Novel G4 / Hemin-tFNA medicine as well as preparation method and application thereof

Abstract

The invention relates to the technical field of kidney disease drugs, in particular to a novel G4 / Hemin-tFNA drug and a preparation method and application thereof.The novel G4 / Hemin-tFNA drug is characterized in that G4 is added into tFNA, incubation is conducted in a TMK buffer solution, and G4-tFNA is obtained; and adding hemin into the G4-tFNA, carrying out incubation, then carrying out centrifugation, and collecting a supernatant so as to prepare the G4 / Hemin-tFNA drug. The G4 / Hemin-tFNA medicine prepared by the invention can efficiently remove ROS (reactive oxygen species), is safe and has kidney targeting property, and solves the problem that the existing medicine for treating kidney diseases cannot have good treatment effect, excellent kidney targeting property and high safety at the same time.

Description

Technical Field

[0001] The present invention relates to the technical field of drugs for kidney diseases, and particularly relates to a novel G4 / Hemin-tFNA drug and its preparation method and application. Background Art

[0002] Kidney diseases seriously threaten human health, and the incidence rate shows an upward trend globally. Among them, acute kidney injury (AKI), as a common and serious kidney disease, is characterized by acute onset and rapid progression. If not treated in time, it is extremely likely to cause chronic kidney disease or even renal failure, resulting in poor prognosis of patients and bringing a heavy economic burden to families and society. At present, the treatment methods for AKI are limited, mainly based on supportive treatment, and there is a lack of effective drugs. Therefore, it is urgent to develop new and effective treatment drugs. Reactive oxygen species (ROS) play a key role in the occurrence and development of kidney diseases. Under normal physiological conditions, the production and clearance of ROS in cells are in dynamic balance, maintaining the normal functions of cells. However, when the kidney is damaged, this balance is broken, and ROS accumulate in large amounts, triggering oxidative stress reactions, resulting in oxidative damage to biological macromolecules such as proteins, lipids, and nucleic acids in cells, and then destroying the normal structure and function of cells, accelerating the progression of kidney diseases.

[0003] Existing drugs for treating kidney diseases have many limitations. The efficacy of some drugs is not good and they cannot effectively inhibit the progression of kidney diseases; although some drugs have certain therapeutic effects, they have serious adverse reactions, limiting their clinical application. In addition, most drugs lack kidney targeting. During systemic distribution, they will not only reduce the effective concentration of drugs in the kidneys, but also produce toxic and side effects on other organs. Summary of the Invention

[0004] The purpose of the present invention is to provide a novel G4 / Hemin-tFNA drug that can efficiently scavenge ROS, is safe and has kidney targeting, and its preparation method and application.

[0005] To achieve the above purpose, the present invention provides the following technical solutions:

[0006] The present invention provides a preparation method of a novel G4 / Hemin-tFNA drug, comprising the following steps:

[0007] S1, adding G4 to tFNA and incubating in TMK buffer to obtain G4-tFNA;

[0008] S2, adding hemin chloride to the G4-tFNA and incubating, then centrifuging and collecting the supernatant to prepare the G4 / Hemin-tFNA drug.

[0009] Further, the tFNA is obtained by dissolving four single strands in TM buffer in equal proportion, heating, storing, and heating.

[0010] Further, the four single strands include T1 shown in SEQ ID NO: 2, T2 shown in SEQ ID NO: 3, T3 shown in SEQ ID NO: 4, and T4 shown in SEQ ID NO: 5. The temperature is raised to 95 °C, heated for 10 min, and stored at 4 °C for 30 min.

[0011] Further, the molar ratio of the tFNA to the G4 is 1:3.

[0012] Further, the concentration of hemin chloride is 3 mM, and the volume ratio of hemin chloride to G4-tFNA is 1:1000.

[0013] Further, the incubation in steps S1 and S2 is carried out at 37 °C for 2 h.

[0014] Further, the centrifugation is carried out at 1000 rpm for 5 min.

[0015] The present invention also includes a novel G4 / Hemin-tFNA drug prepared by any of the above preparation methods.

[0016] The present invention also includes the application of the novel G4 / Hemin-tFNA drug according to the above in the treatment of kidney diseases.

[0017] Compared with the prior art, the G4 / Hemin-tFNA drug prepared by the present invention can efficiently scavenge ROS, is safe and has kidney targeting, and solves the problem that the existing drugs for treating kidney diseases cannot simultaneously have good therapeutic effects, excellent kidney targeting, and high safety.

[0018] Description of the drawings

[0019] Figure 1 It is a schematic diagram of the determination result of the mimetic enzyme activity.

[0020] Figure 2 It is a schematic diagram of the distribution of G4 / Hemin-tFNA in mouse organs.

[0021] Figure 3 It is the distribution of G4 / Hemin-tFNA in renal tissue; green: glomerulus; red: Cy5-G4 / Hemin-tFNA; blue: DAPI.

[0022] Figure 4 It is a schematic diagram of the results of evaluating the pathological damage of mouse kidneys by H&E and PAS staining.

[0023] Figure 5 Changes in renal function indices in mice: creatinine (A), blood urea nitrogen (B), malondialdehyde (C), and superoxide dismutase (D); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0024] Figure 6 In A, it is a schematic diagram of the results of evaluating the change in ROS level using the DCFH-DA fluorescent probe; in B, it is a schematic diagram of the results of evaluating the change in mitochondrial membrane potential using the JC-1 fluorescent probe; in C - E, it is a quantitative comparison of the relative fluorescence intensities of ROS, JC-1 aggregates, and JC-1 monomers; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0025] Figure 7 H&E staining of the major organs of mice on the 1st, 3rd, 7th, 15th, and 30th days after injecting the therapeutic dose of G4 / Hemin-tFNA.

[0026] Figure 8 Dynamic changes in the main biochemical indices (aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), creatinine (CRE), and uric acid (UA)) in serum; ns ≥ 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Detailed implementation manners

[0027] The technical solutions of the present invention will be further described below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to illustrate and explain the present invention exemplarily, and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection of the present invention.

[0028] The sequences involved in the present invention are shown in Table 1 below:

[0029] Table 1

[0030]

[0031] Unless otherwise specified, the G4 / Hemin-tFNA mentioned in Examples 2 - 8 is the G4 / Hemin-tFNA drug prepared in Example 1.

[0032] Example 1

[0033] The G4 / Hemin-tFNA drug was prepared according to the following steps:

[0034] S1. Dissolve the four single strands T1, T2, T3, and T4 in TM buffer in equal proportions, heat to 95 °C, heat for 10 min, and store at 4 °C for 30 min to obtain tFNA;

[0035] S2. Add G4 to tFNA in a molar ratio of 1 (tFNA): 3 (G4), incubate at 37 °C in TMK buffer for 2 h to obtain G4-tFNA;

[0036] S3. Add hemin chloride with a concentration of 3 mM to the above G4-tFNA at a volume ratio of 1:1000, then incubate at 37 °C for 2 h, and then centrifuge at 1000 rpm for 5 min to remove the unbound hemin precipitate. Collect the supernatant to prepare the G4 / Hemin-tFNA drug.

[0037] Example 2

[0038] Determination of the mimic enzyme activity of G4 / Hemin-tFNA

[0039] Take 100 μL of TMB solution and add it to 20 μL of 1 μM Hemin, G4 / Hemin, tFNA, and G4 / Hemin-tFNA respectively. After mixing, react at 37 °C in the dark for 30 min. After taking a photo against a white background, add 100 μL of 2% H 2 SO 4 to terminate the reaction. Use a UV spectrophotometer to record the absorbance at 450 nm in the wavelength range of 200 - 800 nm at room temperature.

[0040] GPX catalyzes the oxidation of GSH by H 2 O 2 to generate GSSG. The remaining GSH can react with DTNB, and the reaction product has an absorption peak at 422 nm. For the four groups of Hemin, G4 / Hemin, tFNA, and G4 / Hemin-tFNA, operate according to the kit instructions. Use an enzyme-labeled instrument to measure the absorbance at 422 nm, and calculate the GPX activity according to the formula provided by the kit.

[0041] The results are shown in Figure 1 A. Alone, Hemin and tFNA basically did not initiate the TMB reaction, while G4 / Hemin and G4 / Hemin-tFNA produced extremely obvious TMB reactions, resulting in changes in the solution color and significant UV absorption peaks; continue to verify their mimic enzyme activities. As Figure 1 shown in B, G4 / Hemin-tFNA also has strong GPX activity, and the activity reaches 47 U / mL. The strong mimic enzyme activity is the basis for G4 / Hemin-tFNA to scavenge ROS.

[0042] Example 3

[0043] Kidney-targeted enrichment test of G4 / Hemin-tFNA

[0044] (1) Anesthetize C57BL / 6 mice by intramuscular injection of Zoletil 50 (15 mg / kg). When the vital signs of the mice are stable, slowly inject Cy5-labeled G4 / Hemin-tFNA via the tail vein at a dose of 100 μL per mouse (concentration: 1 μM).

[0045] (2) Remove the heart, liver, spleen, lungs, and kidneys, arrange them in order, place them on black cardboard, and take pictures using a small animal imaging system (wavelength range: 640 - 690 nm; excitation intensity: 5%; exposure time: 1 s). The obtained images are analyzed for ROI using aura.

[0046] The results are as Figure 2 shown. In addition to the kidneys, some G4 / Hemin-tFNA also accumulated in the liver, indicating that the kidneys are its main metabolic organs. The fluorescence intensity in the kidneys of AKI mice is much greater than that in healthy mice, indicating that when the kidneys are damaged, G4 / Hemin-tFNA has significant retention in the kidneys. The long-term retention of the drug in the AKI kidneys of mice further highlights its potential role in the treatment of AKI.

[0047] Example 4

[0048] Tissue distribution of G4 / Hemin-tFNA in the kidneys

[0049] (1) Anesthetize C57BL / 6 mice by intramuscular injection of Zoletil 50 (15 mg / kg). When the vital signs of the mice are stable, slowly inject Cy5-labeled G4 / Hemin-tFNA via the tail vein at a dose of 100 μL per mouse (concentration: 1 μM).

[0050] (2) Take the kidneys and fix them with 4% paraformaldehyde, then embed them in paraffin.

[0051] (3) Dewax the paraffin sections: sequentially place the sections in xylene I for 15 min, xylene II for 15 min, absolute ethanol I for 5 min, absolute ethanol II for 5 min, 85% alcohol for 5 min, 75% alcohol for 5 min, and wash with distilled water 3 times.

[0052] (4) Antigen retrieval: The tissue sections were placed in a retrieval box filled with sodium citrate antigen retrieval buffer (pH 6.0) and antigen retrieval was carried out in a microwave oven. Medium-high heat for 10 min, stop the fire for 8 min, then turn to medium-low heat for 8 min. During this process, evaporation of the buffer should be prevented and the slides must not be dried out. After natural cooling, the slides were placed in PBS (pH 7.4) and shaken on a shaker for washing 3 times, 5 min each time.

[0053] (5) Spontaneous fluorescence quenching: After the sections were slightly dried by shaking, a circle was drawn around the tissue with a histochemical pen, and a spontaneous fluorescence quenching agent was added to the circle for 5 min, followed by rinsing with running water for 10 min.

[0054] (6) Serum blocking: BSA was added dropwise to the circle and incubated for 30 min.

[0055] (7) Primary antibody incubation: The blocking solution was gently shaken off, and the primary antibody (CD31) was added dropwise to the sections. The sections were placed flat in a wet box and incubated at 4 °C overnight.

[0056] (8) Secondary antibody incubation: The slides were placed in PBS (pH 7.4) and shaken on a shaker for washing 3 times, 5 min each time. After the sections were slightly dried by shaking, a FITC-labeled fluorescent secondary antibody was added dropwise to the circle and incubated at room temperature in the dark for 50 min.

[0057] (9) DAPI nuclear counterstaining: The slides were placed in PBS (pH 7.4) and shaken on a shaker for washing 3 times, 5 min each time. After the sections were slightly dried by shaking, DAPI staining solution was added dropwise to the circle and incubated at room temperature in the dark for 10 min.

[0058] (10) Mounting: The slides were placed in PBS (pH 7.4) and shaken on a shaker for washing 3 times, 5 min each time. After the sections were slightly dried by shaking, they were mounted with an anti-fluorescence quenching mounting agent.

[0059] (11) Microscopic examination and photography: The sections were observed and images were collected under a slide scanner.

[0060] The results are shown in Figure 3 As shown, the green area labeled with CD31 represents glomeruli, and G4 / Hemin-tFNA has a high degree of overlap with the area outside the glomeruli (renal tubules), indicating that G4 / Hemin-tFNA has the property of targeting renal tubular cells, and this property is also beneficial for the treatment of AKI.

[0061] Example 5

[0062] Staining to observe pathological changes in mouse kidneys

[0063] Male C57BL / 6 mice (20 - 22 g) at 6 - 8 weeks of age were selected and randomly divided into a normal saline group, a cisplatin group, a cisplatin + Hemin group, a cisplatin + G4 / Hemin group, a cisplatin + tFNA group, and a cisplatin + G4 / Hemin - tFNA group. In the cisplatin - induced AKI model, mice were intraperitoneally injected with cisplatin at 20 mg / kg, and the normal saline group was injected with the same amount of normal saline. 24 h after cisplatin injection, 100 μL of 1 μM / animal of Hemin, G4 / Hemin, tFNA, and G4 / Hemin - tFNA were injected via the tail vein.

[0064] H&E and PAS staining were used to observe the histopathological changes of the mouse kidneys, and the results are shown in Figure 4 , the normal saline group showed normal kidney histological structure, while the AKI group showed obvious kidney tissue damage, mainly manifested in tubular cast formation, tubular dilation, and renal cell exfoliation. After treatment with Hemin, G4 / Hemin, and tFNA, the damage was alleviated to some extent, but obvious damage still existed. After treatment with G4 / Hemin - tFNA, the kidney damage was effectively reduced, and tubular cast formation and tubular dilation were significantly alleviated, indicating that G4 / Hemin - tFNA can improve renal tissue damage caused by AKI.

[0065] Example 6

[0066] Detection of BUN, CRE in mouse serum and MDA and SOD in renal tissue

[0067] Male C57BL / 6 mice (20 - 22 g) at 6 - 8 weeks of age were selected and randomly divided into a normal saline group, a cisplatin group, a cisplatin + Hemin group, a cisplatin + G4 / Hemin group, a cisplatin + tFNA group, and a cisplatin + G4 / Hemin - tFNA group. In the cisplatin - induced AKI model, mice were intraperitoneally injected with cisplatin at 20 mg / kg, and the normal saline group was injected with the same amount of normal saline. 24 h after cisplatin injection, 100 μL of 1 μM / animal of Hemin, G4 / Hemin, tFNA, and G4 / Hemin - tFNA were injected via the tail vein.

[0068] Anesthetize C57BL / 6 mice with 50 mg / kg of Zoletil, cut off their whiskers, draw blood by enucleating the eyeballs, and pay attention to the operation to prevent hemolysis. After standing at room temperature for 2 h, centrifuge at 4000 rpm for 15 min, take the supernatant, and use the mouse serum samples to measure creatinine (CRE) and blood urea nitrogen (BUN). Operate according to the kit instructions, measure the OD value at the corresponding wavelength using an enzyme-linked immunosorbent assay (ELISA) reader, and substitute it into the formula provided in the instructions for calculation. After euthanizing the mice, open the abdominal cavity to remove the kidneys, accurately separate the cortex, homogenize on ice, and then operate according to the instructions of the malondialdehyde (MDA) detection kit and the superoxide dismutase (SOD) activity detection kit. Measure the OD value at the corresponding wavelength using an ELISA reader, substitute it into the formula provided in the instructions for calculation, and use the BCA method to quantify the protein concentration.

[0069] As Figure 5 shown in Figure 5 A and

[0070] B, the levels of CRE and BUN in the AKI group increased significantly. After treatment with G4 / Hemin and tFNA, both decreased slightly, but still showed a significant difference compared with healthy mice. After treatment with G4 / Hemin-tFNA, the levels of CRE and BUN in the mouse serum decreased significantly and returned to near the healthy level of the mice in the normal saline group, indicating that the renal function of AKI mice was restored, and G4 / Hemin-tFNA could effectively restore the renal function of AKI mice and protect the kidneys. Figure 5 shown in Figure 5 C and

[0071] D, the level of SOD in the AKI group decreased significantly, indicating that during the progression of AKI, the gradually increasing oxidative stress level affected the activity of SOD. In contrast, the level of MDA increased significantly, indicating that lipid peroxidation became more and more severe during AKI. After treatment with G4 / Hemin-tFNA, the level of SOD increased and the level of MDA decreased significantly, indicating that G4 / Hemin-tFNA could alleviate the oxidative stress caused by AKI.

[0071] Example 7

[0072] 1. Detection of cellular ROS level

[0073] After culturing HK-2 cells to an appropriate density, seed the cells in 6-well plates and divide them into 6 groups: normal group, hydrogen peroxide (H 2 O 2 ) group, H 2 O 2 +Hemin group, H2 O 2 + G4 / Hemin group, H 2 O 2 + tFNA group and H 2 O 2 + G4 / Hemin - tFNA group. 500 μM H 2 O 2 After 12 h of injury, 200 nM of Hemin, G4 / Hemin, tFNA, and G4 / Hemin - tFNA were added. After incubation for 24 h, the cells were incubated with DCFH - DA fluorescent probe in a cell culture incubator for 30 min and washed 3 times with PBS, 5 min for each wash. Images were taken using an inverted fluorescence microscope.

[0074] DCFH - DA is a commonly used ROS fluorescent probe. Non - fluorescent DCFH - DA can freely penetrate the cell membrane and then be hydrolyzed by intracellular esterase to produce DCFH. DCFH is subsequently oxidized by intracellular ROS to generate the fluorescent compound DCF. And DCF cannot cross the cell membrane. Therefore, the fluorescence of DCF can accurately reflect the level of intracellular ROS.

[0075] Refer to Figure 6 A and Figure 6 C. Under normal conditions, the ROS level in cultured cells is very low (about 20 nM), but after hydrogen peroxide - induced injury, the cells are under oxidative stress, resulting in an increase in the ROS level. Compare the ability of different materials to scavenge ROS: The fluorescence intensity of the hydrogen peroxide group is the highest, followed by the Hemin group. The fluorescence intensities of the G4 / Hemin group and the tFNA group decreased slightly, but not as significantly as that of the G4 / Hemin - tFNA group, and the fluorescence intensity of the G4 / Hemin - tFNA group is almost equivalent to that of the normal group, indicating that G4 / Hemin - tFNA has a strong ROS scavenging ability.

[0076] 2. JC - 1 staining

[0077] After culturing HK - 2 cells to an appropriate density, the cells were seeded in 6 - well plates and divided into 6 groups: normal group, hydrogen peroxide (H 2 O 2 ) group, H 2 O 2 + Hemin group, H 2 O 2 + G4 / Hemin group, H 2 O 2 + tFNA group and H 2 O 2 + G4 / Hemin - tFNA group. 500 μM H 2 O 2At 12 h after injury, 200 nM of Hemin, G4 / Hemin, tFNA, and G4 / Hemin-tFNA were added. After incubation for 24 h, the cells were incubated with the JC-1 fluorescent probe in a cell culture incubator for 10 min, washed 3 times with PBS, 5 min for each wash. Images were taken using an inverted fluorescence microscope.

[0078] Mitochondria are the most important organelles affected by oxidative stress and are also one of the main sources of ROS. The JC-1 fluorescent probe was used to monitor the health status of mitochondria. JC-1 accumulates in the mitochondrial matrix and produces red fluorescence, while under low MMP conditions, JC-1 monomers cannot accumulate and produce green fluorescence. The health status of mitochondria was evaluated by observing the changes in red and green fluorescence.

[0079] The results are shown in Figure 6 B, Figure 6 D, and Figure 6 E. The green fluorescence was the strongest in the hydrogen peroxide group, while the red fluorescence was the strongest in the normal group. This indicates that hydrogen peroxide damages mitochondria and then reduces the mitochondrial membrane potential. After treatment with G4 / Hemin-tFNA, the green fluorescence was significantly weakened, and the red fluorescence also recovered to a level close to normal, indicating that G4 / Hemin-tFNA can reduce the damage of oxidative stress to mitochondria, stabilize the mitochondrial membrane potential, and protect mitochondria.

[0080] Example 8

[0081] H&E (hematoxylin-eosin) staining method

[0082] (1) Male C57BL / 6 mice (20 - 22 g) at 6 - 8 weeks of age were selected and injected with 100 μL of 1 μM / animal of G4 / Hemin-tFNA via the tail vein once on the 1st, 3rd, 7th, 15th, and 30th days respectively. The first group was injected only on the 1st day, the second group was injected on the 1st and 3rd days, and so on, for a total of 5 groups. The mice in each group were euthanized on the 1st, 3rd, 7th, 15th, and 30th days respectively, and the brain, heart, liver, spleen, lung, and kidney were taken and fixed in 4% paraformaldehyde.

[0083] (2) Dewax the paraffin sections.

[0084] (3) Hematoxylin staining: Immerse the sections in hematoxylin staining solution for 3 - 5 min, rinse with distilled water, differentiate with differentiating solution, rinse with distilled water, blue with bluing solution, and rinse with distilled water.

[0085] (4) Eosin staining: Immerse the sections in 85% and 95% gradient ethanol for dehydration for 5 min each, and then immerse them in eosin staining solution for 5 min.

[0086] (5) Dehydration and mounting: The sections were successively placed in absolute ethanol I for 5 min - absolute ethanol II for 5 min - absolute ethanol III for 5 min - xylene I for 5 min - xylene II for 5 min for clearing, and then mounted with neutral balsam.

[0087] (6) Microscopic examination and photography.

[0088] The tissue damage of the brain, heart, liver, spleen, lung and kidney of mice was analyzed by H&E staining on the 1st, 3rd, 7th, 15th and 30th days, and the results were as Figure 7 shown. After continuous administration for 30 days, no obvious tissue damage was observed in the main organs of the mice, indicating good safety.

[0089] Some key blood biochemical index changes were further evaluated, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), creatinine (CRE) and uric acid (UA), and the results were as Figure 8 shown. The values of these indexes were all within the normal range and showed minimal fluctuations, further demonstrating the good biosafety of G4 / Hemin-tFNA.

[0090] Although the specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments we described are illustrative rather than limiting the scope of the present invention. Equivalent modifications and variations made by those skilled in the art in accordance with the scope of the patent application of the present invention should be covered by the scope of the claims of the present invention.

Claims

1. A method for preparing a novel G4 / Hemin-tFNA drug, characterized in that: The following steps are involved: S1, add G4 to tFNA and incubate in TMK buffer to obtain G4-tFNA; S2, adding hemin chloride to the G4-tFNA and incubating, followed by centrifugation and collecting the supernatant to prepare the G4 / Hemin-tFNA drug.

2. A method for preparing a novel G4 / Hemin-tFNA drug according to claim 1, characterized in that: The tFNA is prepared by dissolving four single chains in TM buffer in equal proportions, raising the temperature, heating, and storing to obtain tFNA.

3. The method for preparing a novel G4 / Hemin-tFNA drug according to claim 2, characterized in that: The four single strands include T1 as shown in sequence number 2, T2 as shown in sequence number 3, T3 as shown in sequence number 4 and T4 as shown in sequence number 5. The temperature is raised to 95°C, the heating is for 10 minutes, and the storage is at 4°C for 30 minutes.

4. The method for preparing a novel G4 / Hemin-tFNA drug according to claim 1, characterized in that: The molar ratio of the tFNA to the G4 is 1:

3.

5. The method for preparing a novel G4 / Hemin-tFNA drug according to claim 1, characterized in that: The concentration of the hemin is 3 mM, and the volume ratio of the hemin to the G4-tFNA is 1:1000.

6. The method for preparing a novel G4 / Hemin-tFNA drug according to claim 1, characterized in that: The incubations in step S1 and step S2 are both performed at 37° C. for 2 h.

7. The method for preparing a novel G4 / Hemin-tFNA drug according to claim 1, characterized in that: The centrifugation was performed at 1000 rpm for 5 min.

8. A novel G4 / Hemin-tFNA drug prepared by the preparation method according to any one of claims 1 to 8.

9. Use of the novel G4 / Hemin-tFNA drug according to claim 9 in drugs for treating kidney diseases.

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