononuclear cells, the cyclosporine-encapsulated PLGA particles exhibited an immunosuppressive effect greater than that of the peripheral blood mononuclear cells themselves, suggesting that the peripheral blood mononuclear cells transfected with cyclosporine-encapsulated PLGA particles also exert an immunosuppressive effect on surrounding untreated mononuclear cells through the release of cyclosporine. Furthermore, it was shown that a lower concentration of cyclosporine was sufficient to exert the effect compared to when cyclosporine was added alone.

[Example 2]
(In vitro angiogenesis evaluation test using mesenchymal stem cells transfected with minoxidil-encapsulated PLGA particles)
When human umbilical vein endothelial cells (endothelial cells) are co-cultured with mesenchymal stem cells, the vein endothelial cells form a blood vessel-like structure. The formation of this blood vessel-like structure is achieved by co-culturing an excess of mesenchymal stem cells relative to the endothelial cells (e.g., three times the amount of endothelial cells). However, when the ratio of endothelial cells to mesenchymal stem cells is equal, the formation of the blood vessel-like structure is almost nonexistent. In the following, a test was conducted under co-culture conditions where the ratio of endothelial cells to mesenchymal stem cells was set to equal.

Minoxidil-encapsulated PLGA particles were prepared by encapsulating minoxidil in PLGA using a method similar to that described in Example 1.

Next, a mesenchymal stem cell suspension was prepared. Human mesenchymal stem cells (ATCC/PCS-500-011 (normal human adipose-derived mesenchymal stem cells)) were expanded in a medium prepared by mixing equal volumes of KBM ADSC-2 (Kohjin Bio/16030030) and 10% FBS-containing DMEM medium (the medium was changed every two days). When the cells had proliferated to an appropriate number, the medium was removed, and the adherent cells were washed with PBS. TrypLE (Gibco/12605010) was added and spread over the front of the adherent cells. The liquid was then removed, and the cells were incubated at 37°C and 5% _CO2 for 4-5 minutes. After confirming that the cells had detached, an appropriate amount of medium was used to obtain a cell suspension.

A suspension of minoxidil-encapsulated PLGA particles (PLGA particle concentration 10 mg/mL) and a suspension of mesenchymal stem cells (concentration 1 x ¹⁰ cells/mL) were mixed at a 1:1 (volume ratio) and treated at 37°C in the presence of 5% _CO2 for 30 minutes to allow the PLGA particles to be incorporated into the cells. Four conditions were prepared, with final PLGA particle concentrations of 5 mg/mL, 1.25 mg/mL, 0.5 mg/mL, and 0 mg/mL, respectively. PLGA particles not incorporated into the cells were then removed by washing twice with PBS.

Endothelial cells with fluorescently labeled cell membranes and mesenchymal stem cells were mixed in equal numbers and seeded into each well of a 96-well plate and cultured for 48 hours at 37°C in ₅ % CO. After 48 hours of culture, the formation of blood vessel-like structures was observed and evaluated using a fluorescence microscope.

As a control, wells were prepared in which 100 μM, 25 μM, 10 μM, and 0 μM minoxidil was added under conditions in which equal amounts of mesenchymal stem cells not treated with PLGA particles (untreated mesenchymal stem cells) and endothelial cells were mixed.

The results of fluorescence microscopy observations under each condition are shown in Figure 2. No blood vessel-like structures were observed when vascular endothelial cells were cultured alone, or when vascular endothelial cells were cultured with 100 μM minoxidil. Furthermore, when vascular endothelial cells were cultured with three times the amount of mesenchymal stem cells, blood vessel-like structures were observed, but when the same amount of mesenchymal stem cells was co-cultured, almost no blood vessel-like structures were observed (Figure 2 (A)).

Furthermore, when vascular endothelial cells and mesenchymal stem cells were co-cultured in equal amounts, the addition of 100 μM or 25 μM minoxidil promoted the formation of blood vessel-like structures in a concentration-dependent manner, while the addition of 10 μM minoxidil almost eliminated this effect (Figure 2(B)). Furthermore, under co-culture conditions in which an equal amount of mesenchymal stem cells into which minoxidil-encapsulated PLGA particles had been introduced was added instead of untreated mesenchymal stem cells, the formation of blood vessel-like structures was observed, and differences in the efficiency of blood vessel structure formation were observed depending on the concentration of minoxidil-encapsulated PLGA particles incorporated (Figure 2(B)).

The concentration of minoxidil in mesenchymal stem cells transfected with minoxidil-encapsulated PLGA particles was measured, and the concentration of minoxidil in the test system was calculated to be 50 nM when 5 mg/mL PLGA particles were incorporated. This demonstrates that the use of mesenchymal stem cells transfected with minoxidil-encapsulated PLGA particles provides sufficient effects at lower concentrations than when minoxidil is added alone.

Claims (4)

comprising a population of cells,
the cells are cells that accumulate at an inflammatory site and into which particles containing a bioabsorbable polymer and a drug have been introduced;
the cells are immune cells or mesenchymal stem cells;
the immune cell is a T cell, a B cell, a natural killer (NK) cell, a macrophage, a monocyte, an eosinophil, a neutrophil, a basophil, or a mast cell;
the bioabsorbable polymer is poly(lactic acid) (PLA), poly(glycolic acid) (PGA), or poly(lactic-co-glycolic acid) (PLGA);
The drug is cyclosporine or minoxidil.
Pharmaceutical compositions. For use in a method comprising administering cyclosporine or minoxidil in a dose less than the dose per dosage unit of cyclosporine or minoxidil,
The pharmaceutical composition of claim 1. A pharmaceutical composition for suppressing an immune response, comprising:
the cells are peripheral blood mononuclear cells;
the drug is cyclosporine;
The pharmaceutical composition according to claim 1 or 2. A pharmaceutical composition for promoting angiogenesis, comprising:
the cells are mesenchymal stem cells,
The drug is minoxidil.
The pharmaceutical composition according to claim 1 or 2.