JP2018197208A - Nitrogen monoxide-containing bubble liposome, and use of the same - Google Patents

Abstract

To provide a technique capable of selectively and efficiently delivering nitrogen monoxide to a given site of a living body.SOLUTION: The present invention relates to nitrogen monoxide-containing bubble liposome contains a lipid molecule in which electric charge at pH 7.3 to 7.5 shows neutrality and/or a lipid molecule in which electric charge at pH 7.3 to 7.5 shows anionic properties as a substantial basic constitution lipid molecule, and contains nitrogen monoxide so as to include it therein. Further, the present invention relates to a system for site-selectively delivering nitrogen monoxide in a living body which contains a nitrogen monoxide-containing bubble liposome.SELECTED DRAWING: Figure 16

Description

  The present invention relates to a nitric oxide-encapsulated bubble liposome capable of stably holding nitric oxide in an in vivo environment. The present invention also relates to a system for site-selective delivery of nitric oxide in vivo, comprising the nitric oxide-encapsulated bubble liposome.

Currently, population dynamics are moving in the direction of aging society in countries around the world. This tendency is particularly remarkable in developed countries including Japan, and the number of serious patients suffering from cardiovascular diseases such as cardiac ischemia and lower limb ischemia due to lifestyle-related diseases associated with an aging society is increasing.
To improve the pathology of these circulatory diseases, administration of nitric oxide (hereinafter sometimes referred to as “NO”), which can be controlled in vivo, provides healthy blood vessel construction and protection of surrounding tissues. It is expected that it will be possible to replay. Here, nitric oxide is a substance that exerts physiological action as a gaseous molecule, and nitric oxide exerts a vascular wall dilating action (antihypertensive action), an anti-arteriosclerotic action, etc., so a healthy circulatory system Recognized as a useful substance for the maintenance of circulatory diseases and the treatment of cardiovascular diseases.
In light of the physiological function of nitric oxide in the living body, if a nitric oxide delivery system capable of selectively delivering nitric oxide to any site is established, It is expected to be an extremely effective treatment for improving the pathological condition. In addition, when nitric oxide can be selectively delivered to any tumor blood vessel or the like, it becomes possible to selectively enhance tumor blood vessel permeability (that is, enhance the EPR effect) in any tumor blood vessel. It is also expected to lead to improvement in treatment efficiency of nano-preparation containing an anticancer agent.

Here, when nitric oxide is directly administered intravenously as a gas molecule, nitric oxide is rapidly oxidized and inactivated, and exhibits the excellent physiological functions of nitric oxide as described above. I can't.
Therefore, as a technique for generating nitric oxide in vivo, a technique for transdermally administering nitroglycerin, which is a precursor of nitric oxide, has been reported (Non-patent Document 1). In this technique, it has been reported that nitroglycerin can be absorbed and administered to capillaries by a transdermal patch, whereby nitric oxide can be generated in the blood vessel via nitrite ions.
However, nitric oxide produced by this technique is oxidized in the blood vessel and becomes inactive immediately, and the nitric oxide concentration is extremely low in the target affected area. The pharmacological action of nitric oxide cannot be expected. In addition, although it is possible to adjust the patch application position in this technology, nitroglycerin and its derivative compounds absorbed percutaneously from the patch are in a state in which the compound itself is directly administered into the blood vessel. It has low affinity with other application technologies for delivery and it is difficult to connect to application technologies.

  As a method different from the above, research has been reported in which nitric oxide is encapsulated in bubble liposomes (Non-patent Document 2). Therefore, the present inventors prepared a liposome having the same lipid composition as the bubble liposome described in Non-Patent Document 2, and prepared a bubble liposome in which nitric oxide was encapsulated in the liposome. Liposomes were remarkably unstable in the intravascular environment, and it was confirmed that encapsulated nitric oxide escaped from the bubble liposomes in only 3 to 5 minutes (see Example 1 of the present specification). That is, it was confirmed that the technique described in Non-Patent Document 2 is a technique that makes it difficult to deliver nitric oxide to a target tissue or the like in a living body.

  As described above, at the present time, a technique that enables selective and efficient delivery of nitric oxide selectively to an arbitrary site in the form of an active gas molecule has not been established.

Bioconjugate Chemistry, 2010, 21 (5), p797-802; Hiroshi Maeda; Tumor-Selective Delivery of Macromolecular Drugs via the EPR Effect: Background and Future Prospects. International Journal of Nanomedicine, 2014, 9, p4671-4683; Sutton et al .; Pulsed ultrasound enhances the delivery of nitric oxide from bubble liposomes to ex vivo porcine carotid tissue. Pharmaceutical Journal 130 (11) 2010, p1489-1496

  The present invention has been made in view of the above-described prior art, and the subject of the present invention is to provide a technology that enables nitric oxide to be selectively and efficiently delivered to any site in the living body. The purpose is to do.

In the situation of the above-mentioned prior art, the present inventors have conducted intensive research. As a result, regarding the lipid molecules constituting the bubble liposome, the charge is neutral or an anion near pH 7.3 to 7.5 corresponding to the pH in blood. The idea was to use bubble liposomes using sex lipid molecules. The inventors of the present invention prepared a liposome containing lipid molecules having the above composition as a basic constituent lipid, and encapsulated it in nitric oxide. The present inventors have found that nitric oxide can be stably retained in the bubble liposome for a long time. That is, it has been found that in the bubble liposome, nitric oxide can be stably administered systemically in the form of gas molecules.
Furthermore, the present inventors have the ability to retain nitric oxide in vivo, especially when bubble liposomes are prepared using lipid molecules that show neutrality in an environment around pH 7.3 to 7.5. It has been found that it can be remarkably improved. In this respect, the function and effect exhibited by the configuration does not become an optimal condition if the charge of the lipid molecule is simply shifted to the anion side, and the details of the mechanism are unknown.

  In addition, the present inventors subcutaneously administer the nitric oxide-encapsulated bubble liposome to an arbitrary tissue site, thereby causing a slow and sustained release of nitric oxide around the administration site. It has been found that the blood vessel wall dilating action is exerted. Furthermore, the present inventors intravenously injected the nitric oxide-encapsulating bubble liposome, and performed ultrasonic irradiation treatment on an arbitrary target site. As a result, nitric oxide was found around the ultrasonic irradiation site. It has been found that a site-selective vascular wall dilation effect is exerted. Here, “blood vessel wall dilation” is one of the physiological functions of nitric oxide, which is detected by visualizing with Evans Blue. In the site where nitric oxide is delivered, other nitric oxide possesses it. It is recognized that the physiologically active function is also exhibited.

Here, as described in the above paragraph, Non-Patent Document 2 describes the research content of encapsulating nitric oxide in bubble liposomes using cationic lipids, and blood vessels in cultured tissues using the bubble liposomes An experiment to measure tension is described. However, the experiment according to Non-Patent Document 2 is an experiment under Ex vivo conditions in which a sample is added to a cultured tissue and the effect is observed with a microscope, and is recognized as a test in a short time after the sample is added. That is, Non-Patent Document 2 does not show an experiment in which the stability and nitric oxide retention ability in an actual long-time intravascular environment are confirmed by an in vivo experiment in which blood is administered into a living body.
Therefore, the present inventors prepared nitric oxide-encapsulated bubble liposomes having the same lipid composition as the cationic lipid-containing bubble liposome according to Non-Patent Document 2, and verified the stability in vivo. Was extremely unstable in the intravascular environment, and it was shown that nitric oxide, which should be encapsulated in only 3 to 5 minutes, immediately escapes from the bubble liposome (see Example 1 of the present specification). That is, the bubble liposome using the cationic lipid described in Non-Patent Document 2 can temporarily hold nitric oxide, but cannot hold nitric oxide in an actual in vivo environment. It was confirmed that this technique cannot deliver nitric oxide to a desired tissue or the like by vascular administration.

Here, the bubble liposome is an object for research and development of a carrier for a drug delivery system in addition to the in vivo delivery of a contrast gas such as perfluorohydrocarbon (Non-Patent Document 3). In particular, bubble liposomes using “cationic lipids” are attracting attention as carriers for drug delivery systems for gene therapy because they can efficiently add or mount nucleic acid molecules on the surface.
The findings of the present inventors regarding the charge due to the constituent lipid composition of bubble liposomes and the ability to retain nitric oxide in the living body are the opposite technology in bubble liposomes of cationic lipids that have accumulated extensive research results as bubble liposomes. It was discovered in the presence of knowledge. In this respect, it is recognized that there was technical common sense that acts in the direction of inhibiting the idea according to the present invention.

  Based on the above findings, the present inventors have completed the present invention. The present invention specifically relates to the invention described below.

[Claim 1]
a lipid molecule having a neutral charge at pH 7.3 to 7.5 and / or a lipid molecule having an anionic charge at pH 7.3 to 7.5 as a substantially basic constituent lipid molecule. Nitrogen oxide-encapsulating bubble liposome, comprising nitric oxide encapsulated.
[Section 2]
Item 2. The nitric oxide-encapsulated bubble liposome according to Item 1, wherein the bubble liposome is capable of retaining nitric oxide in an in vivo environment.
[Section 3]
Item 3. The nitric oxide-encapsulated bubble liposome according to Item 1 or 2, wherein the bubble liposome is substantially free of lipid molecules having a cationic charge at pH 7.3 to 7.5 as a basic constituent lipid molecule.
[Claim 4]
Item 4. The nitric oxide according to any one of Items 1 to 3, wherein the bubble liposome comprises a lipid molecule having a neutral charge at pH 7.3 to 7.5 as a substantially basic lipid molecule. Encapsulated bubble liposome.
[Section 5]
Item 5. The nitric oxide-encapsulated bubble liposome according to any one of Items 1 to 4, wherein the bubble liposome comprises DSPC and / or DPPC as a substantially basic constituent lipid molecule.
[Claim 6]
Item 6. The nitric oxide-encapsulated bubble liposome according to any one of Items 1 to 5, wherein the average particle size is 200 μm or less.
[Claim 7]
Item 7. The nitric oxide-encapsulated bubble liposome according to any one of Items 1 to 6, comprising a lipid molecule modified with polyethylene glycol at 10 mol% or less of the basic constituent lipid molecule.
[Section 8]
Furthermore, the nitric oxide inclusion | inner_cover bubble liposome in any one of claim | item 1 -7 which has a target directivity improvement molecule | numerator.
[Claim 9]
Item 9. A blood vessel wall dilator, an EPR effect improver, a blood pressure antihypertensive agent, an arteriosclerosis inhibitor, or a central nerve protective agent, comprising the nitric oxide-encapsulating bubble liposome according to any one of Items 1 to 8.
[Section 10]
Item 9. A pharmaceutical kit or a pharmaceutical composition comprising the nitric oxide-encapsulating bubble liposome according to any one of Items 1 to 8.
[Section 11]
Item 11. The pharmaceutical kit or pharmaceutical composition according to Item 10, comprising a nano-form preparation having another pharmacological component.
[Claim 12]
Item 9. A system for site-selectively delivering nitric oxide in vivo, comprising the nitric oxide-encapsulating bubble liposome according to any one of items 1 to 8.
[Claim 13]
Item 13. The system according to Item 12, further comprising ultrasonic irradiation means.
[Section 14]
In the production process of bubble liposome encapsulating nitric oxide,
In a liposome comprising a lipid molecule having a neutral charge at pH 7.3 to 7.5 and / or a lipid molecule having an anionic charge at pH 7.3 to 7.5 as a substantially basic constituent lipid molecule. Including a step of including nitric oxide so as to include it.
A method for producing nitric oxide-encapsulating bubble liposomes capable of retaining nitric oxide in an in vivo environment.
[Section 15]
In the production process of bubble liposome encapsulating nitric oxide,
In a liposome comprising a lipid molecule having a neutral charge at pH 7.3 to 7.5 and / or a lipid molecule having an anionic charge at pH 7.3 to 7.5 as a substantially basic constituent lipid molecule. Including a step of including nitric oxide so as to include it.
A method for improving nitric oxide retention ability of bubble liposomes in an in vivo environment.

The present invention makes it possible to provide a technique that enables selective and efficient delivery of nitric oxide to any site in a living body.
Specifically, according to the present invention, it is possible to provide a nitric oxide-encapsulated bubble liposome capable of stably holding nitric oxide in an intravascular environment in a living body. The bubble liposome makes it possible to achieve sustained release of nitric oxide by site-selective local administration and / or local release of nitric oxide by site-selective ultrasonic irradiation after systemic administration. . That is, according to the present invention, it is possible to provide a system that selectively delivers nitric oxide to an arbitrary in vivo site in a gas state that is an active state.

As a result, in the present invention, it becomes possible to exert the physiologically active functions of nitric oxide nitric oxide, such as the vascular wall dilating action, blood pressure antihypertensive action, and arteriosclerosis inhibiting action, at any site in the living body. It is expected that it can be effectively used in the treatment of system diseases. Further, since nitric oxide is recognized to have a central nerve protecting action and the like, it is expected to be usable in brain treatment applications.
Further, in the present invention, the EPR effect improving action utilizing the above-mentioned vasodilation can be exerted at any site in the living body, and the therapeutic efficiency of a nano-ized preparation containing an anticancer drug or an anti-rheumatic drug, It is expected to be able to greatly improve.

In Example 1, it is a result figure which measured the amount of nitric oxide encapsulated in bubble liposome (neutral lipid BL, cationic lipid containing BL) using DAF-2.

In Example 1, it is a result figure which measured the echocardiogram intensity after systemic administration of the nitric oxide inclusion | inner_cover bubble liposome (anionic lipid BL, neutral lipid BL).

In Example 1, it is a figure which shows the echocardiogram after systemic administration of the nitric oxide inclusion | inner_cover bubble liposome (anionic lipid BL, neutral lipid BL). A portion indicated by a broken-line circle in the drawing indicates the position of the heart.

In Example 1, it is a result figure which measured the echocardiogram intensity after systemic administration of nitric oxide inclusion bubble liposome (cationic lipid containing BL).

In Example 1, it is a figure which shows the echocardiogram after systemic administration of the nitric oxide inclusion | inner_cover bubble liposome (cationic lipid containing BL). A portion indicated by a broken-line circle in the drawing indicates the position of the heart.

In Example 2, it is a result figure which measured the echocardiogram intensity after systemic administration of nitric oxide inclusion bubble liposome (DPPC).

In Example 2, it is a figure which shows the echocardiogram after systemic administration of nitric oxide inclusion | inner_cover bubble liposome (DPPC). A portion indicated by a broken-line circle in the drawing indicates the position of the heart.

In Example 3, it is a result figure which measured the echocardiogram intensity after systemic administration of the nitric oxide inclusion bubble liposome (sample from which PEG modification rate differs).

In Example 4, it is a result figure which measured the amount of nitric oxide encapsulated in bubble liposome (bubble liposome before stirring, nanobubble liposome) using DAF-2. “M” in the figure indicates a μm-scale bubble liposome before stirring. “N” in the figure indicates a nanobubble liposome.

In Example 4, it is a result figure which measured the amount of nitric oxide encapsulated in bubble liposome (nanobubble liposome after normal-pressure storage) using DAF-2.

In Example 4, it is a result figure which measured the echocardiogram intensity after systemic administration of the nitric oxide inclusion bubble liposome (nanobubble liposome).

In Example 5, it is a result figure which measured the amount of nitric oxide encapsulated in bubble liposome (bubble liposome after pressure storage) using DAF-2.

In Example 5, it is a result figure which measured the amount of nitric oxide encapsulated in bubble liposome (nano bubble liposome after pressure storage) using DAF-2.

It is the photograph image figure which observed the Evans blue leaking degree in the back part visually after the subcutaneous injection of the nitric oxide inclusion | inner_cover bubble liposome in Example 6. FIG. The high contrast portion near the administration site in the photographic image shows the site that developed blue color due to leakage of Evans blue.

In Example 7, it is a result figure which measured the Evans blue amount in the muscle tissue irradiated with the ultrasonic wave.

FIG. 16A is a conceptual diagram showing a schematic structure of a nitric oxide-encapsulating bubble liposome according to the present invention. FIG. 16B is a conceptual diagram showing an aspect including ultrasonic irradiation means in the nitric oxide delivery system according to the present invention.

  Hereinafter, embodiments of the present invention will be described in detail. It should be noted that the technical scope according to the present invention is not intended to exclude aspects including configurations other than those described below, unless they substantially hinder the operational effects of the technical features according to the present invention. Absent. Further, the technical scope according to the present invention is not limited to an embodiment including all of the following configurations.

In this specification, the abbreviation “NO” may be used as a term indicating nitric oxide. In addition, the abbreviation “BL” may be used as a term indicating bubble liposome.
In the present specification, expressions such as “neutral lipid bubble liposome” indicate properties relating to the charge of lipid molecules constituting the basic constituent lipid of the bubble liposome.
In the present specification, “EPR” is an abbreviation for Enhanced Permeability Retention, and refers to a phenomenon in which a drug of a scale that does not penetrate normal blood vessels accumulates in a gap between new blood vessels around a tumor or an inflammatory site.

1. Nitric oxide encapsulated bubble liposomes present invention relates to stable nitric oxide encapsulated bubble liposomes that can hold the nitric oxide in vivo environment. Specifically, the bubble liposome according to the present invention comprises i) a lipid molecule having a neutral and / or anionic charge in a predetermined solution as a substantially basic constituent lipid molecule, and ii) nitric oxide. It is related with the bubble liposome characterized by including.

  In this specification, “Bubble Liposome” refers to a liposome in which a gas is encapsulated among liposomes formed by spheroidizing a lipid bilayer membrane. When a contrast gas such as perfluorohydrocarbon gas is used as the inclusion gas, the inclusion gas resonates due to ultrasonic irradiation, and bursts when reaching a certain sound pressure. The bubble liposome which concerns on this invention shows the property which can enclose and hold | maintain nitric oxide in a gaseous state as not only contrast gas but inclusion gas.

[Basic lipid molecules]
As the bubble liposome according to the present invention, a lipid molecule having a neutral charge at pH 7.3 to 7.5 and / or a lipid molecule having an anionic charge at pH 7.3 to 7.5 is substantially used. It comprises as a constituent lipid molecule.

The membrane structure of the bubble liposome according to the present invention is constituted by a double membrane of lipid molecules. Here, examples of lipid molecules constituting the membrane include phospholipids, glyceroglycolipids, glycosphingolipids, and the like. In particular, phospholipids that are the main components of cell membrane components are administered to the body. This is preferable in terms of safety.
As the bubble liposome according to the present invention, a phospholipid capable of forming a bilayer structure at room temperature or body temperature can be used as a basic constituent lipid. Specific examples include DSPC (distearoylphosphatidylcholine), DSPG (distearoylphosphatidylglycerol), DSPA (distearoylphosphatidic acid), DSPE (distearoylphosphatidylethanolamine), DPPC (dipalmitoylphosphatidylcholine), DPPG (dipalmitoylphosphatidylglycerol). ), DMPG (dimyristoyl phosphatidylglycerol), DMPC (dimyristoyl phosphatidylcholine), DLPG (dilauryl phosphatidylglycerol), HSPG (hydrogenated soybean phosphatidylglycerol), HEPG (hydrogenated egg phosphatidylglycerol), EPC (yolk phosphatidylcholine), POPC (Palmitoyl oleoylphosphatidylcholine), P (Phosphatidylserine), PG (phosphatidyl glycerol), PI (phosphatidylinositol), and the like can be used, but is not limited thereto.

The bubble liposome according to the present invention comprises a lipid molecule having a predetermined charge state as a substantially basic constituent lipid molecule.
Here, as a pH condition for the lipid molecule to exhibit a predetermined charge state, a mammal blood pH value or a pH in the vicinity thereof, pH 7.3 to 7.5 can be preferably exemplified. A value around pH 7.4, which is more preferably pH 7.35 to 7.45, and particularly preferably pH of human blood, can be preferably mentioned.
Examples of the solution in which the lipid molecule according to the present invention exhibits the predetermined charge state include blood or an aqueous solution such as a salt concentration similar thereto. Specific examples of the solution include serum, plasma, physiological saline, PBS solution, glucose buffer solution and the like. That is, the lipid molecules according to the present invention are preferably those in which the charge exhibits a predetermined charging condition in these solutions in the above pH range.

The bubble liposome according to the present invention comprises a lipid molecule having a neutral charge and / or a lipid molecule having an anionic property in the predetermined solution as a substantially basic constituent lipid molecule. As a particularly preferred embodiment, in view of the stable retention ability of nitric oxide in the living body, the lipid molecule showing neutrality and / or the lipid molecule showing anionic property in the predetermined solution is substantially substituted. What constitutes a basic constituent lipid molecule is preferred.
Here, the “basic constituent lipid molecule” is used as a term indicating a lipid molecule constituting a lipid bilayer membrane which is a basic structure of bubble liposome. It is a concept that excludes other compound molecules embedded in or bound to the membrane of the lipid bilayer structure, that is, proteins and peptides.
Further, in the present invention, “comprising as a substantially basic constituent lipid molecule” means a state in which a double membrane structure of bubble liposome is substantially constituted by a lipid molecule having a predetermined charge state. It is shown. Here, “substantially” means that even when the lipid state is a lipid molecule whose charge state deviates from a predetermined state, the content rate is small and nitric oxide in vivo when bubble liposomes are formed. In the case where the internal retention ability exhibits the following predetermined function, its inclusion can be allowed. The specific content indicating that the substance is substantially constituted is that the lipid composition of the basic constituent lipid molecule has a lipid molecule content of a predetermined charge state according to the present invention of 90 mol% or more, preferably 94 mol%. As mentioned above, the aspect which is 98 mol% or more more preferably, Especially preferably, 99.5 mol% or more can be mentioned.
Particularly preferably, in view of the stable retention ability of nitric oxide in the living body, the basic constituent lipid molecule according to the present invention is an embodiment in which all of the lipid molecules exhibit a predetermined charge state. Is preferred.

Neutral Lipid Molecules In the bubble liposome according to the present invention, as a particularly preferred embodiment, those containing a lipid molecule exhibiting neutral charge in the predetermined solution as a substantially basic lipid molecule are suitable. is there. In an embodiment in which the basic constituent lipid molecules constituting the bubble liposome are substantially neutral lipid molecules, an effect is obtained in which the stable retention ability of nitric oxide in the living body is extremely good. Here, the above paragraphs can be referred to for substantial meanings and preferred embodiments.
Further, particularly preferably, in view of the stable retention ability of nitric oxide in the living body, the basic constituent lipid molecules according to the present invention are all composed of lipid molecules showing a neutral charge state. This embodiment is preferable.

As the neutral lipid molecule constituting the bubble liposome according to the present invention, a lipid molecule which does not show a bias of charge as a whole molecule or substantially does not show a bias of charge in the predetermined solution can be used. Specifically, as in the case of lipid molecules constituting normal liposomes, lipid molecules that are neutral in terms of charge as a whole can be used as the positive and negative charges in the molecule cancel each other. The neutral lipid molecule is acceptable as the neutral lipid molecule according to the present invention if it is recognized as being substantially charge neutral even if there is some charge bias due to pH conditions or salt concentration. . For example, when forming bubble liposomes without surface modification, lipid molecules exhibiting a surface charge bias of about -4 mV to +2 mV, preferably about -1 mV to +1 mV are acceptable as neutral lipid molecules.
As the neutral lipid molecule according to the present invention, a neutral lipid molecule capable of forming a double membrane structure at room temperature or in the body temperature can be used. Examples of the neutral lipid molecule include phosphatidylcholine and phosphatidylethanolamine lipid molecules. More specifically, examples of neutral lipid molecules include DSPC (distearoylphosphatidylcholine), DPPC (dipalmitoylphosphatidylcholine), DMPC (dimyristoylphosphatidylcholine), EPC (egg yolk phosphatidylcholine), POPC (palmitoyloleoylphosphatidylcholine), DSPE (distearoyl phosphatidylethanolamine) or the like can be used.
In particular, considering the stability of nitrogen monoxide retention in bubble liposomes, lipid molecules having an alkyl chain having 14 to 20 carbon atoms, preferably lipid molecules having an alkyl chain having 16 to 18 carbon atoms, may be used. Is preferred. The alkyl chain is preferably a linear molecule.
As an example of the embodiment, it is particularly preferable to use DSPC / or DPPC as the neutral lipid molecule according to the present invention. In particular, it is more preferable to use DSPC.

Anionic lipid molecule As the basic constituent lipid molecule constituting the bubble liposome according to the present invention, it is preferable to use a lipid molecule having neutral charge in the predetermined solution, and the charge in the predetermined solution. That comprise lipid molecules exhibiting anionic properties as a substantially basic constituent lipid molecule. In addition, a lipid molecule having a neutral charge in the predetermined solution and a lipid molecule having an anionic charge in the predetermined solution are included as a substantially basic constituent lipid molecule in a mixed composition. Also included. Note that the above paragraphs can be referred to for the meaning of substantial or the like.

  As the bubble liposome according to the present invention, it is possible to adopt an embodiment in which all or substantially all of the basic constituent lipid molecules are anionic lipid molecules, but considering the stable retention ability of nitric oxide, In an embodiment in which an ionic lipid molecule and an anionic lipid molecule are substantially basic constituent lipid molecules in a mixed composition, the content of the anionic lipid molecules is preferably small. Specifically, in the mixed composition, an anionic lipid molecule content in the lipid composition of the basic constituent lipid molecules is 98 mol% or less, preferably 94 mol% or less, more preferably 90 mol% or less. It is. Particularly preferably, in the mixed composition, the content of anionic lipid molecules in the lipid composition of the basic constituent lipid molecules is 50 mol% or less, more preferably 30 mol% or less, and still more preferably 15 mol% or less. It is.

As the anionic lipid molecule constituting the bubble liposome according to the present invention, a lipid molecule in which the charge of the molecule as a whole is biased to the negative side in the predetermined solution can be used. Specifically, as with the lipid molecules that make up normal liposomes, the use of lipid molecules that have a property of being negatively charged as a whole, with negative charge predominating in the charge of positive and negative charges within the molecule, as a whole. Can do.
As the anionic lipid molecule according to the present invention, an anionic lipid molecule capable of forming a double membrane structure at room temperature or in the body temperature can be used. Examples of the anionic lipid molecule include phosphatidylglycerol-based lipid molecules. More specifically, examples of anionic lipid molecules include DSPG (distearoylphosphatidylglycerol), DPPG (dipalmitoylphosphatidylglycerol), DMPG (dimyristoylphosphatidylglycerol), DLPG (dilaurylphosphatidylglycerol) HSPG (hydrogenated). Soybean phosphatidylglycerol), HEPG (hydrogenated egg phosphatidylglycerol) and the like can be used.
In particular, considering the stability of nitrogen monoxide retention in bubble liposomes, lipid molecules having an alkyl chain having 14 to 20 carbon atoms, preferably lipid molecules having an alkyl chain having 16 to 18 carbon atoms, may be used. Is preferred. The alkyl chain is preferably a linear molecule.
As an example of the embodiment, it is particularly preferable to use DSPG / or DPPG as the anionic lipid molecule according to the present invention. In particular, it is more preferable to use DSPG.

Cationic lipid molecule As the bubble liposome according to the present invention, the bubble liposome substantially comprising as a basic constituent lipid molecule the charge in the above-mentioned predetermined solution is cationic. Nitrogen retention ability is remarkably low, stability is not ensured, and it is not recognized as suitable. That is, it is preferable that the bubble liposome according to the present invention is substantially free of lipid molecules having a cationic charge at pH 7.3 to 7.5 as basic constituent lipid molecules.

However, as the bubble liposome according to the present invention, even if it contains a cationic lipid molecule as a part of the basic constituent lipid molecule, its content is very small and it is one in vivo when the bubble liposome is formed. In the case where the nitrogen oxide inclusion retention ability exhibits the following predetermined function, its inclusion can be allowed.
Here, as a specific content indicating that “the cationic lipid is substantially not included”, in the lipid composition of the basic constituent lipid molecule, the content of the lipid molecule exhibiting a cationic property is 4 mol% or less, preferably Examples include 2 mol% or less, more preferably 1 mol% or less, and particularly preferably 0.5 mol% or less. Moreover, as an aspect acceptable as a bubble liposome containing a cationic lipid, an aspect in which the cationic lipid molecule is uniformly distributed throughout the bubble liposome is more preferable than an aspect in which the cationic lipid molecules are partially localized.
The bubble liposome according to the present invention is particularly preferably an embodiment in which a lipid molecule exhibiting cationic properties is not included as a basic constituent lipid molecule from the viewpoint of the ability to stably hold nitric oxide in vivo.

[PEG-modified lipid]
The bubble liposome according to the present invention is preferably an embodiment in which polyethylene glycol (PEG) is contained on the surface of the lipid bilayer membrane at a predetermined content. That is, the bubble liposome according to the present invention is preferably an embodiment comprising a lipid molecule modified with polyethylene glycol at a predetermined molar content or less with respect to the basic constituent lipid.
Here, the PEG molecule can be a part of the constituent molecule of the bubble liposome according to the present invention as a PEG-modified lipid molecule in which PEG is bound to the basic constituent lipid molecule. As a binding mode between the PEG molecule and the basic constituent lipid molecule, a binding mode that can be used in the preparation of a normal liposome can be adopted.

In the bubble liposome according to the present invention, when the PEG-modified lipid molecule is contained in the basic constituent lipid molecule, a hydrated layer is formed by the interaction between the PEGs on the surface of the bubble liposome, and from macrophages and various degrading enzymes. Protection is possible. As a result, it becomes possible to dramatically improve the stability of the bubble liposome in the in vivo environment such as a blood vessel by the hydrated layer.
The bubble liposome according to the present invention preferably contains a predetermined ratio or more of PEG molecules in order to form a hydrated layer imparting in vivo stability. Specifically, it is preferable that the content of the PEG-modified lipid molecule in the lipid composition of the basic constituent lipid molecule is 0.1 mol% or more, preferably 1 mol% or more, more preferably 2 mol% or more. Here, in the case of bubble liposomes having a PEG-modified lipid molecule content of 0 mol%, a hydrated layer is not formed on the membrane surface of the bubble liposomes, but the stability of the bubble liposomes in the in vivo environment such as in blood vessels is good. Absent. Therefore, it is recognized that it is preferable that the bubble liposome used as the nitric oxide delivery carrier has a PEG modification on the surface.
Moreover, it is recognized that the PEG content of the bubble liposome according to the present invention affects the stable retention ability of nitric oxide, and those having a PEG content that is too high are not suitable. Specifically, the upper limit is preferably such that the content of the PEG-modified lipid molecule in the lipid composition of the basic constituent lipid molecule is 30 mol% or less, preferably 10 mol% or less. Particularly preferred is an embodiment in which the content of PEG-modified lipid molecules in the lipid composition of the basic constituent lipid molecules is 6 mol% or less.

The PEG-modified lipid molecule according to the present invention has a molecular structure in which a PEG chain is bonded to a part of the basic constituent lipid molecule described above. When the bilayer structure is formed, a structure in which PEG molecular chains protrude from the hydrophilic side of the outer surface and / or inner surface of the bubble liposome is provided.
As the PEG-modified lipid molecule according to the present invention, a PEG molecule having a molecular weight suitable for forming a hydrated layer is preferably used. As the PEG average molecular weight, for example, an average molecular weight of 200 to 50000, preferably 500 to 20000, and more preferably 750 to 5000 can be suitably exemplified.
Further, as a PEG molecule for modifying the basic constituent lipid molecule, a PEG derivative can be used in addition to a normal PEG. As the PEG derivative, one provided with a maleimide group, an aldehyde group, an amino group, a hydroxyl group, a carboxyl group or the like, or one having a terminal NHS can be used.

[Target-directed molecules]
The bubble liposome according to the present invention is preferably in a mode having a target directivity-improving molecule in order to improve selectivity and delivery efficiency in nitric oxide delivery to a desired site in a living body.
Here, as the target directivity-improving molecule, preferred examples include ligand molecules and antibodies that can be used for liposome modification used in ordinary drug delivery systems (DDS) and the like. That is, as the bubble liposome according to the present invention, it is possible to adopt a mode in which ligand molecules and / or antibodies are bound. In the present invention, the structure in which these molecules are exposed on the outer surface of the bubble liposome can dramatically improve the directivity of delivery to a target tissue or the like.
Here, examples of antibodies, ligand molecules, and the like include peptides, proteins, glycoproteins, and the like that can bind to or adhere to a predetermined target. More preferred are those with high target specificity. Examples of proteins targeted by antibodies and ligand molecules include, but are not limited to, antigens, receptors, and cell adhesion molecules present in cell membranes and extracellular matrices.

In addition, as the target directivity-improving molecule that can be used in the present invention, proteins other than the above-mentioned antibodies and peptidic polymer compounds can be employed as long as they can be bound to the bubble liposome surface. is there. For example, a mode in which a cell membrane permeable peptide, a signal peptide, an Fc binding peptide of IgG, and the like are bound to the surface of a bubble liposome can be used.
In addition, other compounds other than proteins and peptidic properties can be employed as the target directivity improving molecule. For example, when there is a compound or the like that can be recognized and bound by a cell such as a predetermined target tissue, the compound is employed as a target directivity improving molecule according to the present invention, and is bound to a bubble liposome surface. Is possible.

  As an aspect which has a target directivity improvement molecule | numerator on the bubble liposome surface based on this invention, the aspect which couple | bonds the said molecule | numerator specifically to the PEG molecular chain of a PEG modification lipid molecule can be mentioned. For example, as a specific binding mode in the case of using an antibody, a ligand molecule, or the like, it can be easily performed through a molecule or a functional group serving as a linker. As an example, a mode in which maleimide is added to the end of a PEG molecular chain and cysteine is added to the end of an antibody, a ligand molecule, or the like to bond the SH group of the cysteine and a functional group derived from maleimide. Although it can mention, the coupling | bonding aspects, such as an antibody and a ligand molecule, are not restrict | limited in particular to the said aspect.

[Internal gas]
The bubble liposome according to the present invention comprises nitric oxide encapsulated inside a lipid bilayer spheroid (liposome) formed of basic lipid molecules. Here, “inner packaging” in the present specification includes concepts such as inner packaging and encapsulation.
Here, the realization of the temporary or short-time inclusion state of nitric oxide in the bubble liposome can be performed regardless of the charge state of the basic lipid molecule. In the bubble liposome (Non-Patent Document 2) comprising the above, since nitric oxide contained within 3 to 5 minutes escapes from the bubble liposome in the living body, it is stable in the intravascular environment circulating in the living body. Thus, nitric oxide cannot be retained (see Example 1 of the present specification).
In contrast, in the bubble liposome composed of the basic constituent lipid molecules according to the present invention described above, nitric oxide is in a gaseous state (gas molecule state, gas state) in the bubble liposome even in an intravascular environment circulating in the living body. Can be held stably. Specifically, in the bubble liposome according to the present invention, it is possible to stably maintain nitric oxide for a time sufficient for blood circulation in the body for about 10 minutes to several tens of minutes, so any target tissue in the living body or It becomes feasible to perform nitric oxide delivery to the site.

The bubble liposome according to the present invention is preferably encapsulated as a pure gas of nitric oxide or a gas in a high purity state. Moreover, it can also be included as a mixed gas of nitric oxide and another gas as desired.
In the embodiment in which nitric oxide is included as the mixed gas, the mixing ratio of nitric oxide is arbitrary, but considering the delivery efficiency, for example, 10% or more, preferably 30% or more, more preferably 50% of the volume of the included gas. An embodiment in which nitric oxide is included at a ratio of% or more can be mentioned. Here, the mixed gas that can be included in the bubble liposome according to the present invention is not particularly limited as long as it is a gas that can be included in the bubble liposome and does not exhibit substantial reactivity with nitric oxide. It is possible. As an example, it is possible to enclose perfluorohydrocarbon gas or the like, which is an ultrasound contrast gas, as a mixed gas with nitrogen monoxide.
Specific examples of the perfluorohydrocarbon gas include perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane, perfluoroheptane, and perfluorooctane. it can.

[Bubble liposome particle size]
The bubble liposome according to the present invention is not particularly limited as long as it has a particle size that encloses nitric oxide and can stably maintain the bubble structure in the living body, but is suitable for circulation in blood vessels. Those having a small diameter are suitable. In the present invention, the particle size of the bubble liposome in the state of containing nitric oxide includes, for example, an average particle size of 2.5 μm or less, preferably 2 μm or less, more preferably 1.6 μm or less. it can.

In the bubble liposome according to the present invention, as the particle size is finer, a tendency to increase the amount of nitric oxide that can be stably encapsulated in the bubble structure is observed, and further, stability in the in vivo environment is also improved. Thus, the nitric oxide retention ability is improved. Therefore, as the bubble liposome according to the present invention, it is more preferable to use a finer bubble liposome than the above-described μm-scale bubble liposome. In the present specification, such micronized bubble liposomes are called nanobubble liposomes.
As an embodiment using nanobubble liposomes as bubble liposomes according to the present invention, those having an average particle diameter of 300 nm or less, preferably 200 nm or less can be mentioned. In the bubble liposome, it is recognized that the finer the particle size, the better the migration in the capillary blood vessels and the better the transferability to the deep part of the tissue. Particularly preferred is a scale mode of 100 nm or less which is effective for accumulation in cancer tissues or inflamed sites.

  Although there is no restriction | limiting in particular as a particle size size of the bubble liposome which concerns on this invention, 20 nm or more which is near the lower limit size which can prepare a nano bubble liposome, Preferably 40 nm or more can be mentioned.

[Others]
The bubble liposome according to the present invention may have an aspect including or including a molecule, a compound, or the like other than those described above, unless it is an aspect that substantially hinders the effect of the technical characteristics according to the present invention. It is.
For example, it is possible to adopt a mode in which other compounds, structures, and the like are mounted on, added to, or bonded to the surface of the bubble liposome. Further, it is possible to adopt an embodiment in which other compounds, structures, and the like are added to or combined with PEG molecules or linker molecules bonded to PEG. It is also possible to adopt an embodiment in which other compounds, structures, gas molecules and the like are included in the bubble liposome.
As an example of such an embodiment, it is possible to adopt an embodiment in which a pharmacological component such as a nucleic acid molecule or a compound is mounted on, added to, or bound to the surface of a bubble liposome and used as a carrier for a drug delivery system. is there.

2. Nitric Oxide Delivery System and Various Agent Applications The bubble liposome according to the present invention can be effectively used in the constituent means of the nitric oxide delivery system and various agent applications.

[Nitric oxide delivery system]
The bubble liposome according to the present invention is a technology in which means for selectively releasing nitric oxide at an arbitrary site is established in addition to the stable retention of nitric oxide in the in vivo environment. .
Accordingly, the present invention includes an invention relating to a nitric oxide delivery system (NO delivery system) comprising the bubble liposome as a constituent means. That is, the present invention includes an invention related to a system for delivering nitric oxide site-selectively in a living body.
The expression “part” in the living body here is used as an expression including tissues, organs, and the like.

Spontaneous sustained release In the system according to the present invention, it is possible to construct a nitric oxide delivery system utilizing the spontaneous sustained release action of nitric oxide.
Specifically, in the bubble liposome constituting the system according to the present invention, after a stable retention of nitric oxide for 10 minutes to several tens of minutes after administration to the in vivo environment, an appropriate time lapse of several tens of minutes to several hours Along with this, spontaneous release of nitric oxide occurs. Therefore, when the bubble liposome is administered locally, it becomes possible to selectively deliver nitric oxide at the site and / or the periphery thereof. That is, the physiological function of nitric oxide can be exhibited site-selectively and efficiently by local administration.

As a specific aspect of the administration form, there is no particular limitation as long as the administration form achieves local administration at a desired target site, and various administration forms can be assumed.
As the administration mode, it is preferable to adopt a mode such as subcutaneous administration such as subcutaneous injection, or local vascular administration using a tourniquet. In these aspects, for example, the physiological function of nitric oxide can be exhibited site-selectively only by using simple administration means using an injection device, an infusion device, or the like. In the subcutaneous administration mode, for example, nitric oxide can be selectively and efficiently delivered to tissues and organs such as muscle tissue, subcutaneous tissue, brain, intraocular, tumor, and central nerve. It becomes possible.

Use of Ultrasonic Irradiation Means In the system according to the present invention, it is possible to construct a nitric oxide delivery system utilizing the phenomenon that the encapsulated gas in the bubble liposome is released by ultrasonic irradiation (FIG. 16).
Specifically, in the bubble liposome constituting the system according to the present invention, it is possible to induce the release of nitric oxide from the bubble liposome by performing local ultrasonic irradiation on a desired site after blood vessel administration. It becomes. Therefore, in this aspect, it becomes possible to selectively deliver nitric oxide at an arbitrary site where ultrasonic irradiation is performed and / or the periphery thereof. In other words, local ultrasonic irradiation from outside the body can be performed so that the physiological function of nitric oxide can be exhibited site-selectively and efficiently.

Specific embodiments of the form using the ultrasonic irradiation include: i) administering the bubble liposome into a blood vessel so that the bubble liposome is circulated in the systemic vascular system including the administration site of interest. Is preferred.
Here, when systemic administration is performed by vascular administration, in the case of human beings, delivery to all organs and tissues is possible in about 5 to 20 minutes. It is preferable to carry out after considering the above. That is, in the present invention, it is preferable to irradiate the target site with ultrasonic waves after the time required to sufficiently reach the target tissue by administering the bubble liposome to the blood vessel.
Here, as a vascular administration form for systemic administration in this embodiment, vascular administration to veins, vascular administration to arteries, administration to capillaries, intestinal tract administration, oral administration, and the like are administration to the bloodstream. It is possible to envisage various possible dosage forms.
Preferably, the administration form includes an embodiment in which intravenous blood vessel administration is performed. For example, administration methods such as intravenous injection into a vein or intravenous infusion can be employed.
In addition, as a specific aspect of the form using the ultrasonic irradiation, after performing local administration using a tourniquet or the like depending on a tissue or the like to be a target site, the target site is irradiated with ultrasonic waves. It is also possible to adopt a mode of performing the above. In this case, the ultrasonic irradiation is performed after local administration, not systemic administration.

In the system using ultrasonic irradiation means in the system according to the present invention, the systemic administration mode described in i) above is particularly suitable. In this aspect, nitric oxide can be selectively delivered to any part of the whole body that can be delivered to any organ or tissue such as the brain, heart, muscle, and other internal organs and cannot be delivered by subcutaneous injection or the like. Is possible. It is also possible to selectively deliver nitric oxide to muscle tissue, subcutaneous tissue, brain, intraocular, tumor, central nerve and the like.
In the case of using bubble liposomes bound to directivity-improving molecules such as antibodies and ligand molecules described in the above paragraph, higher nitric oxide can be obtained by performing ultrasonic irradiation after the time for accumulation on the target. The delivery effect can be realized.

In an aspect in which the ultrasonic irradiation means is used in the system according to the present invention, the ultrasonic irradiation means is included as a component of the system. That is, the system according to the present invention includes an invention relating to a nitric oxide delivery system comprising ultrasonic irradiation means as a constituent means.
Here, examples of the release mode of the encapsulated gas in the system include the promotion of leakage from the lipid membrane due to the resonance phenomenon of the encapsulated gas, the burst of the bubble liposome itself, and the like. Any mechanism of action is included as long as it induces the release of nitric oxide induced by the ultrasonic irradiation.

As ultrasonic irradiation conditions in the system, it is possible to employ ultrasonic irradiation with an irradiation frequency (frequency) of 0.2 to 10 MHz, preferably 0.5 to 5 MHz. When an ultrasonic wave having a frequency in the range is irradiated, it acts in a direction in which the induction of release of the encapsulated gas is promoted. When a large amount of perfluorohydrocarbon gas is included as the encapsulated gas of the bubble liposome, it is recognized that resonance rupture is likely to be induced.
It should be noted that, under conditions where the irradiation frequency greatly deviates to the high frequency side or low frequency side of the range, it is difficult to induce the release of the inclusion gas, and thus it is not suitable as the irradiation condition according to the present invention.

As the ultrasonic irradiation conditions in the system, from the viewpoint of easy induction of the release of nitric oxide encapsulated in bubble liposomes, ultrasonic irradiation with a relatively weak intensity as employed in ultrasonic therapy is adopted. It is possible. Specifically, the irradiation intensity can be 0.1 W / cm ² or more, preferably 0.5 W / cm ² or more, more preferably 1 W / cm ² or more.
The upper limit of the irradiation intensity is not particularly limited as long as it does not substantially affect the human body, and can be, for example, 2000 W / cm ² or less as long as it uses focused ultrasonic irradiation. Moreover, if it is the aspect using the ultrasonic wave for treatment with the safer influence on a human body, it is suitable to employ | adopt 20 W / cm < ² > or less, for example, Preferably it is 10 W / cm < ² > or less.

The irradiation time in the system is preferably set according to the irradiation intensity, and it is possible to adopt a range in which the release of the encapsulated gas from the bubble liposome can be induced and the human body is not substantially affected. Is preferred.
Specifically, if a strong irradiation intensity such as focused ultrasonic waves is employed, it is preferable to set a relatively short time in order to avoid the irradiation site from becoming a high temperature. As an example, setting conditions in the range of 1 second to 3 minutes can be given.
In addition, if a weak irradiation intensity for ultrasonic therapy is employed, it is preferable to set a relatively long time in order to sufficiently induce nitric oxide release. As an example, the setting conditions in the range of 10 seconds to 60 minutes can be given.

The ultrasonic irradiation means constituting the system according to the present invention can be used without particular limitation as long as it is an apparatus or an apparatus provided with means capable of irradiating ultrasonic waves that satisfy the above conditions. .
Specifically, in the system according to the present invention, it is possible to use an ultrasonic irradiation apparatus or instrument capable of weak ultrasonic irradiation from the viewpoint of easy release induction of nitric oxide encapsulated in bubble liposomes. is there.
In this aspect, for example, it is possible to use a therapeutic ultrasonic irradiation apparatus used in clinical practice. It is also possible to divert and use a diagnostic ultrasonic diagnostic apparatus. Since these devices are devices that are ordinarily widely used in the medical field or the like, it becomes an aspect that can be easily used by many patients.
In the system according to the present invention, it is also possible to use an apparatus capable of irradiating focused ultrasound with the irradiation area narrowed to a pinpoint. In the aspect using the focused ultrasound device, for example, it is possible to perform the predetermined ultrasound irradiation without attenuating the brain tissue covered with the skull or the visceral tissue deep in the body cavity.
In the system according to the present invention, it is also possible to use a low-frequency ultrasonic device used for fracture treatment or the like.

  Ultrasonic irradiation using the ultrasonic irradiation means can be executed by applying a probe or the like, which is an ultrasonic generator, from outside the body to a target site in the living body. Since the ultrasonic irradiation means in the system according to the present invention does not normally require a surgical operation such as an incision, it is recognized as a treatment that places little burden on the patient.

Dosage The dosage of bubble liposomes in the system according to the present invention can be appropriately determined according to the desired physiological function of nitric oxide, the target tissue, and the like. In one aspect, in terms of the total lipid content of bubble liposomes, administration of about 0.001 to 100 mg, preferably about 0.1 to 50 mg per kg body weight of a subject (non-specimen in the case of animals) is performed. Is possible.

[Applications for various agents]
In the bubble liposome according to the present invention, various physiological functions of nitric oxide can be exhibited selectively at an arbitrary site in the living body. Accordingly, the present invention includes inventions relating to various drugs characterized by comprising the above-mentioned nitric oxide-encapsulating bubble liposomes.

In the present invention, it can be used for medical use utilizing various physiologically active functions of nitric oxide that are exhibited by direct delivery of nitric oxide in a gaseous state to any site (tissue, organ, etc.) in the body. Become. Specifically, in the present invention, a vascular wall dilator, a blood pressure antihypertensive agent, an arteriosclerosis inhibitor and the like comprising the above-mentioned nitric oxide-encapsulating bubble liposome are included. By using these drugs, it is possible to treat and improve symptoms particularly related to cardiovascular diseases.
Moreover, in this invention, utilization for the pharmaceutical use using the central nerve protective effect of nitric oxide is also attained. A central neuroprotective agent comprising the nitric oxide-encapsulated bubble liposome is included. The drug can particularly improve the symptoms of brain treatment and nervous system diseases.

Furthermore, according to the present invention, it is possible to selectively and efficiently exert an EPR effect improving action utilizing vasodilation of nitric oxide at any site in a living body. That is, the present invention includes an EPR effect improving agent comprising the above-mentioned nitric oxide-encapsulated bubble liposome.
Here, “EPR” is an abbreviation for Enhanced Permeability Retention, and a phenomenon in which a drug on the scale of several tens to several hundreds of nanometers that does not penetrate normal blood vessels accumulates in a gap between new blood vessels around a tumor or an inflammation site. Point to.
In the EPR effect enhancer, by selectively using a pharmacological component in combination with a nano-preparation containing, binding, adhering, or mounting, the nano-form preparation passes through the blood vessel wall of the target tissue or the like, and is selectively It is possible to accumulate the nano-ized preparation in With this drug, this EPR effect-improving action can greatly improve the treatment efficiency of the nano-formulation administered together.
Here, as a DDS carrier constituting a nano-preparation to be administered in combination with the EPR effect improver, it usually does not pass through the vascular wall in a vascular state, but can pass through the expanded vascular wall according to the vasodilatory action. If it is a thing, it can be used without a restriction | limiting in particular. Examples include liposomes, micelles, capsule-like protein complexes, virus particles, metal nanoparticles, nanocrystals, dendrimer nanoparticles, etc., which can exhibit the EPR effect improving action according to the present invention. If it is, it will not restrict | limit in particular in these.
In addition, as a drug encapsulated in, combined with, attached to, or mounted in a nano-formulation administered in combination, any drug can be used depending on a desired use, and in particular, an anticancer drug or anti-rheumatic drug is used. In an embodiment in which a pharmacological component such as a drug is included, the therapeutic effect can be greatly improved. Moreover, the effect is also expected in an embodiment including a nucleic acid drug.

As the dosage forms of various drugs according to the present invention, all modes of agents such as liquid, viscous liquid, semi-solid, solid, encapsulated, powder, granule, and excipient mixed type are assumed. However, in the case of use at the time of administration, it is preferably a liquid dosage form that can be vascularly or subcutaneously administered. Preferably, shapes such as ampoule liquid and concentrated liquid are suitable.
In addition, for long-term storage of several days or more in a state of containing nitric oxide, an agent form that is preferably stored in a liquid state pressurized and sealed in a sealed container is suitable.
Moreover, it is also possible to employ | adopt the aspect made into a mixed composition with a pH adjuster, antibiotics, another chemical | medical agent, etc. as needed.

Here, in this invention, invention regarding the pharmaceutical kit or pharmaceutical composition using the EPR improvement effect which the said nitric oxide inclusion bubble liposome has is included. That is, the present invention includes a pharmaceutical kit or a pharmaceutical composition comprising the nitric oxide-encapsulated bubble liposome.
Here, as an example of a pharmaceutical kit, for example, a pharmaceutical product in the form of a kit separately containing the above-mentioned nitric oxide-encapsulating bubble liposome and other pharmacological components can be mentioned. Moreover, as an example of a pharmaceutical composition, the pharmaceutical product of the composition form which contains the said nitric oxide inclusion | inner_cover bubble liposome and another pharmacological component as a mixed composition can be mentioned, for example.
In particular, in the present invention, an embodiment comprising a nano-ized preparation having other pharmacological components is suitable. In this embodiment, the EPR effect of the nano-ized preparation described in the above paragraph can be dramatically improved.

  As dosage forms of various drugs according to the present invention, reference can be made to the dosage forms of the nitric oxide delivery system described in the above paragraph. The dosage can also refer to the dosage form of the nitric oxide delivery system described in the above paragraph.

3. Various methods
The present invention includes inventions relating to the production of the above-described nitric oxide-encapsulating bubble liposomes and various methods relating to the related technology.

[Production method]
In the present invention, it is possible to stably maintain nitric oxide of bubble liposomes in a living environment by using bubble liposomes containing lipid molecules having the above-mentioned predetermined properties as substantially basic constituent lipid molecules. is there.
That is, in the present invention, in the production process of bubble liposome encapsulating nitric oxide, the liposome comprising the lipid molecule having the predetermined property as a substantially basic constituent lipid molecule is allowed to contain nitric oxide. The invention relates to a method for producing a nitric oxide-encapsulating bubble liposome capable of retaining nitric oxide in an in vivo environment.

  In the production process of bubble liposomes according to the present invention, any known or unknown method for producing bubble liposomes is used, except that the characteristics such as the type and composition of basic constituent lipid molecules are those described in the above paragraph. It is possible to use. Moreover, description of Example 1 can be referred as one Embodiment of the manufacturing process of the bubble liposome which concerns on this invention.

  As one aspect of the production method according to the present invention, for example, a liposome having a large particle size is prepared by a conventional method, and then passed through a nanofilter or the like to be miniaturized, and this is sealed with nitrogen monoxide pressurized. Examples of the method include ultrasonic treatment in a reagent bottle of the formula. By performing this operation, it becomes possible to prepare a bubble liposome encapsulating nitric oxide, and the particle size can be obtained as a nitric oxide bubble liposome close to the upper limit of the particle size range. It becomes possible.

  Further, as another aspect of the production method according to the present invention, after preparing the μm-scale nitric oxide-encapsulating bubble liposome described in the above paragraph, this is sealed in a reagent bottle sealed with nitrogen monoxide under pressure, etc. And vigorous suspension with a high-speed stirrer or the like. By performing this operation, it becomes possible to prepare nanobubble liposomes encapsulating nitric oxide, and the particle size is 20 to 300 nm, preferably 40 to 200 nm, more preferably about 50 to 180 nm. It is possible to obtain a nanobubble liposome corresponding to the prepared nanobubble liposome.

[Storage method]
The produced nitric oxide-encapsulated bubble liposomes can be stored in a closed container or the like and stored under pressurized conditions for long-term storage in consideration of production distribution as a product. That is, in this invention, the invention regarding the storage method of the nitric oxide inclusion | inner_cover bubble liposome is included.

As the pressurizing condition for the long-term storage, a pressure higher than 1 atm can be adopted so that leakage of the encapsulated gas from the bubble liposome is prevented. It is preferable to store under pressure conditions of preferably 1.2 atmospheres or more, more preferably 1.5 atmospheres or more, still more preferably 1.8 atmospheres or more, and particularly preferably 2 atmospheres or more. In particular, in order to ensure the maintenance of the storage state for several days or more, it is preferable to increase the pressure condition. As the upper limit of the pressure condition, the range of the pressure resistance condition of the sealed container can be adopted, and for example, 5 atm or less can be mentioned.
In the storage, any closed container having a pressure resistance capable of enclosing a liquid sample can be used without particular limitation. It is also possible to use commercially available glass vials, resin sample containers, and the like. As the gas for pressurization filled in the sealed container, it is preferable to use a gas having the same composition as the encapsulated gas encapsulated in the bubble liposome. Preferably, nitric oxide is used.

[Various methods]
In the present invention, in addition to the above-described production method and storage method, inventions relating to various methods utilizing various functions and effects exhibited by the bubble liposome according to the present invention are included.
For example, the present invention includes a method for improving the ability to retain nitric oxide in bubble liposomes in the in vivo environment, which includes the steps described in the above production method. That is, the present invention includes a method for improving the ability to retain nitric oxide in bubble liposomes, a method for in vivo stabilization of nitric oxide-encapsulated bubble liposomes, and the like.
In addition, the present invention includes a method for site-selective delivery of nitric oxide in vivo, characterized in that it includes the processes and operations described above for the nitric oxide delivery system.
In addition, the present invention includes methods for treating various diseases and improving symptoms using the physiological function of nitric oxide. That is, the present invention includes inventions relating to a vascular wall dilation method, blood pressure reduction method, arteriosclerosis suppression method, central nerve protection method, EPR effect improvement method, and the like.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated, the scope of the present invention is not limited by these.

[Example 1] “Nitrous oxide retention ability of bubble liposome in vivo”
Bubble liposomes encapsulating nitric oxide inside the liposome were prepared, and the ability to retain nitric oxide when administered systemically by intravenous injection was evaluated.

(1) “Preparation of nitric oxide-encapsulated bubble liposome”
Each lipid was dissolved in an organic solvent (chloroform: diisopropyl ether: PBS = 1: 1: 1) so that the lipid composition was as shown in the following table.
Here, as basic constituent lipids, DSPG which is an anionic lipid (1,2-distearoyl-sn-glycero-3-phospho- (1'-rac-glycerol) (sodium salt), manufactured by Avanti Polar Lipid), DSPC, which is a neutral lipid (1,2-distearoyl-sn-glycero-3-phosphocholine, manufactured by NOF Corporation), DPPC, which is a neutral lipid (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, JP Oil Co., Ltd.), DOTAP (N- [1- (2,3-Dioleoyloxy) propyl] -N, N, N-trimethylammonium methyl-sulfate, Avanti Polar Lipids, Inc.), a cationic lipid, The composition was used as shown in the following table. As PEG-conjugated lipids, DSPE-PEG ₂₀₀₀ (Distearoyl phosphatidyl ethanolamine-PEG ₂₀₀₀ -OMe, manufactured by NOF Corporation), DSPE-PEG ₇₅₀ (Distearoyl phosphatidyl ethanolamine-PEG ₇₅₀ -OMe, manufactured by NOF Corporation) are used. Using.
The solution was subjected to ultrasonic treatment using a probe sonicator (20 kHz), and then the organic solvent was distilled off using a rotary evaporator to obtain liposomes.
The obtained liposome was freeze-thawed and then passed through a polycarbonate membrane having a pore size of 0.2 μm using an extruder (Lipex Biomembrane Inc.) to prepare a particle size of about 100 to 200 nm. Thereafter, the solution was sterilized by filtration using a cellulose acetate membrane syringe filter having a pore size of 0.45 μm.

0.8 mL of the prepared liposome solution (lipid conversion concentration: 1 mg / mL) is sealed in a saturated sealed vial with perfluoropropane (C ₃ F ₈ ), and then nitric oxide is added to the vial. Was sealed under pressure. The sample was subjected to ultrasonic treatment for 5 minutes using a bath sonicator (42 kHz) to prepare a suspension of bubble liposomes containing nitric oxide. Here, in the bubble liposome according to the present example, nitric oxide gas and C ₃ F ₈ gas are encapsulated so as to be a 1: 1 mixed gas, but C ₃ F ₈ gas is an ultrasonic wave for detection. It is a gas mixed as a contrast gas. The properties of the bubble liposomes obtained in this example are shown in the following table.

  As a result, it is possible to prepare bubble liposomes with an increased particle size by performing gas encapsulation, in any case using three types of liposomes with different charge-related properties of basic constituent lipids. It has been shown.

(2) “Identification and quantification of nitric oxide”
Regarding the prepared nitric oxide-encapsulated bubble liposome, it was confirmed whether the encapsulated nitric oxide was encapsulated in a gaseous state having physiological activity.

  After adding 100 μL of 10 μM DAF-2 (Diaminofluorescein-2, Goryuka Chemical Co., Ltd.) in PBS to 20 μL of nitric oxide-encapsulated bubble liposomes prepared in (1) above, using a bath sonicator (42 kHz) Sonication was performed for 10 seconds. The relative fluorescence intensity (RFU value) of the obtained solution was measured with a fluorometer (Em: 495 nm, Ex: 515 nm). The results are shown in the table below.

  As a result, for both the neutral lipid bubble liposome (Sample 1-2) and the cationic lipid-containing bubble liposome (Sample 1-3), the same fluorescence intensity derived from the structural change of DAF-2 was detected. . That is, it was confirmed that nitric oxide was encapsulated in the state of gas having physiological activity in the bubble liposomes prepared in the examples.

(3) “Change over time at normal pressure”
About the prepared nitric oxide inclusion | inner_cover bubble liposome, the nitric oxide retention amount after storing for 30 minutes at a normal pressure was measured. Nitrogen monoxide retention was measured by relative quantification using DAF-2 in the same manner as described in (2) above. The results are shown in the following table and FIG.

As a result, when the nitric oxide-encapsulated bubble liposome was stored for 30 minutes under normal pressure, the cationic lipid-containing bubble liposome (sample 1-3) was immediately after the production according to (2) above. It was shown to decrease to about 12% of the amount of nitric oxide. In contrast, in the neutral lipid bubble liposome (Sample 1-2), about 31% of the amount of nitric oxide immediately after production remains, and remains after storage for 30 minutes at normal pressure than the cationic lipid-containing bubble liposome. The rate increased about 2.5 times.
From this, it was shown that the neutral lipid bubble liposome has the property that the nitric oxide retention ability at normal pressure is significantly improved as compared with the cationic lipid-containing bubble liposome. Here, the cationic lipid-containing bubble liposome described in Sample 1-3 is the same as the lipid composition of the bubble liposome described in Non-Patent Document 2 of the prior art.

(4) "Systemic administration and in vivo imaging"
About the prepared anionic lipid bubble liposome (sample 1-1), neutral lipid bubble liposome (sample 1-2), and cationic lipid-containing bubble liposome (sample 1-3), systemic administration by intravenous injection and in vivo Imaging was performed to evaluate nitric oxide retention ability in vivo.

The above-prepared bubble liposome solution 200 μL (lipid equivalent concentration: 1 μg / μL) was systemically administered to 5-6 week-old ICR strain mice by tail vein injection. After administration, echocardiograms of the heart were taken every minute using an ultrasonic diagnostic apparatus. Since the bubble liposome is not ruptured by echo irradiation (12 MHz) of the ultrasonic diagnostic apparatus, the contrast image in the echo detects C ₃ F ₈ gas that is a contrast gas in the bubble liposome that is not ruptured. Thus, the behavior of nitric oxide-encapsulated bubble liposomes is exhibited. The bubble liposome images blood vessels throughout the body by intravenous administration, but the heart was selected and photographed for the purpose of facilitating image analysis.
The luminance of the photographed photographic image was measured using image analysis software (ImageJ), and the ROI value (Region of intensity) was calculated. The ROI value is a value obtained by quantifying a value indicating the intensity of echocardiography. The results are shown in FIGS. A part of the photographed image is shown in FIGS.

  As a result, when nitric oxide is encapsulated in the anionic lipid bubble liposome (sample 1-1) or the neutral lipid bubble liposome (sample 1-2), a high heart rate derived from the contrast gas for a long time after systemic administration. The echo intensity was detected (FIGS. 2 and 3). That is, it was shown that these bubble liposomes have high stability even in an intravascular environment circulating in the living body and can stably hold nitric oxide for at least 21 minutes exceeding the human blood circulation time. . In particular, the neutral lipid bubble liposome (Sample 1-2) has a remarkably high ability to stably hold nitric oxide, indicating that it can stably hold nitric oxide for 29 minutes in vivo. .

  On the other hand, in the case where nitric oxide is encapsulated in the cationic lipid-containing bubble liposome (Sample 1-3), the echocardiographic intensity derived from the contrast gas is drastically reduced in only 3 minutes after systemic administration, and 5 minutes have elapsed after systemic administration. By the time, the signal was not detected (FIGS. 4 and 5). That is, it was shown that the cationic bubble liposome is extremely unstable in the vascular environment circulating in the living body and has the property that it cannot retain the encapsulated nitric oxide for a long time.

(5) "Summary"
From the above results, it is possible to encapsulate nitric oxide in bubble liposomes regardless of the charge of the constituent lipids. In cationic bubble liposomes (with the same lipid composition as in Non-Patent Document 2), It has been shown that nitric oxide cannot be stably maintained in the intravascular environment circulating in the body and cannot be used as a delivery carrier for nitric oxide.
On the other hand, neutral lipid bubble liposomes and anionic lipid bubble liposomes can stably hold nitric oxide even in an intravascular environment that circulates in the living body, and can be suitably used as a nitric oxide delivery carrier. It was shown to be possible. In particular, neutral lipid bubble liposomes have excellent retention time, indicating that they can be suitably used as nitric oxide delivery carriers.

[Example 2] “Effectiveness confirmation test of neutral lipid bubble liposome”
Bubble liposomes were prepared using neutral lipids different from the above examples, and the effect of the ability to retain nitric oxide in the living body of the neutral lipid bubble liposomes was confirmed.

(1) “Preparation of nitric oxide-encapsulated bubble liposome”
Nitrogen oxide-encapsulating bubble liposomes were prepared in the same manner as described in Example 1 except that neutral lipid DPPC was used as the basic constituent lipid. Here, as DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (manufactured by NOF Corporation) was used so as to have the composition shown in the following table.
The bubble liposome according to the present example was also encapsulated so that the ratio of C ₃ F ₈ gas for ultrasonic contrast and nitric oxide gas was 1: 1 as in the case of Example 1. The properties of the bubble liposomes obtained in this example are shown in the following table.
As a result, it was confirmed that even when DPPC was used, it was possible to prepare bubble liposomes with an increased particle size by performing gas encapsulation.

(2) “Systemic administration and in vivo imaging”
About the prepared neutral lipid bubble liposome (sample 2-1), the systemic administration by intravenous injection and in vivo imaging were performed, and the nitric oxide retention ability in the living body was evaluated. Systemic administration to mice and measurement of echocardiographic intensity by in vivo imaging were performed in the same manner as described in Example 1. The results are shown in FIGS.
As a result, even when bubble liposomes are prepared using DPPC as the basic constituent lipid and nitric oxide is encapsulated, the encapsulated nitric oxide can be stably retained in the vascular environment circulating in the living body. It was confirmed that.

From the above results, even when neutral lipid bubble liposomes are prepared using lipid molecules having a different carbon number from the alkyl chain than DSPC, the ability to retain nitric oxide in vivo is excellent. Was confirmed.

[Example 3] “Examination of PEG modification rate”
The effect of modification of lipid PEG molecules constituting bubble liposomes on the ability of bubble liposomes to retain nitric oxide in circulation in blood vessels was examined.

(1) “Preparation of nitric oxide-encapsulated bubble liposome”
Nitric oxide encapsulated bubble liposomes were prepared in the same manner as described in Example 1 so that the lipid composition was as shown in the following table. In the bubble liposome according to this example, DSPC was used as the basic constituent lipid, and DSPE-PEG ₂₀₀₀ was used as the PEG-linked lipid.
The bubble liposome according to the present example was also encapsulated so that the ratio of C ₃ F ₈ gas for ultrasonic contrast and nitric oxide gas was 1: 1 as in the case of Example 1. The properties of the bubble liposomes obtained in this example are shown in the following table.

  As a result, it is shown that, even when any liposome having a PEG modification rate of 2 to 10 mol% is used, it is possible to prepare bubble liposomes having an increased particle size by performing gas encapsulation. It was done.

(2) “Identification and quantification of nitric oxide”
The nitrogen monoxide-encapsulating bubble liposome prepared above was subjected to relative quantification of nitric oxide in vitro to measure the amount of nitric oxide retained during the preparation. The relative quantification of nitric oxide was performed in the same manner as described in Example 1 using DAF-2 which is a nitric oxide detection indicator. The results are shown in the table below.
As a result, in any of the bubble liposomes having a PEG modification rate of 2 to 10 mol%, it was confirmed that the same amount of nitric oxide was encapsulated in the state of a gas molecule having physiological activity.

(3) “Systemic administration and in vivo imaging”
The above-prepared bubble liposomes having different PEG modification rates were systemically administered by intravenous injection and in vivo imaging to evaluate the ability to retain nitric oxide in vivo. Systemic administration to mice and measurement of echocardiographic intensity by in vivo imaging were performed in the same manner as described in Example 1. The results are shown in FIG.

  As a result, any of the bubble liposomes having a PEG modification rate of 2 to 10 mol% is highly stable in an intravascular environment circulating in the living body, and nitric oxide for at least 17 minutes exceeding the human blood circulation time. It was shown that the stable maintenance of In particular, bubble liposomes (sample 3-1 and sample 3-2) having a PEG modification rate of 2 to 6 mol% have a remarkably high ability to stably hold nitric oxide, and nitric oxide for a long period of 30 minutes or more in vivo. It was shown that the stable maintenance of

(4) “Summary”
From the above results, it is possible to encapsulate nitric oxide in bubble liposomes regardless of the difference in the PEG modification rate, and it is possible to stably maintain nitric oxide even in an intravascular environment circulating in the living body. Indicated. That is, it was shown that it can be suitably used as a nitric oxide delivery carrier in vivo. In particular, when the PEG modification rate is in the range of 2 to 6 mol%, the in vivo stability is improved and the leakage of nitric oxide is greatly reduced, and it can be suitably used as a nitric oxide delivery carrier. It was shown that.
In the case of bubble liposomes with a PEG modification rate of 0%, a hydrated layer is not formed on the lipid membrane surface, and it cannot be said that the stability of bubble liposomes in the in vivo environment such as blood vessels is good. Therefore, it is recognized that it is preferable that the bubble liposome used as the nitric oxide delivery carrier has a PEG modification on the surface.

[Example 4] "Preparation of nanobubble liposome"
Further refined bubble liposomes were prepared from the above-prepared μm-scale bubble liposomes, and their properties were verified.

(1) “Preparation of nitric oxide-encapsulated bubble liposome”
Samples 3-1 to 3-3 in Example 3 in which neutral lipid bubble liposomes were prepared and Samples 1-3 in Example 1 in which cationic lipid-containing bubble liposomes were prepared were sealed in vials, respectively. These were vigorously stirred and mixed at 4400 rpm for 20 seconds at room temperature using a high-speed stirrer (AMALGAMATOR TP-103, Goldsmith & Revere, Inc) to obtain nitric oxide-encapsulated bubble liposomes with a refined particle size.
The bubble liposome according to the present example was also encapsulated so that the ratio of C ₃ F ₈ gas for ultrasonic contrast and nitric oxide gas was 1: 1 as in the case of Example 1. The properties of the bubble liposomes obtained in this example are shown in the following table.

  As a result, nanobubble liposomes (sample 4-1 to sample 4-) in which the average particle size was reduced to 50 to 200 nm in neutral lipid bubble liposomes by vigorously stirring the μm-scale bubble liposomes. It was shown that the preparation of 3) becomes possible. On the other hand, in the case of cationic lipid-containing bubble liposomes, even when the same stirring operation is performed, only those having an average particle diameter of about 660 nm (sample 4-4) can be obtained, and it is difficult to reduce the size. It was done.

(2) “Identification and quantification of nitric oxide”
About the prepared nitric oxide inclusion | inner_cover bubble liposome, it was confirmed whether the included nitric oxide was included with the gas which is a physiologically active substance. Nitric oxide was identified using DAF-2 in the same manner as described in Example 1. The results are shown in the following table and FIG.

As a result, the same level of fluorescence intensity derived from the structural change of DAF-2 was detected for all of the neutral lipid nanobubble liposomes (Sample 4-1 to Sample 4-3). That is, it was confirmed that nitric oxide was encapsulated in a gaseous state having physiological activity in the nanobubble liposomes prepared in this example.
Moreover, it was shown that the amount of nitric oxide that can be encapsulated in the neutral lipid nanobubble liposomes (Sample 4-1 to Sample 4-3) is increased as compared with the μm-scale bubble liposome before stirring.

(3) “Change over time at normal pressure”
About the said nitric oxide inclusion | inner_cover bubble liposome, the nitric oxide retention amount after storing for 30 minutes at a normal pressure was measured. Nitrogen monoxide retention was measured by relative quantification using DAF-2 in the same manner as described in (2) above. The results are shown in the following table and FIG.

As a result, in the neutral lipid nanobubble liposomes (Sample 4-1 to Sample 4-3), the fluorescence intensity indicating the amount of retained nitric oxide has a high value even when kept for 30 minutes under normal pressure conditions. Indicated. These values are high values corresponding to 50 to 74% of the value of the above (2) indicating the value immediately after the production, and the neutral lipid bubble liposome (sample 1-2) shown in Example 1 is used. ) Was significantly higher than the gas retention rate (about 31%) under the same conditions.
From this, it was shown that the nanobubble liposomes having an average particle diameter of 50 to 200 nm have the property of improving the nitric oxide retention ability at normal pressure.

(4) "Systemic administration and in vivo imaging"
About the prepared nitric oxide inclusion | inner_cover nanobubble liposome, the systemic administration by intravenous injection and in vivo imaging were performed, and the evaluation regarding the nitric oxide retention ability in the living body was performed. Systemic administration to mice and measurement of echocardiographic intensity by in vivo imaging were performed in the same manner as described in Example 1. The results are shown in FIG.

As a result, any of the nanobubble liposomes having a PEG modification rate of 2 to 10 mol% has high stability even in an intravascular environment circulating in the living body, and at least 30 minutes or more that greatly exceeds human blood circulation time. It has been shown that stable retention of nitric oxide is possible. The ROI value detected in this test is generally echocardiographic, compared with the results of a similar test with μm scale bubble liposomes with a PEG modification rate of 2-10 mol% prepared in Example 3. The strength was strong, and it was shown that the nitric oxide retention ability was improved by miniaturization of bubble liposomes.
In particular, nanobubble liposomes (sample 4-2) with a PEG modification rate of 6 mol% show particularly high values of detected echocardiogram, and can stably hold nitric oxide very stably in vivo. It has been shown.

(5) "Summary"
From the above results, it is possible to prepare nitric oxide-encapsulated nanobubble liposomes of 200 nm or less that are effective for accumulation in cancer tissues and inflammatory sites by vigorously suspending nitric oxide-encapsulated bubble liposomes on the μm scale. It was shown that there is.
In the nanobubble liposome, it was shown that the amount of nitric oxide that can be encapsulated is larger than that of the bubble liposome of μm scale, and the ability to retain nitric oxide in the in vivo environment is also improved. In particular, nanobubble liposomes with a PEG modification rate of 6 mol% have very high in vivo stability, and it has been shown that they can be particularly suitably used as nitric oxide delivery carriers.

[Example 5] “Pressurized storage test of nitric oxide-encapsulated bubble liposome”
The above-prepared nitric oxide-encapsulated bubble liposomes were examined for means for enabling long-term storage in consideration of production distribution and the like.

(1) “Preparation of nitric oxide-containing bubble liposome”
The bubble liposome in the micrometer scale prepared in Example 3 was used as Sample 5-1 to Sample 5-3. Moreover, the nanobubble liposome in the 50-200 nm scale prepared in Example 4 was used as the sample 5-4 to the sample 5-6.

(2) “Change with time under pressure”
About the said nitric oxide inclusion | inner_cover bubble liposome, it stored for 60 minutes on the conditions which pressurized the pressure in a vial bottle to 2 atmospheres, and the nitric oxide retention amount was measured. The measurement of nitrogen monoxide retention was performed by relative quantification using DAF-2 in the same manner as described in Example 1. The results are shown in the following table, FIG. 12 and FIG.

  As a result, under storage conditions in which the pressure in the vial was increased to 2 atm, bubble liposomes (sample 5-1 to sample 5-3) and nanobubble liposomes (sample 5-4 to sample 5) on a micrometer scale were used. In any of the cases -6), no decrease in fluorescence intensity was detected after storage for 60 minutes, indicating that the amount of nitric oxide immediately after production was maintained as it was.

From the above results, it was shown that the nitric oxide-encapsulated bubble liposome can be stored for a long time in consideration of the production distribution and the like by storing the inside of a vial or the like under a pressurized condition. As specific pressurizing conditions, it was shown that pressurizing conditions of 2 atm or more are effective.

[Example 6] “Evaluation of physiological functions in the body”
It was verified whether the nitric oxide-encapsulated bubble liposome prepared above can exert site-selective bioactive functions of nitric oxide by local subcutaneous administration into the body.

(1) "Subcutaneous administration test"
Using Evans Blue, the presence or absence of vasodilatory effect of nitric oxide derived from nitric oxide-encapsulating bubble liposomes was detected. Here, Evans blue is a compound that binds with serum albumin and develops a blue color, and it is possible to detect a site where the vascular wall dilates and the compound permeates outside the blood vessel.

Evans blue solution (10 μg / μL) 10 mg / kg body weight equivalent was systemically administered to 5-6 week old ICR strain mice by tail vein injection. Subsequently, the amount of the bubble liposome solution (concentration in lipid: 1 μg / μL) prepared as Sample 1-2 in Example 1 was subcutaneously injected into the back of the mouse subcutaneously. Here, sample 1-2 is a neutral lipid bubble liposome (DSPC: DSPE-PEG2000 = 94: 6) containing nitric oxide having a particle diameter of μm scale. After 180 minutes from the subcutaneous administration, the administration site was visually observed to visually observe the degree of leakage of Evans Blue from the microvessel, and evaluated according to the following criteria.
As a comparative test, a bubble liposome containing only C ₃ F ₈ gas, which is a contrast gas, was prepared in the same manner as in Sample 1-2, and the same as above except that the bubble liposome was administered subcutaneously. A test was conducted. As a control, the test was conducted in the same manner as above except that only PBS was administered subcutaneously. The test results are shown in the following table and FIG.

[Standard for visual evaluation]
No leakage was confirmed: −
Slight leakage was confirmed: ±
Leakage confirmed: +
A large amount of leakage was confirmed: ++

  As a result, leakage of Evans blue from the capillary was confirmed at the site where the nitric oxide-encapsulating bubble liposome was subcutaneously administered (Test 6-1 to Test 6-3). On the other hand, no leakage of Evans blue from capillaries was observed at the site where only contrast gas was administered subcutaneously (Test 6-4 to Test 6-6).

From the above results, it was shown that by administering the nitric oxide-encapsulating bubble liposome subcutaneously, the encapsulated nitric oxide is gradually and gradually released, and the blood vessel wall in the vicinity of the administration site can be expanded. . That is, according to the results of this example, the nitric oxide-encapsulating bubble liposome according to the present invention is locally administered subcutaneously to an arbitrary site, whereby the vasodilatory action that is the physiological function of nitric oxide is applied to an arbitrary site of administration. It has been shown that it can be selectively exerted.

[Example 7] "Selective nitric oxide release by ultrasonic irradiation"
It was verified whether or not nitric oxide can be selectively released to an arbitrary site from the above-prepared nitric oxide-encapsulating bubble liposome by performing ultrasonic irradiation from outside the body.

(1) "Systemic administration and ultrasonic irradiation"
200 μL of bubble liposome solution prepared as Sample 1-2 in Example 1 (lipid equivalent concentration: 1 μg / μL) was systemically administered to 5-6 week-old ICR strain mice by tail vein injection. Here, sample 1-2 is a neutral lipid bubble liposome (DSPC: DSPE-PEG2000 = 94: 6) containing nitric oxide having a particle diameter of μm scale.
After administration, a probe of a therapeutic ultrasonic irradiation device (Sonitron 2000, Neppagene Co., Ltd.) was applied to the lower limb muscle tissue to perform ultrasonic irradiation from outside the body. The ultrasonic irradiation was performed under irradiation conditions of a frequency of 1 MHz, an irradiation intensity of 2 W / cm ² and an irradiation time of 60 seconds. Subsequently, an Evans blue solution (10 μg / μL) equivalent to 10 mg / kg body weight was systemically administered by tail vein injection. After 180 minutes from the intravenous administration, the muscle tissue at the ultrasonic irradiation site was collected, and the amount of Evans blue contained in the muscle tissue was quantified.
As a control, the test was performed in the same manner as described above except that ultrasonic irradiation was not performed. The test results are shown in the following table and FIG.

  As a result, the amount of Evans Blue accumulated in the muscle tissue compared to the case where the ultrasound irradiation was not performed by locally irradiating the muscle tissue after the systemic administration of the nitric oxide-encapsulated bubble liposome. Was shown to increase significantly. In addition, the ultrasonic irradiation in the said test could be performed using weak ultrasonic irradiation using the therapeutic ultrasonic irradiation apparatus.

From the above results, nitric oxide-encapsulating bubble liposomes according to the present invention were administered systemically, and then local ultrasonic irradiation was performed on a desired site, whereby nitric oxide was selectively applied to any tissue or site. It has been shown that it can be delivered. That is, it has been shown that the vasodilatory action, which is the physiological function of nitric oxide, can be selectively exerted at any ultrasonic irradiation site.

The present invention is expected to be a technology that is effectively used in the medical field, the medical field, the life science research field, and the like.

1. 1. Nitric oxide encapsulated bubble liposome 2. Lipid bilayer membrane PEG
4). Nitric oxide

11. Heart position 12. Administration site 13. Midline

21. Subject 22. Injection device 23. Ultrasonic wave generating means 24. Ultrasound 25. Vascular system 26. Target site, target tissue 27. Medicinal solution

Claims (15)

  a lipid molecule having a neutral charge at pH 7.3 to 7.5 and / or a lipid molecule having an anionic charge at pH 7.3 to 7.5 as a substantially basic constituent lipid molecule. Nitrogen oxide-encapsulating bubble liposome, comprising nitric oxide encapsulated.   The nitric oxide-encapsulated bubble liposome according to claim 1, wherein the bubble liposome is capable of retaining nitric oxide in an in vivo environment.   The nitric oxide-encapsulated bubble liposome according to claim 1 or 2, wherein the bubble liposome substantially does not contain a lipid molecule having a cationic charge at pH 7.3 to 7.5 as a basic constituent lipid molecule. .   The monoxide according to any one of claims 1 to 3, wherein the bubble liposome comprises a lipid molecule having a neutral charge at pH 7.3 to 7.5 as a substantially basic lipid molecule. Nitrogen encapsulated bubble liposomes.   The nitric oxide-encapsulated bubble liposome according to any one of claims 1 to 4, wherein the bubble liposome comprises DSPC and / or DPPC as a substantially basic constituent lipid molecule.   The nitric oxide-encapsulated bubble liposome according to any one of claims 1 to 5, having an average particle size of 200 µm or less.   The nitric oxide-encapsulated bubble liposome according to any one of claims 1 to 6, comprising a lipid molecule modified with polyethylene glycol at 10 mol% or less of a basic constituent lipid molecule.   Furthermore, the nitric oxide inclusion bubble liposome in any one of Claims 1-7 which has a target directivity improvement molecule | numerator.   A vascular wall dilator, an EPR effect improver, a blood pressure antihypertensive agent, an arteriosclerosis inhibitor, or a central nerve protective agent, comprising the nitric oxide-encapsulating bubble liposome according to any one of claims 1 to 8.   A pharmaceutical kit or a pharmaceutical composition comprising the nitric oxide-encapsulating bubble liposome according to any one of claims 1 to 8.   The pharmaceutical kit or pharmaceutical composition according to claim 10, comprising a nano-form preparation having other pharmacological components.   A system for site-selectively delivering nitric oxide in vivo, comprising the nitric oxide-encapsulating bubble liposome according to any one of claims 1 to 8.   The system according to claim 12, further comprising ultrasonic irradiation means. In the production process of bubble liposome encapsulating nitric oxide,
In a liposome comprising a lipid molecule having a neutral charge at pH 7.3 to 7.5 and / or a lipid molecule having an anionic charge at pH 7.3 to 7.5 as a substantially basic constituent lipid molecule. Including a step of including nitric oxide so as to include it.
A method for producing nitric oxide-encapsulating bubble liposomes capable of retaining nitric oxide in an in vivo environment. In the production process of bubble liposome encapsulating nitric oxide,
In a liposome comprising a lipid molecule having a neutral charge at pH 7.3 to 7.5 and / or a lipid molecule having an anionic charge at pH 7.3 to 7.5 as a substantially basic constituent lipid molecule. Including a step of including nitric oxide so as to include it.
A method for improving nitric oxide retention ability of bubble liposomes in an in vivo environment.