Application of light-excited riboflavin in the preparation of a drug for treating leukemia

Abstract

The present invention relates to the field of biomedicine technology, and more particularly to the use of light-excited riboflavin in the preparation of a drug for treating leukemia. By combining UVA and UVB with specific wavelengths to light-excite riboflavin, the survival rate of leukemia cells, especially M3 type, can be effectively inhibited, and the drug can be used for the clinical treatment of leukemia.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to the application of light-excited riboflavin in the preparation of a drug for treating leukemia. Background Art

[0002] Riboflavin (RF), also known as vitamin B2, is an essential micronutrient for the human body. It is a safe and non-toxic vitamin that is essential for the body to carry out normal life activities.

[0003] The riboflavin photochemical method refers to mixing riboflavin with the sample to be processed and then subjecting it to light treatment. It is commonly used for blood treatment, such as regulating lymphocytes in the blood or inactivating pathogens in the blood. For example, the patent document (CN112316166A) discloses a riboflavin photochemical inactivation method for pathogens in a biological liquid sample, including the steps of adding riboflavin to the biological liquid sample and irradiating the biological liquid sample with light, wherein the light is a narrow spectrum ultraviolet light with a wavelength range selected from 360-370nm and / or 390-400nm. In addition, the patent document actually discloses that the wavelength of light has a great influence on the inactivation effect. For example, for Escherichia coli in the plasma of blood donors, ultraviolet light with a wavelength of 365nm and 395nm has the effect of inhibiting the growth of Escherichia coli, while wavelengths near it, such as 375, 390, and 400, have no inhibitory effect on Escherichia coli. However, the riboflavin photochemical method has certain limitations. For example, it requires direct irradiation of the blood or even the human body, which may affect normal proteins in the blood or the human body.

[0004] The inventor has previously demonstrated in research (CN115944730A) that, unlike the traditional riboflavin photochemical method, the excited riboflavin product, when in contact with tumor cells, has an inactivating effect on tumor cells, and can become a new tumor treatment method. However, the above patent does not disclose research on the effect of excited riboflavin on leukemia cells.

[0005] Clinically, leukemia is divided into acute and chronic categories according to the degree of immaturity of leukemia cells and the natural course of leukemia. Acute leukemia includes acute lymphocytic leukemia and acute myeloid leukemia (also known as "acute non-lymphocytic leukemia"). The classification of acute non-lymphocytic leukemia includes 8 types, including M0, M1, M2, M3, M4, M5, M6 and M7. Specifically, M0 (acute myeloid leukemia microdifferentiation), M1 (acute granulocytic leukemia undifferentiated), M2 (acute granulocytic leukemia partially differentiated), M3 (acute promyelocytic leukemia), M4 (acute granulocytic-monocytic leukemia), M5 (acute monocytic leukemia), M6 (erythroleukemia) and M7 (acute megakaryocytic leukemia). Leukemia seriously endangers human health, and it is very important to find safe and effective treatment methods. At the same time, there are differences in treatment methods for different types of leukemia.

[0006] Therefore, the present invention unexpectedly discovered that the effects of stimulated riboflavin on leukemia cells of different types are different. Summary of the invention

[0007] In a first aspect of the present invention, there is provided a use of riboflavin stimulated by light in the preparation of a drug for treating leukemia, wherein the light dose rate of the light stimulated by light is 1-60 J / cm 2 , for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 J / cm 2 .

[0008] Preferably, the light excitation includes using visible light and / or invisible light.

[0009] Preferably, the visible light includes blue light; and the invisible light includes ultraviolet light.

[0010] Preferably, the ultraviolet light includes one or more of UVA, UVB or UVC.

[0011] In some embodiments, the light stimulation comprises the use of blue light. In some embodiments, the light stimulation comprises the use of UVA. In some embodiments, the light stimulation comprises the use of UVB. In some embodiments, the light stimulation comprises the use of UVC. In some embodiments, the light stimulation comprises the combined use of UVA and UVB. In some embodiments, the light stimulation comprises the combined use of UVA and UVC. In some embodiments, the light stimulation comprises the combined use of UVB and UVC. In some embodiments, the light stimulation comprises the combined use of UVA, UVB and UVC. In some embodiments, the light stimulation comprises the combined use of blue light and UVA. In some embodiments, the light stimulation comprises the combined use of blue light and UVB. In some embodiments, the light stimulation comprises the combined use of blue light and UVC. In some embodiments, the light stimulation comprises the combined use of blue light, UVA and UVB. In some embodiments, the light stimulation comprises the combined use of blue light, UVB and UVC. In some embodiments, the light stimulation comprises the combined use of blue light, UVA and UVB. In some embodiments, the light stimulation comprises the combined use of blue light, UVB and UVC. In some embodiments, the light stimulation comprises the combined use of blue light, UVA and UVC. In some embodiments, the light stimulation comprises the combined use of blue light, UVA, UVB and UVC.

[0012] Preferably, riboflavin is configured to any value in the range of 0.01 μmol / L-8000 μmol / L, for example, 0.01 μmol / L, 0.1 μmol / L, 1 μmol / L, 5 μmol / L, 10 μmol / L, 12.5 μmol / L, 25 μmol / L, 50 μmol / L, 100 μmol / L, 200 μmol / L, 500 μmol / L, 1000 μmol / L, 1500 The protein was excited by light after the concentration of 2000 μmol / L, 2000 μmol / L, 2500 μmol / L, 3000 μmol / L, 3500 μmol / L, 4000 μmol / L, 4500 μmol / L, 5000 μmol / L, 5500 μmol / L, 6000 μmol / L, 6500 μmol / L, 7000 μmol / L, 7500 μmol / L or 8000 μmol / L was adjusted.

[0013] In a specific embodiment of the present invention, riboflavin or a pharmaceutically acceptable salt solution thereof is diluted to a concentration of any value between 0.01 μmol / L and 8000 μmol / L. The dilution is performed, for example, by culture medium.

[0014] In a specific embodiment of the present invention, the light excitation includes the combined use of UVA (wavelength 365nm) and UVB (wavelength 313nm) for light excitation, and the light dose rate of the light excitation includes 1-60J / cm2 .

[0015] In a specific embodiment of the present invention, the light excitation comprises the combined use of UVA (wavelength 365nm) and UVB (wavelength 313nm) light excitation for 20 minutes, and the light dose rate is 12J / cm 2 Preferably, the leukemia includes one or more of acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia or chronic myeloid leukemia.

[0016] Preferably, the acute myeloid leukemia includes one or more of acute myeloid leukemia microdifferentiation type (M0 type), acute myeloid leukemia undifferentiated type (M1 type), acute myeloid leukemia partially differentiated type (M2 type), acute promyelocytic leukemia (M3 type), acute myeloid-monocytic leukemia (M4 type), acute monocytic leukemia (M5 type), erythroleukemia (M6 type) or acute megakaryocytic leukemia (M7 type).

[0017] In a specific embodiment of the present invention, the leukemia includes acute promyelocytic leukemia (M3 type).

[0018] Preferably, the method for treating leukemia comprises administering to the subject an effective amount of light-stimulated riboflavin.

[0019] Preferably, the application comprises treating leukemia cells with riboflavin after light excitation.

[0020] Preferably, the leukemia cells are, for example, NB4, THP-1 or Kasumi-1, preferably NB4.

[0021] The second aspect of the present invention provides a riboflavin derivative, wherein the riboflavin derivative is obtained by subjecting riboflavin to light excitation. Preferably, the light excitation is defined in the same manner as in the first aspect of the present invention.

[0022] The third aspect of the present invention provides a method for preparing a riboflavin derivative, the method comprising subjecting the riboflavin to light excitation. Preferably, the light excitation is defined in the same manner as in the first aspect of the present invention.

[0023] The fourth aspect of the present invention provides a drug, wherein the drug comprises riboflavin after light excitation, and / or the riboflavin derivative described in the second aspect, and / or the riboflavin derivative obtained by the preparation method described in the third aspect. Preferably, the limitation on light excitation is the same as that of the first aspect of the present invention.

[0024] Preferably, the drug further comprises pharmaceutically acceptable excipients.

[0025] Preferably, the pharmaceutically acceptable excipients include conventional excipients in the art, such as one or more of diluents, excipients, fillers, absorption enhancers, surfactants, suspending agents, flavoring agents, wetting agents, binders, disintegrants, lubricants, pH adjusters, antioxidants, metal ion chelators, antibacterial agents or isotonicity adjusters.

[0026] Preferably, the drug can be in any suitable dosage form, including but not limited to tablets, pills, powders, granules, capsules, lozenges, syrups, emulsions, suspensions, injections, sprays, aerosols, powder sprays, lotions, ointments, pastes, iron preparations, eye drops, nasal drops, suppositories, effervescent tablets, pills or gels, etc.

[0027] Preferably, the drug can be administered by any suitable route, such as enteral administration (e.g. oral administration) or parenteral administration (e.g. intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, nasal drops, eye drops, intrathecal administration or transdermal administration, etc.).

[0028] Preferably, the drug can be a human drug or a veterinary drug.

[0029] Preferably, the drug may contain 0.01-99.5% (e.g., 0.01, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 99 or 99.5%) of active substance (e.g., riboflavin after light excitation, and / or, the riboflavin derivative described in the second aspect above, and / or, the riboflavin derivative obtained by the preparation method described in the third aspect above) by weight or volume.

[0030] The fifth aspect of the present invention provides a method for treating and / or preventing a disease, the method comprising administering to a subject an effective amount of riboflavin after light excitation, and / or the riboflavin derivative described in the second aspect, and / or the riboflavin derivative obtained by the preparation method described in the third aspect.

[0031] A sixth aspect of the present invention provides a use of light-excited riboflavin in the preparation of a drug for inducing apoptosis of leukemia cells or in inactivating leukemia cells in a sample.

[0032] Preferably, the sample comprises whole blood, plasma or leukemia cell suspension.

[0033] Preferably, the sample is derived from a human or a non-human animal.

[0034] Preferably, the leukemia cells are, for example, NB4, THP-1 or Kasumi-1, preferably NB4.

[0035] Preferably, the relevant limitations on light excitation are the same as those in the first aspect of the present invention.

[0036] As used herein, "treating" means slowing, interrupting, preventing, controlling, stopping, alleviating or reversing the progression or severity of a sign, symptom, disorder, condition or disease after the disease has begun to develop, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions or disorders.

[0037] The "effective amount" of the present invention refers to the amount or dosage of the product of the present invention that provides the desired treatment after administration to a subject in single or multiple doses.

[0038] The "subject" described in the present invention can be a human or a non-human animal, or a cell, tissue or organ of a human or non-human animal, and the non-human animal can be a wild animal, a zoo animal, an economic animal, a pet or an experimental animal, etc. Preferably, the non-human animal includes but is not limited to pigs, cattle, sheep, donkeys, foxes, minks, jackals, camels, dogs, cats, rabbits, mice (such as rats, mice, guinea pigs, hamsters, gerbils, chinchillas or squirrels, etc.) or monkeys, etc.

[0039] The "method" or "application" described in the present invention may be for therapeutic purposes or non-therapeutic purposes.

[0040] The term “include” or “comprising” described in the present invention is an open description, which includes the specified components or steps described and other specified components or steps that will not be substantially affected.

[0041] The "and / or" described in the present invention includes all combinations of items connected by the term, and each combination should be deemed to have been listed separately in the present application. For example, "A and / or B" includes "A", "B" and "A and B"; another example is "A, B and / or C", including "A", "B", "C", "A and B", "A and C", "B and C" and "A and B and C".

[0042] The term "pharmaceutically acceptable" as used herein means that the agent neither significantly stimulates the organism nor inhibits the biological activity and properties of the active substance of the administered product. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0044] Figure 1 : Cell survival rates of different leukemia cells after treatment with riboflavin or riboflavin after light excitation. DETAILED DESCRIPTION

[0045] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0046] Example 1 Effect of UVA+UVB-induced riboflavin on leukemia cells

[0047] 1. Treatment of leukemia cells: Take leukemia cells growing in the logarithmic phase, resuspend them in RPMI-1640 complete medium, take 100 μL of the suspension from the treatment group and the control group, add them to a 96-well plate, inoculate 5,000 cells per well, and set up 3 replicates for each group. After culturing in a 37°C, 5% CO2 incubator for 24 hours, add 10 μL of CCK-8 reagent to each well, incubate in the incubator for 1-4 hours, and detect the absorbance value at a wavelength of 450 nm on an ELISA reader.

[0048] 2. Illumination treatment of riboflavin: In this embodiment, 365nm UVA and 313nm UVB ultraviolet lamps are used to irradiate riboflavin for 20 minutes, and the total light source dose rate is 12J / cm 2 Specific treatment methods include:

[0049] The riboflavin sodium phosphate injection (initial riboflavin concentration of 8000 μmol / L) was dissolved in RPMI-1640 complete medium for dilution, and a 10 mL irradiation system was prepared according to the different concentrations required for the experiment. The system was injected into a specially customized transparent irradiation bag (made of ethylene-vinyl acetate copolymer, size 5×4 cm, capacity 10 mL) and irradiated with ultraviolet light (UVA 365 nm and UVB 313 nm, total light source dose rate 12 J / cm 2 ) irradiation treatment for 20 min.

[0050] 3. Incubate the irradiated riboflavin with the leukemia cells obtained in step 1.

[0051] This example uses three acute myeloid leukemia cell lines as examples to verify the effects of riboflavin stimulated by UVA+UVB light on different leukemia cells. The results are shown in Table 1 and Figure 1 .

[0052] Table 1

[0053] Cell lines IC50 NB4(M3 type) 32.76μmol / L THP-1(M5 type) 79.02μmol / L Kasumi-1(M2 type) 71.21μmol / L

[0054] The IC50 value of NB4 leukemia cells (M3 type) treated with riboflavin after ultraviolet irradiation can reach 32.76μmol / L, while the IC50 values ​​of Kasumi-1 (M2 type) or THP-1 (M5 type) leukemia cells are greater than 70μmol / L, and the sensitivity is significantly reduced. It can be seen that riboflavin stimulated by light is more sensitive to M3 type leukemia (acute promyelocytic leukemia), and can kill M3 type leukemia cells at extremely low concentrations, which is expected to provide a new reference drug for the treatment of M3 type leukemia.

[0055] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0056] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A use of light-excited riboflavin in the preparation of a drug for treating leukemia, characterized in that: The light dose rate of the light excitation is 1J / cm 2 -60 J / cm 2 .

2. The use according to claim 1, characterized in that: The leukemia includes one or more of acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia or chronic myeloid leukemia.

3. The use according to claim 1, characterized in that: The leukemia includes acute promyelocytic leukemia (M3 type).

4. The use according to claim 1, characterized in that: The light excitation includes using visible light and / or invisible light.

5. The use according to claim 4, characterized in that: The visible light includes blue light; the invisible light includes ultraviolet light.

6. The use according to claim 5, characterized in that: The ultraviolet light includes one or more of UVA, UVB or UVC.

7. The use according to claim 1, characterized in that: The light excitation includes the combined use of UVA and UVB.

8. The use according to claim 1, characterized in that: The light dose rate of the light excitation is 5-20J / cm 2 .

9. The use according to any one of claims 1 to 8, characterized in that: After riboflavin is prepared to a concentration of 0.01 μmol / L-8000 μmol / L, light excitation is performed.

10. The use according to any one of claims 1 to 9, characterized in that: The light excitation includes the combined use of UVA with a wavelength of 365nm and UVB with a wavelength of 313nm.

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