PEG-modified beta-nicotinamide mononucleotide structure, preparation method and application
By modifying β-nicotinamide single nucleotide (NMN), the synthesis of NMN-PEG and NMN-PEG-FITCs is solved, and targeted treatment is achieved to achieve effective therapeutic effects at small doses.
Patent Information
- Application Number
- CN202411798544.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-09
- Publication Date
- 2025-03-21
AI Technical Summary
Existing beta-nicotinamide single nucleotides (NMN) for acute renal injury (AKI) treatment has low bioavailability, resulting in the need for high doses and frequent administration, which may cause unnecessary side effects.
By performing PEG modification on NMN, NMN-PEG and NMN-PEG-FITC are synthesized, and the distribution of drugs in cortical renal tubular epithelial cells is increased, thereby achieving targeted therapy.
It improves the targeting of NMN in the kidney, can achieve ideal therapeutic effects under the use of smaller doses, and reduces the occurrence of side effects.
Smart Images
Figure CN119661832A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of medicine, and specifically relates to a PEG-modified β-nicotinamide mononucleotide structure, a preparation method and a use thereof. Background Art
[0002] Acute kidney injury (AKI) is a common clinical emergency with a high incidence and poor prognosis. The probability of AKI in patients in intensive care units exceeds 50%. About 2 million people die from AKI and its complications each year worldwide, and there is currently no specific treatment to promote or accelerate kidney recovery. AKI is a common condition in all countries in the world, regardless of their economic conditions. Despite further understanding of the etiology and pathogenesis of acute kidney injury, there are relatively few measures for the diagnosis, treatment and prevention of acute kidney injury.
[0003] In recent years, β-nicotinamide mononucleotide (NMN) has become a popular raw material for health products. Its beneficial physiological functions such as delaying aging, treating diabetes, and treating neurological diseases have attracted widespread attention. NMN is an important coenzyme in the body, NAD + (nicotinamide adenine dinucleotide, also known as coenzyme I) and NADP + One of the precursors of nicotinamide adenine dinucleotide phosphate (NADP), also known as coenzyme II. + and NADP + It is widely involved in many important biochemical reactions in the body and plays a very important role in human health. Studies have shown that NMN can effectively increase the level of NAD in tissues and cells and protect rodents from kidney damage. Due to the nonspecific assimilation of peripheral tissues, conventional NMN treatment for AKI has low bioavailability and cannot relieve AKI in the short term. In order to achieve the relief effect, high doses and frequent administration are often required, which may lead to unnecessary side effects. Summary of the invention
[0004] In view of the defects of the prior art, the present invention modified NMN with PEG to synthesize NMN-PEG and NMN-PEG-FITC, which can increase the distribution of drugs in cortical tubular epithelial cells to play a targeted therapeutic role, solving the problem of rapid metabolism of drugs in the kidneys and low bioavailability.
[0005] The NMN-PEG provided by the present invention has a structural formula as shown in Formula I:
[0006]
[0007] In Formula I, the molecular weight of the PEG group ranges from 500-5000.
[0008] Preferably, the NMN-PEG is NMN-PEG2000.
[0009] The NMN-PEG-FITC provided by the present invention has a structural formula as shown in Formula II:
[0010]
[0011] In Formula II, the molecular weight of the PEG group ranges from 500-5000.
[0012] Preferably, the NMN-PEG-FITC is NMN-PEG2000-FITC.
[0013] The present invention also provides a method for preparing the NMN-PEG shown in the above formula I, comprising the following steps:
[0014] 1) reacting β-nicotinamide mononucleotide and succinic anhydride in the presence of pyridine and DMAP to obtain β-nicotinamide mononucleotide-COOH as shown in formula III;
[0015] 2) Make β-nicotinamide mononucleotide-COOH and NH2-PEG n -NH2 is subjected to amidation reaction to obtain NMN-PEG shown in formula I, wherein the NH2-PEG n The definition of n in -NH2 is the same as that of n in Formula I.
[0016]
[0017] In step 1) of the above method, the molar ratio of β-nicotinamide mononucleotide, succinic anhydride and DMAP is 1:(1.0-1.2):(0.4-0.7).
[0018] In step 1) of the above method, the reaction conditions of the reaction are: reacting at room temperature for 24 hours.
[0019] In step 1) of the above method, after the reaction is completed, the following steps are further included: concentrating the solution of the obtained reaction system by rotary evaporation under reduced pressure, pouring a large amount of acetone into it for precipitation, filtering and collecting the product, and washing it with acetone for multiple times to obtain β-nicotinamide mononucleotide-COOH.
[0020] In step 2) of the above method, the amidation reaction is carried out in the presence of PyBOP and DIPEA, and the amidation reaction is carried out in a solvent, which may be DMF.
[0021] Furthermore, the β-nicotinamide mononucleotide-COOH, NH2-PEGn The feeding molar ratio of -NH2, PyBOP and DIPEA is 1:(1.0-1.2):(1.2-1.4):5.
[0022] In step 2) of the above method, the reaction conditions of the amidation reaction are: reaction at room temperature for 0.5 hours.
[0023] In step 2) of the above method, after the amidation reaction is completed, the following steps are further included: the obtained reaction system is subjected to reduced pressure distillation to concentrate the reaction solution, a large amount of glacial ether is poured into the precipitate, the product is filtered and collected, and the glacial ether is washed for multiple times to obtain β-nicotinamide mononucleotide-PEG.
[0024] The present invention also provides a method for preparing NMN-PEG-FITC shown in the above formula I, comprising the following steps:
[0025] a) reacting β-nicotinamide mononucleotide and succinic anhydride in the presence of pyridine and DMAP to obtain β-nicotinamide mononucleotide-COOH represented by formula III;
[0026] b) subjecting β-nicotinamide mononucleotide-COOH to an amidation reaction with NH2-PEG-FITC to obtain NMN-PEG-FITC as shown in formula II.
[0027] In step a) of the above method, the molar ratio of β-nicotinamide mononucleotide, succinic anhydride and DMAP is 1:(1.0-1.2):(0.4-0.7).
[0028] In step a) of the above method, the reaction conditions of the reaction are: reaction at room temperature for 24 hours.
[0029] In step a) of the above method, after the reaction is completed, the following steps are further included: concentrating the solution of the obtained reaction system by rotary evaporation under reduced pressure, pouring a large amount of acetone into it for precipitation, filtering and collecting the product, and washing it with acetone for multiple times to obtain β-nicotinamide mononucleotide-COOH.
[0030] In step b) of the above method, the amidation reaction is carried out in the presence of PyBOP and DIPEA, and the amidation reaction is carried out in a solvent, which may be DMF.
[0031] Furthermore, the molar ratio of β-nicotinamide mononucleotide-COOH, NH2-PEG-FITC, PyBOP and DIPEA is 1:(1.0-1.2):(1.2-1.4):5.
[0032] In step b) of the above method, the reaction conditions of the amidation reaction are: reaction at room temperature for 0.5 hours.
[0033] In step b) of the above method, after the amidation reaction is completed, the following steps are further included: distilling the obtained reaction system under reduced pressure to concentrate the reaction solution, pouring a large amount of glacial ether into precipitation, filtering and collecting the product, and washing with glacial ether for multiple times to obtain β-nicotinamide mononucleotide-PEG-FITC.
[0034] The present invention also provides a NMN-PEG polymer micelle.
[0035] The NMN-PEG polymer micelles provided by the present invention are obtained by dispersing NMN-PEG in water and self-assembling.
[0036] The present invention also provides applications of the above-mentioned NMN-PEG, NMN-PEG-FITC and NMN-PEG polymer micelles.
[0037] The application provided by the present invention is the use of NMN-PEG or NMN-PEG-FITC or NMN-PEG polymer micelles in the preparation of products for preventing and / or treating acute kidney injury (Acute kidney injury, AKI).
[0038] The product may be a medicine or a pharmaceutical preparation or a health product.
[0039] The present invention also protects a product for preventing and / or treating acute kidney injury.
[0040] The product for preventing and / or treating acute kidney injury provided by the present invention comprises the above-mentioned NMN-PEG or NMN-PEG polymer micelles as an active ingredient.
[0041] Compared with the prior art, the present invention has the following beneficial effects:
[0042] Conventional NMN for the treatment of AKI is mainly high-dose (500 mg / kg) and multiple intraperitoneal injections (7 days). In the early stage of the study, we used NMN (500 mg / kg) for injection and found that its effect on AKI-related indicators in the short term was weak. The present invention improves the kidney targeting of NMN by PEG modification of NMN, and this scheme can achieve ideal therapeutic effects using smaller doses. BRIEF DESCRIPTION OF THE DRAWINGS
[0043] Figure 1 The synthetic reaction flow chart of NMN-PEG2000.
[0044] Figure 2 This is the structural identification map of NMN-PEG2000.
[0045] Figure 3 This is the formation process of NMN-PEG2000 polymer micelles.
[0046] Figure 4 Transmission electron microscopy (TEM) image of NMN-PEG2000 polymer micelles.
[0047] Figure 5 This is the particle size distribution diagram of NMN-PEG2000 polymer micelles.
[0048] Figure 6 This is the distribution diagram of NMN-PEG-FITC polymer micelles in mice.
[0049] Figure 7 The immunofluorescence images are of drugs and aquaporin 1 (AQP1).
[0050] Figure 8 In vivo validation of drug efficacy; A: Schematic diagram of model construction and drug administration process; B: Actual kidney image; C: HE staining image of kidney and statistics of injury degree; D: Serum SCR and BUN levels; E: Expression levels of renal injury factors KIM-1 and NGAL mRNA; F: Relative expression of TNF-α, IL-6 and IL-1β mRNA in mouse kidney tissue; G: Representative images of transmission electron microscopy observation of mouse kidney mitochondria (scale bar on the left = 2.0 μm; scale bar on the right = 1.0 μm); H: Prototype glutathione (GSH) content, oxidized glutathione (GSSG) content and GSH / GSSG ratio in kidney tissue; I: Total iron content and ferrous ion content in mouse kidney tissue; J: Malondialdehyde content in mouse kidney tissue.
[0051] Fig. 9 It is the result of hemolysis rate test.
[0052] Fig.10 This is the result of HK-2 cell activity detection. DETAILED DESCRIPTION
[0053] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.
[0054] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.
[0055] Example 1. Synthesis of NMN-PEG2000
[0056] according to Figure 1The reaction flow chart described above synthesizes NMN-PEG2000, and the specific method is as follows:
[0057] Weigh 100 mg of β-nicotinamide mononucleotide and dissolve it in 5 mL of pyridine, add succinic anhydride (1.0-1.2 eq.) and DMAP (0.4-0.7 eq.) to dissolve completely, react at room temperature overnight, concentrate the solution by rotary evaporation under reduced pressure, pour in a large amount of acetone to precipitate, filter and collect the product, wash with acetone several times to obtain β-nicotinamide mononucleotide-COOH. Weigh 70 mg of β-nicotinamide mononucleotide-COOH and dissolve it in 2 mL of water, add NH2-PEG2000 (1.0-1.2 eq.), PyBOP (1.2-1.4 eq.) and DIPEA (5.0 eq.) to dissolve completely in 5 mL of DMF, react at room temperature for 0.5 h, concentrate the reaction solution by distillation under reduced pressure, pour in a large amount of icy ether to precipitate, filter and collect the product, wash with icy ether several times to obtain β-nicotinamide mononucleotide-PEG2000. The structural identification spectrum is as follows Figure 2 shown.
[0058] 5 mg of β-nicotinamide mononucleotide-PEG2000 was dispersed in 1 mL of water and self-assembled to form β-nicotinamide mononucleotide-PEG2000 polymer micelle solution. The formation process of NMN-PEG2000 polymer micelle is as follows Figure 3 shown.
[0059] The morphological characteristics of β-nicotinamide mononucleotide-PEG2000 were observed using transmission electron microscopy (TEM). Figure 4 As shown. Figure 4 It can be seen that the drug has a spherical structure.
[0060] The particle size of β-nicotinamide mononucleotide-PEG2000 was detected by nanometer particle size potential analyzer (DLS). Figure 5 As shown. Figure 5 It can be seen that the drug particle size is about 200nm.
[0061] Example 2. Synthesis of NMN-PEG-FITC
[0062] The reaction process is as follows Figure 2As shown. The specific method is as follows: weigh 100 mg of β-nicotinamide mononucleotide and dissolve it in 5 mL of pyridine, add succinic anhydride (1.0-1.2 eq.) and DMAP (0.4-0.7 eq.) to dissolve completely, react at room temperature overnight, concentrate the solution by rotary evaporation under reduced pressure, pour a large amount of acetone to precipitate, filter and collect the product, wash with acetone for many times, and obtain β-nicotinamide mononucleotide-COOH. Weigh 70 mg of β-nicotinamide mononucleotide-COOH and dissolve it in 2 mL of water, add NH2-PEG2000-FITC (1.0-1.2 eq.), PyBOP (1.2-1.4 eq.) and DIPEA (5.0 eq.) to dissolve completely in 5 mL of DMF, react at room temperature for 0.5 h, concentrate the reaction solution by distillation under reduced pressure, pour a large amount of icy ether to precipitate, filter and collect the product, wash with icy ether for many times, and obtain β-nicotinamide mononucleotide-PEG2000-FITC.
[0063] Example 3, NMN-PEG-FITC / NMN-PEG pharmacodynamics experiment (improvement of acute renal injury induced by ischemia-reperfusion in mice)
[0064] In vivo validation:
[0065] Experimental methods: Male C57BL / 6J mice aged 7 to 9 weeks were anesthetized and the renal arteries on both sides were clamped with non-traumatic artery clamps. After 30 minutes of ischemia, the artery clamps were released to induce the AKI model. The experiment was divided into sham operation group, operation group, NMN treatment group after operation and NMN-PEG2000 treatment group after operation, with 6 mice in each group. The serum SCR and BUN levels, HE staining pathological damage, KIM-1, and NGAL renal injury factor mRNA levels of mice in the operation group were significantly higher than those of mice in the normal group, indicating that the model was successfully established. The sham operation group was exposed to the kidneys of mice for the same time but without ischemia treatment, and then intravenously injected with the same volume of saline as the drug. NMN 500mg / kg or NMN-PEG2000 20mg / kg was intravenously injected at 2h and 24h. After 48h, the mice were anesthetized and blood was collected from the eyeballs, and plasma and kidneys were collected for index detection.
[0066] Results: The heart, liver, spleen, lung, kidney and intestine of mice were collected at specific times and then imaged using the LASER600 animal in vivo imaging system to observe the in vivo distribution of the drug. Figure 6 . Figure 6 The upper middle picture shows normal mice after a single administration of 20 mg / kg of the drug. The mice were killed at different time points (0, 3, 6, 12, 24, 48, 72 h), and the kidneys were collected to observe the fluorescence intensity. The fluorescence intensity of the drug in the kidneys at different time points was observed. It can be seen from the figure that the fluorescence intensity of the drug reached a peak in the kidneys at the 12th hour and remained for 48 hours. Figure 6The middle and lower figures are a comparison of normal administration of NMN-PEG2000-FITC alone and NMN-PEG2000-FITC after the induction model. The drugs were administered at 2 and 24 hours, and the mice were killed at 48 hours to collect the heart, liver, spleen, lung, kidney and intestine for observation. The fluorescence intensity of the main organs of mice in different groups was observed. As can be seen from the figure, the drug targets the kidneys of AKI mice very well.
[0067] Immunofluorescence showed that NMN-PEG2000-FITC had a good targeting effect on renal tubular epithelial cells. The results are shown in Figure 7 .
[0068] In the AKI model induced by IRI (renal ischemia-reperfusion injury), HE staining, biochemical detection, RT-PCR, mitochondrial electron microscopy and other experiments were used to observe the in vivo efficacy of the drug. Figure 8 .Depend on Figure 8 It can be seen that (A) IRI model induction and drug treatment process. (B) Mice in different treatment groups were killed at 48 hours, kidney tissues were taken, and photos were taken on the same plane. Drug treatment reduced the degree of congestion and swelling of mice, (C) Representative images of H&E staining (n=6, upper scale bar=1000μm; lower scale bar=20μm). Tubular injury was evaluated based on lumen dilation, epithelial necrosis, cast formation, and loss of brush edges. 0, 1, 2, 3, 4, and 5 points were normal injury, <10%, 10-25%, 26-50%, 51-75%, and >75%, respectively. The drug effect alleviated the above conditions. (D) Drug treatment significantly reduced the levels of serum urea nitrogen (BUN) and serum creatinine (SCR) in mice (n=6), (E) the relative expression of kidney injury molecule (KIM-1) and neutrophil gelatinase-associated lipocalcin (NGAL) mRNA, and (F) the relative expression of TNF-α, IL-6, and IL-1β mRNA in mouse kidney tissue (n=6). (G) Representative images of transmission electron microscopy observation of mouse kidney mitochondria (left figure scale = 2.0μm; right figure scale = 1.0μm). The drug treatment reduced the degree of mitochondrial damage (mitochondrial atrophy, mitochondrial membrane rupture, and cristae disappearance). (H) The original glutathione (GSH) in the kidney tissue of mice treated with the drug was restored, the increase of oxidized glutathione (GSSG) was inhibited, and the GSH / GSSG ratio increased (n=6). (I) The total iron content and ferrous ion content in the kidney tissue of mice decreased after drug treatment (n=6), and (J) the malondialdehyde content decreased (n=6)
[0069] In vitro validation:
[0070] Hemolysis experiment: The whole blood of healthy mice was collected in an anticoagulant tube, centrifuged at 3000r for 15 minutes, and blood cells were obtained and suspended with normal saline. Diluted with normal saline to a 4% red blood cell suspension. 1 ml of NMN-PEG2000 solution of different concentrations (25-100ug / ml) was mixed with 200 microliters of blood cells respectively. 1 ml of ultrapure water was used as the positive control, and 1 ml of PBS was used as the negative control. The sample was incubated at 37°C for 2 hours and centrifuged at 3000r for 15 minutes. The OD value was then measured at 542nm using a microplate reader (BioTek, VT, USA). Hemolysis rate = (NMN-PEG2000 OD value-PBS OD value) / (OD value of H2O-OD value of PBS) × 100%.
[0071] Results Fig. 9 .Depend on Fig. 9 It can be seen that the drug has good biocompatibility.
[0072] HK2 cell activity test: Human renal proximal tubular cells (HK2) were subjected to hypoxia in a 1% O2 concentration incubator for 24 hours to induce an in vitro hypoxia model. After reoxygenation for 2 hours, different concentrations (1μg / ml-200μg / ml) of NMN-PEG2000 were applied for 24 hours, and the cells were collected for indicator detection.
[0073] Results Fig.10 .Depend on Fig.10 It can be seen that the drug has good in vitro safety.
[0074] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.
Claims
1. A NMN-PEG, the structural formula of which is shown in Formula I: In Formula I, the molecular weight of the PEG group ranges from 500-5000.
2. The NMN-PEG according to claim 1, characterized in that: The NMN-PEG is NMN-PEG2000.
3. A NMN-PEG-FITC, whose structural formula is shown in Formula II: In Formula II, the molecular weight of the PEG group ranges from 500-5000.
4. The NMN-PEG-FITC according to claim 3, characterized in that: The NMN-PEG-FITC is NMN-PEG2000-FITC.
5. The method for preparing NMN-PEG according to claim 1 or 2, comprising the following steps: 1) reacting β-nicotinamide mononucleotide and succinic anhydride in the presence of pyridine and DMAP to obtain β-nicotinamide mononucleotide-COOH as shown in formula III; 2) Make β-nicotinamide mononucleotide-COOH and NH2-PEG n -NH2 is subjected to amidation reaction to obtain NMN-PEG shown in formula I; the NH2-PEG n The molecular weight range of the PEG group in -NH2 is the same as that of Formula I.
6. The preparation method according to claim 5, characterized in that: In the step 1), the molar ratio of β-nicotinamide mononucleotide, succinic anhydride and DMAP is 1:(1.0-1.2):(0.4-0.7); And / or, in the step 1), the reaction conditions of the reaction are: reacting at room temperature for 24 hours; And / or, in the step 1), after the reaction is completed, the following step is further included: concentrating the solution of the obtained reaction system by rotary evaporation under reduced pressure, pouring acetone into it for precipitation, filtering and collecting the product, and washing it with acetone to obtain β-nicotinamide mononucleotide-COOH.
7. The preparation method according to claim 5 or 6, characterized in that: In the step 2), the amidation reaction is carried out in the presence of PyBOP and DIPEA, and the amidation reaction is carried out in a solvent, and the solvent is DMF; Furthermore, the β-nicotinamide mononucleotide-COOH, NH2-PEG n -The molar ratio of NH2, PyBOP and DIPEA is 1:(1.0-1.2):(1.2-1.4):5; And / or, in the step 2), the reaction conditions of the amidation reaction are: reaction at room temperature for 0.5 hours; And / or, in the step 2), after the amidation reaction is completed, the following step is further included: distilling the obtained reaction system under reduced pressure to concentrate the reaction solution, pouring into glacial ether for precipitation, filtering and collecting the product, and washing with glacial ether to obtain β-nicotinamide mononucleotide-PEG.
8. A NMN-PEG polymer micelle, which is obtained by dispersing the NMN-PEG described in claim 1 or 2 in water and self-assembling.
9. Use of the NMN-PEG described in claim 1 or 2, the NMN-PEG-FITC described in claim 3 or 4, or the NMN-PEG polymer micelle described in claim 8 in the preparation of a product for preventing and / or treating acute kidney injury.
10. A product for preventing and / or treating acute kidney injury, wherein the active ingredient comprises the NMN-PEG described in claim 1 or 2 or the NMN-PEG polymer micelle described in claim 8.