WO2022092411A1 - Liposome for phagocytosis-tracing pet imaging, and nanoprobe comprising same - Google Patents

Abstract

The radioactive liposome nanoparticles for macrophage tracing, derived via the present invention, are considered to be very usefully employed in the diagnosis of a tumor or brain inflammation by quantifying, using PET, activated macrophages (microglial cells) spread in the area of inflammation in the body. In addition, a method using late-stage radioisotopic labeling minimizes the exposure of a worker to radiation, allows mass production, and thus can be easily put to practical use in the medical industry.

Description

Liposomes for phagocytosis tracking PET imaging and nanoprobes containing the same

It relates to a liposome for phagocytosis tracking PET imaging and a nanoprobe including the same.

Microglial cells of the central nervous system contribute to activation of the nervous system and maintenance of homeostasis, and secrete neurotrophin, nitric oxide, or cytokines that induce inflammation to maintain or kill nerve cells (apoptosis) It has the function of causing the back. In fact, activation of microglia has been reported in various degenerative neurological diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, cerebral infarction or injury, and brain infection. It is also known that the deposition of beta-amyl, which is a factor in the onset and progression of Alzheimer's disease, induces activation of microglia.

[Prior literature]

Domestic patent application 10-2009-0042361

Impaired phagocytosis of apoptotic cells causes accumulation of bone marrow-derived macrophages in aged mice. BMB reports. 2017. 43-48.

One object of the present invention is to provide a radiopharmaceutical for diagnosing degenerative brain disease or tumor.

Another object of the present invention is to provide a liposome comprising PIP ₃ capable of tracking phagocytosis.

Another object of the present invention is to provide a liposome for radioactive imaging, including PIP ₃ .

Another object of the present invention is to provide a nanoprobe for diagnosing a degenerative brain disease or tumor comprising a liposome.

In order to achieve the above object,

One aspect of the present invention provides a radiopharmaceutical for diagnosing degenerative brain disease or tumor, wherein the radiopharmaceutical is a nanoparticle-type PET radiopharmaceutical containing F-18 radioisotope in a size of 50 to 200 nm.

One aspect of the present invention provides a liposome comprising PIP ₃ capable of tracking phagocytosis.

Another aspect of the present invention provides a liposome for radioactive imaging, including PIP ₃ .

Provided is a nanocompound in which an anchoring compound such as [ 18 F]HFB containing an F-18 isotope is labeled on a liposome composed of POPC and PIP ₃ .

In this case, the nano-compound may include cholesterol or PEGylate phospholipid.

The above radioisotope-labeled nanomaterials are used for PET images of tumor tissues or PET images of brain inflammation.

Another aspect of the present invention provides a nanoprobe for diagnosing a degenerative brain disease or tumor comprising a liposome.

It is believed that the macrophage tracking radioliposome nanoparticles derived through the present invention will be very useful for diagnosing brain inflammation or tumors by quantifying activated macrophages (microglia) distributed in inflammatory sites in the body with PET. In addition, the method using the terminal stage radioisotope label has the advantage of being easy to put into practical use in the medical industry because it minimizes the exposure of workers and enables mass production.

1 shows a nanoprobe including a liposome for radioactive images according to an embodiment.

Figure 2 shows the manufacturing process of POPC:PIP ₃ liposome.

3 shows the results of DLS analysis of PIP ₃ coated liposomes.

4 shows a TEM image of the liposome (scale var=100 nm).

5 shows a summary of various application ranges of liposomes.

Figure 6 is a schematic diagram showing the apoptosis process and "eat me" signal.

Figure 7 is a pictorial representation of the liposome in the basal state and apoptosis.

8 is a schematic diagram showing "eat-me signal" of PIP ₃ .

9 is a graphical representation of the tumor specificity of PIP ₃ -coated liposome.

10 shows the [ ¹⁸ F]HFB synthesis process.

11 is a pictorial representation of M1 and M2 of microglia.

12 shows DMF images of FDG PET (top) and F-18-labeled liposome PET images (bottom) in a tumor model (a model in which the B16F1 melanoma cell line was transplanted into C57BL/6 mice).

FIG. 13 shows a photograph of the tissue of the same entity as that of FIG. 12 .

Hereinafter, the present invention will be described in detail.

the present invention

POPC (Phosphatidylcholine) as a main component, PIP ₃ (Phosphoinositol-3,4,5-trisphosphate) as a secondary component;

It provides a liposome having a lipid bilayer structure.

The liposome may be anchored with radioisotope-labeled [ 18 F]HFB ([ 18 F]hexadecyl-4-[ 18 F]fluorobenzoate) or an analog thereof.

The liposome may be characterized in that it specifically binds to macrophages in which phagocytosis is activated.

The ratio of POPC and PIP ₃ may be 2:1 to 100:1.

The present invention has an OPC (Phosphatidylcholine) of claim 1 as a main component, and PIP ₃ (Phosphoinositol-3,4,5-trisphosphate) as a secondary component;

To provide a nanoprobe for tracking PET (positron emission tomography, position emission tomography) images including liposomes having a lipid bilayer structure.

The nano-probe may be 60 nm to 150 nm.

The present invention provides a composition for diagnosing degenerative brain disease comprising the nanoprobe.

The present invention provides a composition for diagnosing tumors comprising the nanoprobe.

The diagnostic composition may further include at least one selected from the group consisting of DSPE-PEG (2000) amines, cholesterol, and pegylated phospholipids.

Dissolving the present invention POPC (Phosphatidylcholine) and PIP ₃ (Phosphoinositol-3,4,5-trisphosphate) in a solvent;

sonicating for 10 to 50 minutes;

repeating the freeze-and-thaw process 2 to 10 times;

adjusting the pH to 6.5 to 7.5; and

The POPC (Phosphatidylcholine) comprising; centrifuging as a main component, and PIP ₃ (Phosphoinositol-3,4,5-trisphosphate) as a secondary component;

It provides a method for preparing a liposome, having a lipid bilayer structure.

In the present invention, it is intended to develop a PET radiopharmaceutical in the form of nanoparticles composed of liposomes that can be easily used in patients in the clinic for the purpose of diagnosing cranial nerve inflammatory diseases and TAM (tumor associate macrophage)-activated cancer.

Macrophages (or microglia) found in tumor tissues or central nervous system inflammation can find macrophages in higher concentrations than normal tissues, and cause active macrophage. In macrophage, “eat me” mechanism plays a very important role in which cells in the apoptosis process are killed and progresses to phagocytosis by macrophages.

In the case of phosphatidylserine and phytate, which are present in a large number in the exposed inner cell membrane of dead cells, it is known as an important chemical factor recognized by these macrophages as an “eat me signal”, and the gadolinium-phytate complex using this action can be used to treat colon cancer, etc. It has been reported as a diagnostic MRI contrast agent.

Although positron emission tomography (PET) is a medical imaging device with somewhat lower resolution than MRI, it has a very high sensitivity and is a diagnostic imaging device that helps in diagnosis by observing very small lesions with high contrast.

For non-invasive in vivo imaging of activated macrophages (or microglia) highly distributed in lesions of tumors or degenerative brain diseases, diagnostic radioisotope (F-18) is applied to liposomes carrying “eat me signal”. If it can be labeled and used, it is considered to be a diagnostic PET radiopharmaceutical with high sensitivity. Until now, radiopharmaceuticals that image macrophages in the body in relation to inflammation have not been available in the medical market, so they are expected to be used in clinical practice for various purposes.

As is known in various literatures, various radiopharmaceuticals have been studied using the small size of liposomes to flow into the loosened blood vessel gap around the tumor tissue (Enhanced Permeability and Retention effect, EPR effect), but in almost all cases, very It is manufactured using radioactive isotopes such as Cu-64 and I-124 with long half-lives, and it is known that the waiting time from radiopharmaceutical administration to imaging time is very long to be used in patients. However, in the case of using the macrophage action of macrophages, the uptake of the tumor tissue is fast and irreversible. can be expected

Liposomes in the present invention are nanoparticles having a size of 50 to 200 nm having a lipid bilayer structure containing POPC as a main component and POPS or POP-inositol as a secondary component, freezing and thawing or ultrasound It can be manufactured by controlling the size and uniformity of particles through methods such as sonication or extrusion.

For PET imaging, the radioisotope F-18 (half-life 109 minutes) must be introduced into the liposome. In this case, [18F]HFB (hexadecyl-4-[18F]fluorobenzoate) or an analog that can be easily anchored to the liposome is used. In order to improve the safety of the liposome in the body, a small amount of a steroid-like compound such as cholesterol may be included, or a certain amount of a pegylated phospholipid such as DSPE-PEG(2000) amine may be included to inhibit metabolism.

As a chemical element of “eat me signal”, phosphatidylserine (PS) or phosphoinositides (Phosphorylated forms of phosphatidylinositol; one or more phosphorylated phosphatidylinositols) are used, and liposome nanoparticles are manufactured to consist of a certain proportion of liposomes.

In the example, also through terminal anchoring using an F-18 label containing a long chain (for example, [18F]hexadecyl-4-[18F]fluorobenzoate; [18F]HFB) for radioisotope labeling A method for synthesizing an end-purpose radiopharmaceutical is provided. In the existing method, there are many cases in which radioisotopes are included from the beginning of liposome production, but the terminal anchoring method minimizes the handling of radioisotopes and minimizes the radiation exposure of workers in mass production (high activity). It is commercially advantageous as it is very suitable for repetitive mass production by remote automation devices.

It is expected that the liposome loaded with the above-listed "eat me signal" will accumulate at the site of the lesion related to inflammation by the inherent radioisotope, so that the lesion can be distinguished and found through PET image.

In various brain diseases accompanied by encephalitis, such as Alzheimer's, Parkinson's, and stroke, it is expected that the diagnosis through PET will be possible at an early stage of the disease.

In addition, since it is different from the mechanism of action of existing PET radiopharmaceuticals for diagnosing tumors, it may be used as an alternative radiopharmaceutical for tumors not found in conventional tumor diagnosis.

Liposomes are the only drug delivery nanoparticles that have been approved and marketed with FDA approval, and when loaded with a therapeutic agent, they are valuable to be used as a tool that can perform treatment and diagnosis at the same time.

<Example 1> Preparation of liposomes equipped with "eat me signal"

1) After dissolving POPC and PIP3 in CHCl3, volatilize all the solvents using nitrogen gas for 20 minutes to spread evenly on the walls of the container.

2) Remove all excess CHCl3 under reduced pressure for 3 hours.

3) Add citrate buffer of pH 4.0 and perform sonication for 30 minutes.

4) Repeat freeze-and-thaw in 5 cycles.

5) Add pH 8.2 HEPES buffer until pH is 7.0.

6) Centrifuge for 90 minutes at 13,000 rpm through a centrifuge, remove the upper layer, add buffer, and wash twice.

7) Add DPBS buffer to the pellet and use it for the radioisotope labeling reaction.

<Example 2> Synthesis of [ 18 F]HFB

1) The [18F]fluorine anion aqueous solution generated from cyclotron was passed through an ion exchange QMA cartridge (Water) to adsorb the radioactivity, and 34uL of 1M TBAOH (tetrabutylammonium hydroxide) and D.W. 500uL of the mixed solution is passed, and the solution collected by passing 700uL of acetonitrile immediately is put in a glass reaction vessel, and the solvent is slowly removed for about 60 minutes at 100°C using nitrogen gas. Add 200 uL of acetonitrile to remove the solvent for about 5 minutes to completely remove moisture.

2) Dissolve hexadecyl-4-(N,N,N-trimethylalmino)benzene triflate (4 mg) in 400uL of DMSO, add it to the reaction vessel, and heat at 95°C for 20 minutes to react.

3) After completion of the reaction, 0.1% TFA aqueous solution is added, and separation and purification are performed by semi-prep-HPLC. (Solvent conditions: acetonitrile 70%, 0.1% TFA aqueous solution 30%, semi prep-HPLC reversed-phase column C ₅ )

4) After adsorbing [ 18 F]HFB using the SEP-PAK cartridge (C18) for solid phase extraction, the aliquoted solution is passed through 2 mL of absolute ethanol and collected in a glass vial.

5) Dry the collected [18F]HFB ethanol solution at 100°C using nitrogen gas in a moisture-free state.

<Example 3> Radioisotope labeling of liposomes using [18F]HFB

1) Add 1 mL of the liposome solution prepared in the buffer to the glass vial coated with [ 18 F]HFB.

2) While heating to about 40~50℃, stir quickly with vortex for 10 minutes.

3) A liposomal PET probe with a labeling efficiency of 10 to 15% is prepared and ready for use.

Advantages of the present invention are as follows.

1. It is possible to PET imaging of macrophages (microglia).

2. Since macrophages are widely distributed in tumor tissues and brain inflammation, they can be used for tumor imaging PET and brain inflammation PET imaging.

3. TSPO PET imaging is a macrophage image in the research stage, but the limitation is that there are true-negative patients due to polymorphism.

4. The M1 status of macrophages can be traced. In the case of brain disease, the M1 status provides a pathological cause. M2 is also an activated macrophage and is not related to disease.

Claims (10)

1. POPC (Phosphatidylcholine) as a main component, PIP ₃ (Phosphoinositol-3,4,5-trisphosphate) as a secondary component;

   Liposomes having a lipid bilayer structure.
2. According to claim 1,

   The liposome is a radioisotope-labeled [18F]HFB ([18F]hexadecyl-4-[18F]fluorobenzoate) or an analog thereof, characterized in that anchored, liposome.
3. According to claim 1,

   The liposome is characterized in that it specifically binds to macrophages in which phagocytosis is activated, liposomes.
4. According to claim 1,

   The ratio of the POPC and PIP ₃ liposomes, characterized in that 2:1 to 100:1.
5. A nanoprobe for tracking PET (positron emission tomography, position emission tomography) images comprising the liposome of claim 1.
6. 6. The method of claim 5,

   The nano-probe is characterized in that 60 nm to 150 nm, the nano-probe.
7. A composition for diagnosing degenerative brain disease comprising the nanoprobe of claim 5 .
8. A composition for diagnosing tumors comprising the nanoprobe of claim 5 .
9. 9. The method according to claim 7 or 8,

   The diagnostic composition further comprises at least one selected from the group consisting of DSPE-PEG(2000) amines, cholesterol, and pegylated phospholipids.
10. Dissolving POPC (Phosphatidylcholine) and PIP ₃ (Phosphoinositol-3,4,5-trisphosphate) in a solvent;

    sonicating for 10 to 50 minutes;

    repeating the freeze-and-thaw process 2 to 10 times;

    adjusting the pH to 6.5 to 7.5; and

    centrifuging; including,

    The liposome production method of claim 1.

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