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WO2021098725A1 - Procédé de synthèse d'un dérivé du nmn et applications médicales du nmn et de son dérivé - Google Patents

Procédé de synthèse d'un dérivé du nmn et applications médicales du nmn et de son dérivé Download PDF

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WO2021098725A1
WO2021098725A1 PCT/CN2020/129788 CN2020129788W WO2021098725A1 WO 2021098725 A1 WO2021098725 A1 WO 2021098725A1 CN 2020129788 W CN2020129788 W CN 2020129788W WO 2021098725 A1 WO2021098725 A1 WO 2021098725A1
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nmnh
liver
nmn
nicotinamide mononucleotide
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Haiteng DENG
Yan Liu
Zhaoyun ZONG
Ting Li
Jing Liu
Wenhao Zhang
Yuling Chen
Xiaohui Liu
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Tsinghua University
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Tsinghua University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7084—Compounds having two nucleosides or nucleotides, e.g. nicotinamide-adenine dinucleotide, flavine-adenine dinucleotide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom

Definitions

  • the present invention relates to the technical field of pharmaceutical chemistry, in particular, relates to use of nicotinamide mononucleotide for the preparation of a medicament for preventing or alleviating liver injury or liver fibrosis in a subject, and a method for preventing liver injury or liver fibrosis in a subject.
  • the present invention also relates to the synthetic method of a new NAD precursor, named dihydronicotinamide mononucleotide (NMNH) , and its outstanding NAD boosting effect and other beneficial biological functions.
  • NMNH dihydronicotinamide mononucleotide
  • Liver fibrosis characterized by the deposition of extracellular matrix (ECM) is a reversible wound-healing response triggered by acute or chronic liver injury (Hernandez-Gea and Friedman, 2011) . More recent studies have revealed that alcoholic liver disease (ALD) , nonalcoholic fatty liver disease (NAFLD) and viral hepatitis are the primary causes of liver injury worldwide (Brunt et al., 2015; Seitz et al., 2018) . In the early stages, liver injury can induce hepatic cell death, and is a vital trigger for hepatic inflammation, generation of reactive oxygen species (ROS) and liver fibrosis (Wick et al., 2013) .
  • ALD alcoholic liver disease
  • NAFLD nonalcoholic fatty liver disease
  • ROS reactive oxygen species
  • liver fibrosis Wick et al., 2013
  • liver fibrosis In the later stages, sustained liver damage will promote the progression of liver fibrosis to liver cirrhosis and hepatocellular carcinomas (HCCs) (Affo et al., 2017; Ringelhan et al., 2018) . Liver injury plays crucial roles in the development and progression of liver fibrosis. Therefore, preventing liver injury has been considered as a therapeutic strategy for liver fibrosis (Wattacheril et al., 2018) .
  • ROS including the superoxide (O 2 - ) , hydrogen peroxide (H 2 O 2 ) and hydroxyl radical (OH ⁇ ) are highly reactive molecules mainly produced from mitochondrial electron transport chain (ETC) and NADPH oxidases (NOXs) (Apel and Hirt, 2004; Bedard and Krause, 2007; Sies et al., 2017; Zorov et al., 2014) .
  • ETC mitochondrial electron transport chain
  • NOXs NADPH oxidases
  • GSH and reduced TRXs are main electron donors for GPX and PRX to reduced H 2 O 2 (Lei et al., 2007; Shadel and Horvath, 2015) .
  • GSH can deoxidize many reactive electrophilic compounds via glutathione-S-transferases (GSTs) which also prevents DNA, protein and lipid damage (Hayes et al., 2005; Perkins et al., 2015) .
  • Nicotinamide adenine dinucleotide (NAD + ) an important coenzyme required for over 500 enzymatic reactions, is well known for its roles in oxidation and reduction (Ansari and Raghava, 2010; Rajman et al., 2018; Stein and Imai, 2012) .
  • NAD + can be synthesized using tryptophan in the de novo biosynthesis pathway, using nicotinic acid (NA) in the thals-handler pathway and using nicotinamide (NAM) , nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN) in the salvage pathway (Canto et al., 2015; Chiarugi et al., 2012; Johnson and Imai, 2018) .
  • NA nicotinic acid
  • NAM nicotinamide
  • NR nicotinamide riboside
  • NNMN nicotinamide mononucleotide
  • NAM NAM
  • NR N-oxide-semiconductor
  • NMN neuropeptide-like molecule-like molecule-like molecule-like molecule-like molecule-like molecule-like molecule-like molecule-like molecule-like molecule-like molecule-like molecule-like molecule-like molecule-like molecule-like molecule-like molecule-like molecule-like molecule-like molecule-like molecule-like kinases.
  • boosting NAD + levels by supplementing the intermediates promotes health and extends lifespan (Fang et al., 2016)
  • the pharmacokinetics and metabolic fates of them in different organs under different disease conditions are still under study.
  • it is unclear yet whether supplementing NAD + precursors can prevent liver injury or liver fibrosis by maintaining redox homeostasis.
  • NAD supplement The key NAD intermediates like NMN, which are usually called “NAD supplement” , has drawn researchers’ , investors’ and consumers’ attention to their remarkable effects related to diseases and aging (Gariani et al., 2016; Guan et al., 2017; Mills et al., 2016; Mukherjee et al., 2017) .
  • Traditional NAD supplements like NMN are oxidation form NAD precursors.
  • NAD precursor -dihydronicotinamide mononucleotide (NMNH) which has a better NAD boosting effect than NMN and other biological functions like increasing cellular anti-oxidation capacity, decreasing fat accumulation, decreasing inflammatory response and repressing tumor cell growth.
  • NMNH is a health promotion reagent which has a remarkable commercial potential.
  • the present invention is directed to use of nicotinamide mononucleotide for the preparation of a medicament for preventing or alleviating liver injury or liver fibrosis in a subject.
  • the nicotinamide mononucleotide prevents or alleviates liver injury or liver fibrosis by increasing the levels of GSH in liver.
  • the present invention is directed to a method for preventing liver injury or liver fibrosis in a subject.
  • the method comprises administering nicotinamide mononucleotide to a subject in an amount effective for increasing the levels of GSH in liver.
  • the present invention is directed to a method for producing dihydronicotinamide mononucleotide (NMNH) and uses of NMNH.
  • NMNH dihydronicotinamide mononucleotide
  • Fig. 1 NMN Prevents Liver Fibrosis in CCl 4 Treated Mice.
  • H&E staining and Masson staining of the livers from CCl 4 treated mice n 5/group.
  • Arrow in H&E staining indicates histopathological damage and inflammatory cell infiltrate in hepatic perisinusoidal space.
  • Arrow in Masson staining indicates the collagen deposition in hepatic perisinusoidal space.
  • the length of the black lines in the H&E staining and Masson staining pictures is 2 mm.
  • (B) to (E) Changes in the levels of intermediate metabolites in the GSH metabolism pathway in this study.
  • Fig. 5 NMN Improves Liver Injury both in CCl 4 Treated Mice and in TAA Treated Mice.
  • Fig. 6 Prevents Liver Fibrosis in TAA Treated Mice.
  • Fig. 7 Data Processing and Heatmap Analysis of the Liver Metabolomics Profiling in NMN Supplemented Mice.
  • the green columns represent the frequency of variability from 5%to 100%.
  • the red broken line represents the cumulative frequency of variation.
  • Fig. 14 Cellular GSH concentration fold changes after NMNH treatment for 24 h in 786-O cells.
  • Fig. 15 Relative TAG abundance in 3T3 cells after NMNH treatment for 24 h.
  • Fig. 16 IL-6 mRNA levels under different treatment.
  • Fig. 17 72 h growth rates of 786-O and HK-2 cells under NMNH treatment.
  • the inventors of the present invention found that NMN prevented liver fibrosis in both CCl 4 and TAA induced mouse liver fibrosis models, NMN supplementation boosted the levels of GSH in mouse liver, and the liver injury was significantly reduced by supplementing NMN.
  • the present invention proposes a new therapeutic strategy for liver injury and liver fibrosis by supplementing NMN.
  • the present invention provides use of nicotinamide mononucleotide for the preparation of a medicament for preventing or alleviating liver injury or liver fibrosis in a subject.
  • the nicotinamide mononucleotide prevents or alleviates liver injury or liver fibrosis by increasing the levels of GSH in liver.
  • the medicament is suitable for intraperitoneal injection.
  • the subject is a mammal, such as primates, mice, dogs, cats, horses, and cows, and preferably, is a human.
  • the present invention provides a method for preventing liver injury or liver fibrosis in a subject, comprising administering nicotinamide mononucleotide or a composition comprising nicotinamide mononucleotide to the subject.
  • the nicotinamide mononucleotide or the composition comprising nicotinamide mononucleotide is administered to the subject in an amount effective for increasing the levels of GSH in liver.
  • the nicotinamide mononucleotide or the composition comprising nicotinamide mononucleotide is administered to the subject by intraperitoneal injection.
  • the amount of nicotinamide mononucleotide is in the range of 400 to 600 mg/kg body weight of the subject. In some preferred embodiments, the amount of nicotinamide mononucleotide is 500 mg/kg body weight of the subject. In some preferred embodiments, the subject is a mammal, such as primates, mice, dogs, cats, horses, and cows, and preferably, is a human.
  • the present invention provides a pharmaceutical kit comprising a container, a composition comprising nicotinamide mononucleotide within the container, and a label on or associated with the container that indicates that the composition is for use in preventing or alleviating liver injury or liver fibrosis.
  • the present invention provides a method for increasing the level of GSH in liver in a subject comprising administering nicotinamide mononucleotide or a composition comprising nicotinamide mononucleotide to the subject.
  • the nicotinamide mononucleotide or the composition comprising nicotinamide mononucleotide is administered to the subject by intraperitoneal injection.
  • the amount of nicotinamide mononucleotide is in the range of 400 to 600 mg/kg body weight of the subject.
  • the amount of nicotinamide mononucleotide is 500 mg/kg body weight of the subject.
  • the subject is or will be under oxidative stress.
  • the subject is a mammal, such as primates, mice, dogs, cats, horses, and cows, and preferably, is a human.
  • the present invention provides nicotinamide mononucleotide for use in preventing or alleviating liver injury or liver fibrosis.
  • the present invention provides a pharmaceutical composition comprising nicotinamide mononucleotide for use in preventing or alleviating liver injury or liver fibrosis.
  • the present invention provides a method for producing NMNH by chemically reducing NMN with a reductant.
  • the reductant is thiourea dioxide (TDO) .
  • TDO thiourea dioxide
  • the reducing reaction with TDO is carried out in 5%-50% (wt) ammonia solution at 20-60°C.
  • the present invention provides uses of NMNH or dihydronicotinic acid mononucleotide (NAMNH) .
  • a method is provided for increasing total NAD + /NADH concentration in a cell comprising contacting the cell with an effective amount of NMNH or NAMNH.
  • a method is provided for increasing total NAD + /NADH level in a subject comprising administrating an effective amount of NMNH or NAMNH to the subject.
  • a method is provided for increasing glutathione (GSH) concentration in a cell comprising contacting the cell with an effective amount of NMNH or NAMNH.
  • GSH glutathione
  • a method is provide for increasing GSH level in a subject comprising administrating an effective amount of NMNH or NAMNH to the subject.
  • a method is provided for losing weight or reducing weight gain in a subject in need thereof comprising administrating an effective amount of NMNH or NAMNH to the subject.
  • a composition is provided for losing weight or reducing weight gain comprising an effective amount of NMNH or NAMNH.
  • a method is provided for treating an inflammatory disorder in a subject in need thereof comprising administrating an effective amount of NMNH or NAMNH to the subject.
  • a method for decreasing IL-6 level in a subject in need thereof comprising administrating an effective amount of NMNH or NAMNH to the subject.
  • an anti-inflammatory composition is provided comprising an effective amount of NMNH or NAMNH.
  • a method for treating a tumor in a subject in need thereof comprising administrating an effective amount of NMNH or NAMNH to the subject.
  • an anti-tumor composition is provided comprising an effective amount of NMNH or NAMNH.
  • use of NMNH or NAMNH in the preparation of a medicament for treating a tumor in a subject is provided.
  • the tumor is a kidney tumor.
  • subject as used herein include both human and non-human mammals, such as mice, dogs, cats, horses, and cows.
  • treating include abating or ameliorating at least one symptom of a disease or condition (e.g., liver injury or liver fibrosis) , inhibiting the disease or condition, e.g., arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the disease or condition.
  • a disease or condition e.g., liver injury or liver fibrosis
  • inhibiting the disease or condition e.g., arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the disease or condition.
  • liver injury or liver fibrosis refers to inhibiting partial or full development of liver injury or liver fibrosis, for example, in a subject who is known to have a predisposition to liver injury or liver fibrosis.
  • total NAD + /NADH concentration refers to the total concentration of NAD + and NADH in a cell.
  • total NAD + /NADH level refers to the total level of NAD + and NADH in a subject, which can be conveniently assessed, for example, by determining NAD + and NADH concentrations in a blood sample taken from the subject.
  • an effective amount of a compound means an amount, when administered to a cell or a subject, adequate to accomplish desired effects in the cell or the subject.
  • the effective amount will be dependent on the compound administered, the subject, the severity and type of the affliction, and the administration route of the compound.
  • C57BL/6J mice Six to eight-week-old male C57BL/6J mice were housed under specific pathogen-free conditions at Laboratory Animal Research Central, Tsinghua University.
  • C57BL/6J mice were injected intraperitoneally with CCl 4 diluted 3: 10 in corn oil or vehicle (corn oil) at 2 ⁇ L/g body weight once every two days for a period of 32 days. Nicotinamide mononucleotide (500 mg/kg body weight) was administered by IP at the same time as CCl 4 injection.
  • mice were injected intraperitoneally with TAA dissolved in PBS or vehicle (PBS) at 200mg/kg body weight once every two days for a period of 40 days. Nicotinamide mononucleotide (500 mg/kg body weight) was injected intraperitoneally at the same time as TAA injection. Mice were sacrificed 24 hours after the last CCL 4 or TAA injection, and then blood and liver samples were collected for follow-up study including histological, biochemical and metabolomics analysis.
  • PBS PBS
  • Nicotinamide mononucleotide 500 mg/kg body weight
  • Mouse livers were isolated and divided into small pieces and were fixed in 4%paraformaldehyde solution for 12 hours. Then, the tissues were embedded in paraffin and cut into sections (5 ⁇ m) . All the samples were stained with hematoxylin-eosin (H&E) and Masson’s trichrome for pathological evaluation and collagen detection using standard procedures.
  • H&E hematoxylin-eosin
  • Masson Masson
  • RNA from cells lysis was isolated using TRIzol extraction.
  • Complementary DNA synthesis was performed with reverse transcription system according to the manufacturer’s protocols.
  • Quantitative RT-PCR was performed by using SYBR Green reagent. The relative standard curve method was used for quantitation and 2 (- ⁇ Ct) method was used for gene expression calculation.
  • Tgf- ⁇ 1 forward primer, SEQ ID NO: 1 : CTTTGTACAACAGCACCCGC
  • Tgf- ⁇ 1 reverse primer, SEQ ID NO: 2
  • TGCTTCCCGAATGTCTGACG reverse primer, SEQ ID NO: 3
  • Col1a1 forward primer SEQ ID NO: 4
  • AAGTTCCGGTGTGACTCGTG AAGTTCCGGTGACTCGTG.
  • Untargeted quantitative metabolomics assay based on LC-MS/MS was performed on a Q Exactive orbitrap mass spectrometer (Thermo-Fisher Scientific) coupled to Ultimate 3000 with BEH amide column (Waters) .
  • metabolites were separated by a 30 min gradient elution in an elution buffer containing mobile phase A (10mM ammonium acetate in 95%acetonitrile) and mobile phase B (10mM ammonium acetate in 95%acetonitrile) .
  • Formic acid was applied to adjust the pH of the mobile phase A and the mobile phase B to 3.0.
  • an ammonium hydroxide solution was used to adjust the pH of the mobile phase A and B to 9.0.
  • Data-dependent acquisition mode was performed in the analysis and the mass resolutions of precursors and fragments were set to 70,000 and 17, 500.
  • a TSQ Quantiva mass spectrometer with Ultimate 3000 (Thermo-Fisher Scientific) was employed for targeted quantitative metabolites assay in positive and negative ion switching mode.
  • an aqueous mobile phase (10Mm trimethylamine and 15mM acetic acid in H 2 O) and an organic phase (100%acetonitrile) were used in this analysis.
  • Tracefinder software version 3.2, Thermo-Fisher Scientific
  • embedded an in-house database created by LibraryManager version 2.0, Thermo-Fisher Scientific
  • AST aspartate aminotransferase
  • ALT alanine aminotransferase
  • ALB albumin
  • TP total protein
  • One-way ANOVA was employed to analyze the metabolomics data normalized (z-score algorithm) by using the package of MetaboAanlyst (Chong et al., 2018) .
  • the top 50 of overall changed metabolites (p ⁇ 0.05) were selected for heatmap clustering analysis by using the package of ComplexHeatmap (Ding et al., 2016) .
  • Increased levels of metabolites (fold change >1.2 and p ⁇ 0.05) and decreased levels of metabolites (fold change ⁇ 0.83 and p ⁇ 0.05) were selected for metabolite sets enrichment analysis (KGEE) by using the MetaboAanlyst 4.0 (Chong et al., 2018) .
  • the package of igraph was applied to metabolite-pathway analysis.
  • Example 1 Prevents Liver Fibrosis
  • a classical liver fibrosis mouse model was induced by carbon tetrachloride (CCl 4 ) at a dose of 0.4ml/kg body weight by intraperitoneal injection once every two days for a period of 32 days (Fig. 1A) . Then, H&E staining and Masson staining were performed to assess the levels of liver fibrosis, and the results revealed that collagen deposition was decreased in NMN+CCl 4 treated mice as compared with CCl 4 treated mice (Fig. 1B) .
  • CCl 4 carbon tetrachloride
  • thioacetamide (TAA) induced liver fibrosis model was used to verify our finding from CCl 4 induced mouse model (Fig. 6A) .
  • the Masson staining result exhibited that the collagen deposition in NMN+TAA treated mice was also less than that in TAA treated mice (Fig. 6B) .
  • the expression of marker genes Tgfb1 and Col1a1 was also inhibited in NMN+TAA treated mice as compared to TAA treated mice (Figs. 6C and 6D) . To sum up, the data demonstrated that NMN prevented liver fibrosis in mice.
  • NMN as an important NAD + precursor is used to synthesize NAD + through the salvage pathway in vivo and NAD + plays vital roles in redox reactions and metabolic regulation (Rajman et al., 2018) .
  • metabolites were extracted from the liver tissue by using 80%methanol (v/v) and analyzed by untargeted and targeted liquid chromatography-tandem mass spectrometry liquid chromatography (LC-MS/MS) -based metabolomics analysis (Fig. 2A) .
  • LC-MS/MS liquid chromatography-tandem mass spectrometry liquid chromatography
  • the frequency of variability was calculated by using the data of three technical replications from the same quality control (QC) sample and found that the variation of 90 percent metabolites was less than 20 percent (Fig. 7A) .
  • the top 50 of the overall metabolites were performed by one-way ANOVA analysis after data normalization (Figs. 7B and 7C) , and heatmap clustering analysis of the top 50 metabolites showed that CCl 4 treated mice, NMN treated mice and NMN+CCl 4 treated mice were different from each other in terms of metabolite change patterns (Fig. 7D) .
  • a comprehensive metabolome change resource to study how did NMN supplementation alleviate liver fibrosis from the perspective of metabolomics.
  • the overall proteome changes were different in NMN treated mice and NMN+CCl 4 treated mice.
  • the metabolite changes in NMN treated mice were first analyzed by comparison with PBS treated mice, and it was found that the levels of 19 metabolites were increased (fold change >1.2 and p ⁇ 0.05) and the levels of 19 metabolites were decreased (fold change ⁇ 0.83 and p ⁇ 0.05) in NMN treated mice as compared with PBS treated mice (Fig. 2B) .
  • metabolite set enrichment analysis revealed that the changed metabolites were principally enriched in NAD + metabolism, amino sugar and nucleotide sugar metabolism and amino acid metabolism (Fig. 2C) .
  • MSEA metabolite set enrichment analysis
  • metabolite-pathway network analysis was performed to reveal the complicated relationship between the changed metabolites and the enriched metabolic pathway (Fig. 2D) .
  • the changed metabolites in NMN+CCl 4 treated mice were assessed by comparison with CCl 4 treated mice and it was found that 15 metabolite levels were increased (fold change >1.2 and p ⁇ 0.05) and 13 metabolite levels were decreased (fold change ⁇ 0.83 and p ⁇ 0.05) in NMN+CCl 4 treated mice by comparison with CCl 4 treated mice (Fig. 2E) . Then, the enrichment analysis revealed that the changed metabolites were mainly enriched in glutathione metabolism, NAD + metabolism and Sphingolipid metabolism (Fig. 2F) .
  • NMN+CCl 4 treated mice showed the complicated relationship between the changed metabolites and the enriched metabolic pathway in NMN+CCl 4 treated mice as compared with CCl 4 treated mice (Fig. 2G) .
  • NMN+CCl 4 treated mice As compared with NMN treated mice (Figs. 3B-3E; Figs. 8A-8E) .
  • NMN supplementation increased the levels of NMN, NAD + and NADP + in liver and the increased NAD + might be consumed to resist oxidative damage.
  • Example 5 Increases Liver GSH Levels under Oxidative Stress
  • NMN supplementation improved GSH levels in mouse liver under oxidation stress.
  • levels of Glu-Cys were also increased in NMN+CCl 4 treated mice as compared with CCl 4 treated mice, indicating that NMN might enhance the synthesis of GSH under oxidative stress (Fig. 4E) .
  • NMN might improve liver injury in CCl 4 treated mice.
  • ALT, AST, TP and ALB major liver injury markers
  • NMN improved liver injury by increasing the levels of GSH and the abundances of NAD (P) + -dependent oxidoreductases, which prevented liver fibrosis (Fig. 5E) .
  • Nicotinamide adenine dinucleotide (NAD + ) is a key cofactor related to various essential biological processes such as DNA repair, gene expression regulation and metabolism (Magni et al., 2012; Canto et al., 2015) , which was discovered in 1906 in yeast fermentation by Harden and Young. Recent studies indicated that NAD + metabolism is involved in aging and healthy regulation, which make it a popular star molecule for research and commerce (Yoshino et al., 2018) .
  • NAD + is synthesized through three pathways in mammals including the de novo pathway from tryptophan, the Preiss-Handler pathway from nicotinic acid (NA) and the salvage from nicotinamide (NAM) (Yaku et al., 2018) .
  • NAD + can also be synthesized from nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN) (Bieganowski et al., 2018) .
  • NAD + intermediates like NMN which are usually called “NAD + supplement”
  • NAD + supplement Traditional NAD + supplements like NMN are oxidation form NAD + precursors.
  • NAD + precursor -dihydronicotinamide mononucleotide NMNH
  • NAD + precursor -dihydronicotinamide mononucleotide NMNH
  • NMN NAD + precursor -dihydronicotinamide mononucleotide
  • NMNH Dihydronicotinamide mononucleotide
  • NPN nicotinamide mononucleotide
  • TDO thiourea dioxide
  • NDH dihydronicotinamide riboside
  • our method for NMNH production demands a much more simple and mild reaction condition, has a lower cost and is much easier for production scale expansion (Yang et al., 2019) .
  • reaction procedure is shown below. For 1 milliliter scale reaction, 100-500 mg NMN and 50-300 mg TDO are dissolved in 5-50%ammonia solution. The reaction mix is then incubated in 20-60°C water bath for 0.1-10 hour and NMNH is then purified by HPLC using an amide column.
  • NMNH is purified using an amide column (Waters, XBridge, 10 ⁇ 250 mm) .
  • the elution gradient is shown in table 1.
  • Mobile phase A is H 2 O (pH 10) and mobile phase B is acetonitrile (pH10) .
  • Mass spectrometry analysis is used for correct product identification.
  • the obtained NMNH has a m/z of 335.06 under negative mode (MS1) , indicating that NMNH has 2 hydrogen atoms more than NMN.
  • the elemental composition of NMNH is C11 H17 N2 O8 P1, which identifies with the MS result.
  • NADH and 13 ⁇ g NudC are dissolved in water with 500 mM ammonium acetate and 3 mM magnesium chloride. The reaction mix is then incubated in 48°C water bath for 1 hour. The reaction procedure of NADH decomposition catalyzed by NudC is shown below.
  • Fig. 11 shows the negative mode MS1 spectrum of Nudc reaction mix. NADH is catalyzed to form NMNH and AMP, as former studies suggested (Friedlos et al., 1992) .
  • TDO reduction method was more effective than NudC method because of high product concentration, mild reaction conditions as well as free of metal ion and proteins.
  • NMNH is a better NAD + booster than NMN
  • NMNH is a precursor of NAD + /NADH so we verified the NAD boosting effect of NMNH.
  • Intracellular total NAD concentration was measured using NAD + /NADH detecting kit (Beyotime, S0175) following user’s instruction. Briefly, 786-O cells were cultured in 6-well plates until 90%coverage and then treated with 500 ⁇ M NMNH or NMN for 6 hours. Then media were removed and cells were washed with 1 mL PBS. 200 ⁇ L cold NAD + /NADH extraction buffer was added to cells for cell lysis. After centrifuge (12,000 g, 10 min, 4°C) , supernatant was collected for total NAD measurement. 20 ⁇ L sample supernatant was added into 96-well plate and mixed with 90 ⁇ L ethanol dehydrogenase working solution and incubated in 37°C for 10 min. 10 ⁇ L color reagent was then added to the mix and the absorbance at 450 nM was measured after incubation in 37°C. The total NAD concentration was calculated according to standard curve.
  • NMNH increases cellular total NAD + concentration by 3 folds compared to untreated group in 786-O cell.
  • NMN only increases cellular NAD + concentration by 1.2 folds in 786-O cell. The result clearly shows that NMNH is a better NAD + booster than NMNH.
  • NMNH is better NAD + supplement than widely used NMN.
  • GSH and GSSG detecting kit (Beyotime, S0053) following user’s instruction. Briefly, 786-O cells were cultured in 6-cM dishes until 80%coverage and then treated with 500 ⁇ M NMNH for 24 hours. Then media were removed and cells were washed with 1 mL PBS. Cells were collected and resuspended in solution M and freezed and thawed in liquid nitrogen and water. After centrifugation, 10 ⁇ L cell lysis was mixed with 150 ⁇ L total GSH detecting solution and 50 ⁇ L 0.5 mg/mL NADPH. The absorbance at 412 nM was measured after incubation. Total GSH concentration was calculated according to standard curve. GSH concentration was calculated by subtracting GSSG concentration from total GSH concentration.
  • NMNH is a potential weight loss reagent
  • Cellular TAG accumulation was measured using Free Glycerol Reagent (Sigma, F6428) and Triglyceride Reagent (Sigma, T2449) following user’s instruction. Briefly, 3T3 cells were cultured in 6-well plates until 90%coverage and then treated with 200 ⁇ M oleic acid to induce fat accumulation with or without 0.5 mM NMNH at same time for 24 hours. Next, media were removed and cells were washed with 1 mL PBS. Then cells were resuspended in Rapa buffer and lysed by ultra-sonication. 20 ⁇ L cell lysis was mixed with 100 ⁇ L free glycerol buffer or triglyceride buffer. After 37°C incubation for 15 min, absorbance at 540 nM was measured and cellular TAG concentration was calculated.
  • monocyte THP-1 cell with 100 ng /mL Phorbol-12-myristate-13-acetate (PMA) for 24 h to induce macrophage formation which was then treated with 100 ng/mL lipopolysaccharide (LPS) to induce inflammation.
  • PMA Phorbol-12-myristate-13-acetate
  • LPS lipopolysaccharide
  • mRNA was extracted and reverse-transcripted into cDNA (CWBio, CW2019) .
  • the IL-6 expression level was measured by qPCR (CWBio, CW0957) .
  • NMNH is able to repress specific kinds of tumor cells, such as 786-O cell.
  • NMNH treatment represses 786-O’s growth, which is a kidney tumor cell line.
  • NMNH has no obvious effect on HK-2 cell at the same concentration, which is a kidney normal cell line (Ryan et al., 1994) .
  • the result indicates that NMNH has a preferential inhibiting effect on cancers cells rather than normal cells, which makes it a potential anti-tumor reagent.
  • NAD + as an important coenzyme, plays vital roles in oxidation and reduction (Rajman et al., 2018) . Numerous studies have shown that the levels of NAD + are decreased during aging process and boosting NAD + equivalents can obviously improve multi-organ functions and lifespan (Gomes et al., 2013; Massudi et al., 2012; Mills et al., 2016; Zhao et al., 2015) .
  • NMN can be used to synthesize NAD + via the salvage pathway, and it was indicated in more and more studies that NMN supplementation improved health maintenance by boosting the levels of NAD + in mice (Canto et al., 2015; Mills et al., 2016) . Although NMN supplementation can improve liver functions (Amano et al., 2019; Rajman et al., 2018) , it is not clear yet whether it can prevent liver injury and liver fibrosis by maintaining redox homeostasis.
  • NMN supplementation improved liver fibrosis by alleviating liver injury. It was indicated by metabolomics analysis that NMN supplementation increased the levels of GSH and the ratio of GSH/GSSG under oxidative stress, which was believed to be the major reason why NMN improved liver injury.
  • GSH can reduce H 2 O 2 to H 2 O via GPXs and deoxidize reactive electrophilic compounds via GSTs (Hayes et al., 2005; Lei et al., 2007) .
  • NMN may be used as a safer drug to boost the levels of GSH in liver for liver injury treatment.
  • NMN supplementation prevents liver injury by boosting the levels of GSH in liver, which in turn improves liver fibrosis.
  • present application presented a panoramic view of metabolome changes after NMN supplementation in mouse livers and proposed a new therapeutic strategy for liver injury and liver fibrosis by supplementing NMN.
  • NMNH is a potential anti-inflammatory agent for its ability to decrease IL-6 expression.
  • NMNH is a health promotion reagent which has prominent commercial potential and we have created a procedure to produce it in a simple, cheap and rapid way and we explored its biological function.
  • tumor repression function we suggest that NMNH is able to repress other kinds of tumor cells besides 786-O, which is a kidney tumor cell line.
  • NAMNH dihydronicotinic acid mononucleotide
  • Nicotinamide adenine dinucleotide phosphate oxidase in experimental liver fibrosis GKT137831 as a novel potential therapeutic agent. Hepatology 56, 2316-2327.
  • ROS as signalling molecules mechanisms that generate specificity in ROS homeostasis. Nat Rev Mol Cell Biol 8, 813-824.
  • NOX nicotinamide adenine dinucleotide phosphate oxidase
  • Peroxiredoxins guardians against oxidative stress and modulators of peroxide signaling. Trends Biochem Sci 40, 435-445.
  • NAD-Boosting Molecules The In Vivo Evidence. Cell Metab 27, 529-547.
  • HK-2 an immortalized proximal tubule epithelial cell line from normal adult human kidney [J] . Kidney international, 45 (1) : 48-57.
  • NASH Nonalcoholic Steatohepatitis
  • Hepatic Fibrosis Emerging Therapies. Annu Rev Pharmacol Toxicol 58, 649-662.
  • Dihydronicotinamide riboside is a potent NAD (+) concentration enhancer in vitro and in vivo [J] . Journal of Biological Chemistry, 294 (23) : 9295-307.
  • clusterProfiler an R package for comparing biological themes among gene clusters. OMICS 16, 284-287.
  • ROS reactive oxygen species

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Abstract

L'invention concerne l'utilisation du nicotinamide mononucléotide, en particulier, l'utilisation du nicotinamide mononucléotide pour la préparation d'un médicament destiné à prévenir ou soulager une lésion hépatique ou une fibrose hépatique chez un sujet, et un procédé pour prévenir une lésion hépatique ou une fibrose hépatique chez un sujet. L'invention concerne également un procédé de production de dihydronicotinamide mononucléotide (NMNH) et des utilisations du NMNH.
PCT/CN2020/129788 2019-11-19 2020-11-18 Procédé de synthèse d'un dérivé du nmn et applications médicales du nmn et de son dérivé Ceased WO2021098725A1 (fr)

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CN119978042B (zh) * 2023-11-10 2025-09-09 音芙医药科技(上海)有限公司 还原型β-烟酰胺单核苷酸钙盐的无定型及其制法和用途
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CN120424148B (zh) * 2024-02-02 2025-10-17 音芙医药科技(上海)有限公司 还原型β-烟酰胺单核苷酸盐及其组合物、制法和用途
CN118319935A (zh) * 2024-04-17 2024-07-12 音芙医药科技(上海)有限公司 一种还原型β-烟酰胺单核苷酸的药物用途
CN118546191A (zh) * 2024-07-29 2024-08-27 邦泰生物工程(深圳)有限公司 还原型β-烟酰胺单核苷酸一钠盐晶型及其制备方法和应用

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WO2023160405A1 (fr) 2022-02-23 2023-08-31 音芙医药科技(上海)有限公司 FORME POLYMORPHE DE SEL DISODIQUE DE β-NICOTINAMIDE MONONUCLÉOTIDE RÉDUIT, SON PROCÉDÉ DE PRÉPARATION ET SON UTILISATION
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WO2024251966A1 (fr) * 2023-06-07 2024-12-12 Bohan & Co As. Nicotinamide mononucléotide cristallin réduit (nmnh) et son procédé

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