Liposome preparation of deazaflavin

Abstract

Provided is a technology for formulating deazaflavin as a liposome preparation that is characterized by containing deazaflavin and a polyethylene glycol-modified phospholipid.

Description

Liposome formulation of deazaflavin

[0001] The present invention relates to liposomal formulations of deazaflavins.

[0002] Deazaflavin (5-Deazaflavin) is a general term for a group of compounds with a structure in which the nitrogen at the 5th position of the flavin skeleton is replaced by carbon, and which have a wide range of chemical properties, such as being involved in a variety of metabolic reactions, oxygen activation, and chemiluminescence as coenzymes in the body.

[0003] The pharmacokinetics and chemical properties of deazaflavin are NAD (nicotinamide adenine dinucleotide) ＋ Deazaflavins are similar to FAD (flavin adenine dinucleotide) and stimulate intracellular energy production (ATP). This makes them unique in that they have a wide range of beneficial effects.

[0004] The present inventors have also discovered deazaflavins that are excellent at activating ATP in mitochondrial complexes, and have filed a patent application for this (Patent Document 1).

[0005] Although deazaflavin has the potential to be applied to the treatment of various diseases, it has the problem that it has extremely low solubility and is difficult to formulate into various commonly used pharmaceutical forms.

[0006] Therefore, there is a need for a technology to formulate deazaflavin.

[0007] Patent No. 6717989

[0008] Therefore, an objective of the present invention is to provide a technique for formulating deazaflavin.

[0009] As a result of intensive research to solve the above problems, the present inventors discovered that an excellent phospholipid preparation can be obtained by using polyethylene glycol-modified phospholipids, and thus completed the present invention.

[0010] That is, the present invention relates to a liposome preparation comprising deazaflavin and a polyethylene glycol-modified phospholipid.

[0011] The liposome preparation of the present invention is a new formulation of deazaflavin. This liposome preparation is stable and has permeability through the blood-brain barrier, making it particularly suitable for the treatment of brain diseases.

[0012] 1 is a diagram showing the results of a storage test in Test Example 1. FIG. 2 is a diagram showing an apparatus used in a blood-brain barrier permeability test in Test Example 2.

[0013] The liposome preparation of the present invention contains deazaflavin and a polyethylene glycol-modified phospholipid.

[0014] Deazaflavin, an essential component of the liposome preparation of the present invention, has a structure similar to that of naturally occurring vitamin B2 and is similar to the coenzyme NAD synthesized from NMN. While any known deazaflavin can be used in the liposome preparation of the present invention, 5-deazaflavin (5-Deazaflavins: Pyrimido[4,5-b]quinoline-2,4(3H,10H)-diones) represented by the following formula (I) is preferred. This 5-deazaflavin is described in detail in Japanese Patent No. 6717989 (Patent Document 1). This 5-deazaflavin is excellent at activating ATP in mitochondrial complexes.

[0015] (In the formula, R １ represents a hydrogen atom, an alkyl group, a halogen-substituted alkyl group, a carboxy-substituted alkyl group, or a phenyl group; R ２ represents an alkyl group, a cycloalkyl group, a phenyl-substituted lower alkyl group, a phenyl group, a phenyl group substituted with one of a halogen atom, a lower alkyl group, or a lower alkoxy group, or a lower alkyl-disubstituted phenyl group; R ３ and R ４ represents a hydrogen atom, a lower alkyl group, a halogen atom, a hydroxyl group, a nitro group, a cyano group, a lower alkoxy group, a phenyl-substituted lower alkoxy group, a lower alkylamino group, a phenyl-substituted lower alkylamino group, or a lower alkylsulfonyl group.

[0016] Among these 5-deazaflavins, R １ is a methyl group and R ２is an ethyl group, and R ３ and R ４ The compound named NMN-T, which has hydrogen atoms, is managed by ChemiTerrace Corporation as TND1128JP and is available from ChemiTerrace Corporation under the trade name "5-DEAZAFLAVIN REDOX" or the like.

[0017] The polyethylene glycol-modified phospholipid, which is another essential component of the liposome preparation of the present invention, is not particularly limited, but examples thereof include phospholipids used in known liposome preparations to which polyethylene glycol is bound.

[0018] Examples of phospholipids include natural phospholipids such as phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol, phosphatidylethanolamine (PE), dimyristoylphosphatidylcholine (DMPC), phosphatidic acid (PA), cardiolipin (CL), sphingomyelin (SPH), egg yolk lecithin, soybean lecithin, and lysolecithin; hydrogenated natural phospholipids such as hydrogenated soybean phospholipid (HSPC) prepared by standard methods; and derivatives such as DSPE (1,2 distearoyl-sn-glycerol-3-phosphatidylethanolamine). Among these phospholipids, DSPE is preferred. These phospholipids may be used singly or in combination.

[0019] The phospholipids described above are modified with polyethylene glycol by known methods. The molecular weight of the polyethylene glycol may be appropriately selected, but for example, the average molecular weight is 1,000 to 5,000, preferably 1,000 to 3,000, and more preferably 2,000. This average molecular weight is a value measured by a differential refractive index detector or size exclusion chromatography (SEC) equipped with a single quadrupole MS.

[0020] Preferred examples of the polyethylene glycol-modified phospholipids mentioned above include those represented by the following formula, in which the polyethylene glycol has the above molecular weight.

[0021] As shown in the following formula, the terminal OH of the polyethylene glycol is COOH, NH ２ The substituents are preferably the following.

[0022] Specific examples of the polyethylene glycol-modified phospholipids include DSPE-PEG ２０００、 DSPE-PEG ２０００ -COOH, DSPE-PEG ２０００ -NH ２ Among these, DSPE-PEG ２０００ -COOH, DSPE-PEG ２０００ -NH ２ is preferred, and DSPE-PEG ２０００ --COOH is more preferred.

[0023] These polyethylene glycol-modified phospholipids are commercially available from various companies for use in liposome formation. ２０００ For -COOH, Avanti Polar Lipids' 880135P (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (sodium salt)), DSPE-PEG ２０００ -NH ２ Examples include Avanti Polar Lipids' 880128P (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt)).

[0024] The liposome preparation of the present invention can be produced by using deazaflavin and polyethylene glycol-modified phospholipid, and then incorporating other components as necessary, and encapsulating deazaflavin using a known method for forming liposomes.

[0025] The method for forming liposomes is not particularly limited, but examples thereof include thin-film hydration, detergent removal, solvent injection, and reverse-phase evaporation methods. Among these, the solvent injection method using ethanol is preferred.

[0026] Other components used in forming liposomes include, for example, solvents, surfactants, emollients, stabilizers, buffers, solution-state stabilizers, phospholipids, etc.

[0027] Examples of solvents / surfactants include water, physiological saline, benzyl alcohol, ethanol, methanol, α-tocopherol, triolein, tricaprylin, propylene glycol, polyoxyethylene sorbitan oleate, etc. The above-mentioned phospholipids and polyethylene glycol-modified phospholipids are sometimes used as surfactants. Examples of buffers / solution state stabilizers include disodium succinate, glycine, calcium chloride, sodium citrate, citric acid, sodium phosphate, sodium chloride, triethanolamine, histidine, ammonium sulfate, sucrose, etc. Examples of softeners / stabilizers include cholesterol, etc. These components can be used alone or in combination of two or more.

[0028] Preferred compositions for forming the liposome preparation of the present invention include the following: Deazaflavin: 0.0001 to 5% (w / v), preferably 0.01 to 0.10% (w / v) Polyethylene glycol-modified phospholipid: 0.01 to 5% (w / v), preferably 0.1 to 1% (w / v) If necessary, the following additives may be contained: Solvent / surfactant: 70 to 98% (w / v), preferably 80 to 92% (w / v) Softener / stabilizer: 0.01 to 5% (w / v), preferably 0.1 to 1% (w / v) Buffer / solution state stabilizer: 1 to 20% (w / v), preferably 7 to 14% (w / v)

[0029] The shape of the liposome preparation of the present invention is not particularly limited and may be any known shape, such as a spherical or elliptical shape. Liposome structures are classified into multilamellar vesicles (MLVs) and unilamellar vesicles, with unilamellar vesicles being preferred. It is preferable to further miniaturize these unilamellar vesicle liposomes.

[0030] The method for miniaturization is not particularly limited, but examples thereof include the membrane extrusion method, the high shear homogenization method, the sonication method, etc. Among these methods, the membrane extrusion method is preferred.

[0031] In the liposome preparation of the present invention, the polyethylene glycol-modified phospholipid is DSPE-PEG. ２０００ -COOH and / or DSPE-PEG ２０００ -NH ２ In the case of (1), it is preferable to further bind transferrin receptor T12 peptide and / or transferrin, and DSPE-PEG ２０００In the case of -COOH, it is preferable to further bind a transferrin receptor T12 peptide and / or transferrin. Transferrin receptor T12 peptide and / or transferrin are commercially available from various companies. Examples of these commercially available products include BBB-penetrated transferrin receptor (TfR) binding peptide (Cat. No.: HY-P2297A) from MedChemExpress. The COOH group and / or NH ２ When the transferrin receptor T12 peptide and / or transferrin are bound via a group, the polyethylene glycol-modified phospholipid and the transferrin receptor T12 peptide and / or transferrin are bound via an -NHCO- and / or -CONH- peptide bond.

[0032] Transferrin receptor T12 peptide and / or transferrin are encapsulated in DSPE-PEG. ２０００ -COOH and / or DSPE-PEG ２０００ -NH ２ The order of binding is not particularly limited, but for example, ２０００ -COOH and / or DSPE-PEG ２０００ -NH ２ The transferrin receptor T12 peptide and / or transferrin may be bound to the liposome before the formation of the liposome, and then the liposome may be formed. ２０００ -COOH and / or DSPE-PEG ２０００ -NH ２ After liposomes are formed with DSPE-PEG, the transferrin receptor T12 peptide and / or transferrin may be bound to the liposomes. ２０００ -COOH and / or DSPE-PEG ２０００ -NH ２ The method for binding to the nucleotides is not particularly limited, and any known method may be used.

[0033] The properties of the final liposome formulation of the present invention are not particularly limited as long as it is stable, free from contamination with foreign substances that affect the human body, and has pH, osmotic pressure, etc. that are similar to those of serum and body fluids, but include, for example, the following. It is sufficient for the formulation to have one or more, preferably all, of these properties. The average particle size, polydispersity, and zeta potential are values ​​measured by diluting the formulation 5-fold with buffer and using a Litesizer 500. The concentration of deazaflavin in the in vitro permeability evaluation is a value measured by HPLC-UV. The encapsulation efficiency is also calculated from the value measured by HPLC-UV. pH: 6-8, preferably 7-8 Assay: 55-110%, preferably 90-110% Entrapment efficiency of deazaflavin: more than 40%, preferably more than 60.0% Average particle size: less than 110 nm, preferably less than 100 nm, more preferably 50 nm to less than 100 nm Polydispersity index: less than 0.30, preferably 0.05 to less than 0.30 Zeta potential: in the range of ±30 mV, preferably in the range of ±20 mV

[0034] The liposome preparation of the present invention described above contains deazaflavin, and therefore, the NAD possessed by deazaflavin can be effectively used. ＋ and FAD and have a redox (oxidation-reduction) function similar to that of FAD, and can stimulate ATP production in cells. Therefore, the liposome preparation of the present invention can be used as an activator of ATP production in cells.

[0035] Specifically, the liposome preparation of the present invention can stimulate ATP production, and therefore can be used as a therapeutic or preventive agent for diseases that can be treated or prevented by activating ATP production.

[0036] Examples of the above diseases include diabetes, renal failure, obesity, Alzheimer's disease, Parkinson's disease, neurodegenerative diseases associated with cerebral hemorrhage and cerebral infarction, and depression.

[0037] The liposome preparation of the present invention is particularly excellent in ATP activation in mitochondrial complexes, and is therefore preferred as a therapeutic or preventive agent for brain diseases such as Alzheimer's disease, Parkinson's disease, neurodegenerative diseases associated with cerebral hemorrhage and infarction, and depression, among others. In addition, the liposome preparation of the present invention is preferred as a therapeutic or preventive agent for the above-mentioned brain diseases because it can pass through the blood-brain barrier.

[0038] The dose range of the liposome preparation of the present invention for mammals including humans is appropriately selected depending on the efficacy of the deazaflavin contained in the liposome preparation of the present invention, the administration form, the administration route, the characteristics of the subject (body weight, age, medical condition, use of other medications, etc.), the judgment of the attending physician, etc., and the dose of deazaflavin may be, for example, 1 to 100 mg / day, preferably 10 to 50 mg / day, which may be administered in multiple divided doses.

[0039] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples in any way.

[0040] Example 1 Preparation of liposome formulation: A liposome formulation was prepared by the solvent injection method using the components listed in Table 1 below, and then miniaturized by the membrane release method. First, a buffer solution was prepared by mixing ammonium sulfate, sucrose, and histidine, which are buffering agents / solution state stabilizers, with ethanol, water, and saline, which are solvents, in a round flask. In a separate container, benzyl alcohol, which is a solvent, and DSPE-PEG, which is a polyethylene glycol-modified phospholipid, were mixed. ２０００ -COOH, HSPC as a surfactant, cholesterol as a softener / stabilizer, and NMN-T as a drug were added to obtain a mixture. The mixture was injected with a syringe into a buffer solution that was stirred at 65°C. The ethanol was then evaporated to prepare a suspension containing the liposome formulation.

[0041]

[0042] The suspension containing this liposome preparation was subjected to miniaturization of the liposome preparation by a membrane release method: First, the suspension was passed through a 100 nm polycarbonate membrane filter 10 times while maintained at 65°C, and then passed through a 30 nm polycarbonate membrane filter 10 times.

[0043] The pH of the liposome preparation thus obtained was measured, and the results are shown in Table 1. The appearance of the suspension before and after miniaturization was also evaluated. These results are also shown in Table 1.

[0044] Test Example 1 Storage Test: The appearance of the liposome preparation prepared in Example 1 before and after miniaturization (Nano) and after storage at 2-8°C or 25°C for 5, 7, and 15 days is shown in Figure 1.

[0045] From this result, DSPE-PEG ２０００ HY8448, a liposome formulation containing -COOH, was found to be stable even after miniaturization.

[0046] Example 2 Preparation of liposome preparation: Using the components listed in Table 2 below, liposome preparations were prepared in the same manner as in Example 1, up to miniaturization.

[0047]

[0048] The pH, assay (%) before and after release, encapsulation efficiency (%), average particle size, and polydispersity of the liposome formulation obtained in this manner were measured, and the results are shown in Table 2. In addition, the appearance of the suspension before and after miniaturization was evaluated. These results are also shown in Table 2.

[0049] The total drug analysis (assay (%)) and encapsulation efficiency (%) before and after release were measured by HPLC-UV. (1) Total Drug Analysis: An Agilent 1100 HPLC (Agilent Technologies) equipped with a diode array detector was used. Drugs were separated at 50°C using an XDB-C18 (4.6 x 50 mm, 1.8 μm) column equipped with a C18 (4.6 x 5 mm) precolumn. The mobile phase consisted of 0.1% FA in water (A) and 0.1% FA in acetonitrile (B). The flow rate was 1.50 mL / min and the injection volume was 5.00 μL in gradient mode (0 min: 5%, 5 min: 5%, 10 min: 80%, 12 min: 95%, 14 min: 95%, 14.1 min: 5%, 15 min: 4%). The detector wavelength was 328 nm.

[0050] (2) Entrapment Efficiency. 150 μL (or 200 μL) of the formulation sample was injected into a 30K cutoff filter (Amicon Ultra 0.5 mL centrifugal filter, Ultracel 30K (30,000 Da cutoff), regenerated cellulose 30,000 nominal molecular weight cutoff (NMWL)) and then centrifuged at 9,700 rpm for 10 minutes at room temperature. This procedure allowed the free drug to pass through the filter, while the liposomes remained on top of the filter. The free drug that passed through the filter was diluted 10-fold with ethanol and analyzed by HPLC-UV to determine the concentration of the free drug in the formulation. The entrapment efficiency was calculated using the following formula:

[0051]

[0052] The average particle size, polydispersity and zeta potential were measured by diluting the preparation 5-fold with a buffer solution and using a Litesizer 500 (manufactured by Anton Paar).

[0053] Test Example 2 Blood-brain barrier permeability test: The liposome preparation prepared in Example 2 was subjected to a blood-brain barrier permeability test. The blood-brain barrier permeability test was performed using a Parallel Artificial Membrane Permeability Assay-BBB Kit (Creative Bioarray (Cat. No.: DPK-YS003, Lot No.: 022209001YS)). The test was performed according to the Creative Bioarray protocol. As shown in Figure 2, the donor well was placed in the acceptor well, and the absorbance of the test compound solution that had passed through the membrane pretreated with brain lipids and migrated to the acceptor well was measured, and the membrane permeability was calculated. The test was performed at 37°C for 18 hours. For the high-permeability and low-permeability controls, UV absorbance at 250 nm and 270 nm was measured using a NanoDrop microspectrophotometer. The NMN-T concentration in the formulation was measured by HPLC-UV. The permeability (Pe) was calculated using the following formula. The results are shown in Table 3.

[0054] OD during the ceremony Ａ is the absorbance (concentration) of the acceptor solution, OD Ｅ is the absorbance (concentration) of the parallel standard, and for an incubation time of 18 hours, C is 7.72 × 10 －６ is.

[0055]

[0056] From the above results, it was found that the liposome preparation obtained in Example 2 has excellent permeability through the blood-brain barrier, allowing NMN-T (deazaflavin) to reach the brain.

[0057] Example 3 Preparation of liposome formulation: DSPE-PEG ２０００-COOH (10.0 mg, 3.58 μmol) and EDC HCl (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride: 6.87 mg, 35.8 μmol) were dissolved in 3 mL of 10% THF-containing 2-(N-morpholino)ethanesulfonic acid (MES) buffer (0.01 M), pH 6.0, and stirred at room temperature for 2 hours. Then, TfR-T12 TFA peptide (4.01 mg, 2.69 μmol; MedChemExpress BBB-penetrated transferrin receptor (TfR) binding peptide (Cat. No. HY-P2297A); hereafter, this may be referred to as TfR-T12) was added. The reaction mixture was gently stirred overnight at room temperature. Buffer exchange into 10% ethanol in MilliQ water was performed using a Satorius centrifugal concentrator (MWCO 2000 Da). Four filtration cycles were performed, and the concentrated reaction mixture was lyophilized to obtain the DSPE-PEG. ２０００ -CONH-TfR-T12 was obtained (11 mg, 2.58 μmol, yield: 96%). ２０００ The fact that -CONH-TfR-T12 was obtained was confirmed by LC / Q-TOF.

[0058] <References> Mu, LM., Bu, YZ., Liu, L. et al. Lipid vesicles containing transferrin receptor binding peptide TfR-T12 and octa-arginine conjugate stearyl-R8 efficiently treat brain glioma along with glioma stem cells. Sci Rep 7, 3487 (2017). https: / / doi.org / 10.1038 / s41598-017-03805-7.

[0059] DSPE-PEG prepared above ２０００A liposome preparation was prepared in the same manner as in Example 1, using the components listed in Table 4 below, including -CONH-TfR-T12, and was then subjected to miniaturization. The pH, assay (%) before and after release, encapsulation efficiency (%), average particle size, and polydispersity of the obtained liposome preparation were measured, and the results are also shown in Table 4. In addition, the appearance of the suspension before miniaturization was evaluated. These results are also shown in Table 4.

[0060]

[0061] From this result, DSPE-PEG ２０００ HY8456, a liposomal formulation containing -CONH-TfR-T12, was found to be stable even after miniaturization. Furthermore, because this formulation contains TfR-T12, it may have better blood-brain barrier permeability than formulations that do not contain TfR-T12.

[0062] Example 4 Preparation of liposome preparation: In Example 1, DSPE-PEG ２０００ DSPE-PEG instead of —COOH ２０００ -NH ２ A liposome preparation was prepared in the same manner up to miniaturization except for using the above.

[0063] The liposome preparation of the present invention allows deazaflavin to be used in the treatment and prevention of various diseases.

Claims

1. A liposome preparation comprising deazaflavin and a polyethylene glycol-modified phospholipid.

2. Polyethylene glycol-modified phospholipids are DSPE-PEG ２０００ -COOH and / or DSPE-PEG ２０００ -NH ２ The liposome preparation according to claim 1, 3. Polyethylene glycol-modified phospholipids are DSPE-PEG ２０００ -COOH and / or DSPE-PEG ２０００ -NH ２ 2. The liposome preparation according to claim 1, further comprising a transferrin receptor T12 peptide and / or transferrin bound thereto.

4. The liposome preparation according to any one of claims 1 to 3, which stimulates intracellular ATP production.

5. The liposome preparation according to any one of claims 1 to 3, which is used for treating or preventing a brain disease.

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