WO2019029064A1 - 果糖-1,6-二磷酸与其血药浓度稳定剂组合物的医药用途 - Google Patents

果糖-1,6-二磷酸与其血药浓度稳定剂组合物的医药用途 Download PDF

Info

Publication number
WO2019029064A1
WO2019029064A1 PCT/CN2017/114371 CN2017114371W WO2019029064A1 WO 2019029064 A1 WO2019029064 A1 WO 2019029064A1 CN 2017114371 W CN2017114371 W CN 2017114371W WO 2019029064 A1 WO2019029064 A1 WO 2019029064A1
Authority
WO
WIPO (PCT)
Prior art keywords
fbp
fructose
diphosphate
group
acid
Prior art date
Application number
PCT/CN2017/114371
Other languages
English (en)
French (fr)
Inventor
连晓媛
张治针
胡誉怀
毕洪运
朱荣遥
钱恒
池彬彬
Original Assignee
浙江大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 浙江大学 filed Critical 浙江大学
Priority to US16/637,766 priority Critical patent/US20200246364A1/en
Publication of WO2019029064A1 publication Critical patent/WO2019029064A1/zh

Links

Images

Classifications

    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4985—Pyrazines or piperazines ortho- or peri-condensed with heterocyclic ring systems
    • 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
    • A61K31/7024—Esters of saccharides
    • 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
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2072—Pills, tablets, discs, rods characterised by shape, structure or size; Tablets with holes, special break lines or identification marks; Partially coated tablets; Disintegrating flat shaped forms
    • A61K9/2086—Layered tablets, e.g. bilayer tablets; Tablets of the type inert core-active coat
    • A61K9/209—Layered tablets, e.g. bilayer tablets; Tablets of the type inert core-active coat containing drug in at least two layers or in the core and in at least one outer layer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5026—Organic macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
    • 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
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • 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
    • 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
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the invention belongs to the field of pharmacy and relates to fructose-1,6-diphosphate (also known as fructose-1,6-diphosphate and fructose diphosphate) and blood concentration stabilizer composition thereof for preparing diseases related to prevention and treatment of metabolic diseases and metabolic disorders
  • Applications in medicines include tumors, fatty liver, diabetes, hyperlipidemia, cardiovascular disease, peripheral neurological diseases, and central diseases.
  • Fructose-1,6-bisphosphate is an intermediate of sugar metabolism present in the body.
  • Exogenous FBP produces pharmacological action by regulating the activity of several enzymes in sugar metabolism (fructose diphosphate) Sodium tablets instructions, the second batch of chemical specifications published by the State Drug Administration in 2002).
  • Exogenous FBP can increase the concentration of intracellular adenosine triphosphate and phosphocreatine, promote potassium influx, increase the content of diphosphoglycerate in red blood cells, inhibit the release of oxygen free radicals and histamine, and reduce the body's causes. Damage caused by ischemia and hypoxia, especially for ischemic heart disease, shows a good protective effect.
  • FBP farnesoid fever
  • coronary atherosclerotic heart disease coronary heart disease
  • angina pectoris acute myocardial infarction
  • heart failure heart failure
  • arrhythmia other adjuvant therapy
  • FBP has the effect of treating diabetes or diabetic and combined cardiovascular and cerebrovascular diseases (Chinese invention patent: CN00112023.9).
  • the applicant also disclosed the role of FBP in anti-epilepsy (Chinese invention patent: ZL201310498212.2) and anti-tumor effect (Chinese invention patent: ZL201110066413.6).
  • the anti-epileptic effect of FBP is significantly better than the existing anti-epileptic drugs, which can be used to repair the function of epileptic brain while controlling the effect of epilepsy, and significantly improve the cognitive ability of epileptic animals and sustainable after stopping the drug.
  • the anti-epileptic effect has a broad-spectrum and significant anti-cancer effect while being highly safe against normal cells. It can be seen that FBP has great potential medicinal value.
  • exogenous FBP in vivo limit the medicinal value of existing FBP formulations.
  • the existing fructose-1,6-diphosphate preparation is used in a large dose (recommended oral preparation: 1 g each time, 3-4 times a day; intravenous treatment: 10 g per day, divided into 2 intravenous infusions).
  • oral doses are currently not recommended to significantly increase blood levels of FBP, so it is recommended to increase the clinical dose (Acta Pharm. 65 (2015) 147–157).
  • Exogenous FBP is rapidly degraded in vivo. Intravenous infusion of this product (250mg/kg) to healthy volunteers, the blood concentration can reach 770mg/L within 5 minutes, and the half-life is about 10 ⁇ 15 minutes.
  • the dose range of anti-epileptic and anti-cancer effects of FBP is small, and the long-term effective dose of chronic epilepsy in rats is intragastrically administered 100-200 mg/kg/day, and the effective dose of mouse tumor model is intraperitoneal injection of 400-450 mg/ Kg/time. It can be seen that it is impossible to increase the blood concentration by increasing the dose of FBP, which limits the medical use of FBP. Therefore, methods and substances for elucidating the metabolic mechanism of exogenous FBP in vivo and searching for stable blood levels of exogenous FBP are of great significance for the extensive medical use of FBP.
  • FBP far from explaining that FBP has such a broad range of pharmacological activities. Is there a common biological activity that supports the broad pharmacological activity of FBP, which can lead to a wide range of clinical therapeutic effects? More and more studies have shown that metabolic dysfunction or disorder is a common pathological mechanism of many diseases. These diseases include many major diseases, such as Diabetes and its complications, cardiovascular diseases, neurological disorders (epilepsy, schizophrenia, depression, etc.) and neurodegenerative diseases (senile dementia, vascular dementia, Parkinson's disease, multiple sclerosis, etc.) And tumors, etc. Alzheimer's disease is now also known as type 3 diabetes (Biochem Pharmacol. 2014 Apr 15; 88 (4): 548-59.
  • Mitochondrial dysfunction or malfunction is a common metabolic feature of the above diseases.
  • mitochondrial dysfunction occurs in a variety of neuropathic pains caused by different causes, including chemotherapy-induced neuropathy, diabetic neuropathy, and traumatic neuropathy (Mol Pain. 2015; 11:58.), and mitochondrial function.
  • the malignant chain reaction induced by the disorder includes the deficiency of oxidative phosphorylation (ATP), the increase of oxygen free radicals (ROS), and the reduction of oxidative stress and inflammatory reaction caused by anticancer drugs and other causes.
  • ATP oxidative phosphorylation
  • ROS oxygen free radicals
  • Common pathological mechanisms of neuralgia (Pain. 2013 Nov; 154 (11): 2432-40. Neurosci Lett. 2015 Jun 2; 596: 90-107. Curr Neuropharmacol. 2016; 14 (6): 593-609.)
  • Common pathological events of the above other diseases (Nature. 2006 Oct 19; 443 (7113): 787-95; Neurobiol Dis. 2013 Mar; 51: 27-34; Biomed Pharmacother. 2015 Aug; 74: 101-10. Biochim Biophys Acta.
  • tumor metabolic reprogramming is a core feature of cancer, and is closely related to tumorigenesis and development and cancer treatment resistance.
  • cancer cells can use common nutrients, especially glucose and glutamine, to meet energy requirements, redox balance and highly active biosynthesis, thus ensuring the rapid division and immortality of tumor cells.
  • Tumor epigenetic abnormalities are closely related to anti-cancer gene quiescence and overexpression of oncogenic genes. Recent studies have demonstrated that the characteristic metabolism of tumors also maintains the epigenetic characteristics of tumors.
  • Fructose-1,6-diphosphate as a sugar catabolism and sugar Xenobiotic intermediates may have extensive metabolic regulation and/or reverse the pathological metabolic pattern back to normal metabolic patterns, thereby potentially having a wide range of medical uses for the prevention and treatment of metabolic diseases and metabolic related diseases.
  • FBP exogenous fructose-1,6-diphosphate
  • the object of the present invention is to provide a pharmaceutical use of fructose-1,6-diphosphate and a blood concentration stabilizer composition thereof, which is a composition composed of fructose-1,6-diphosphate and a blood concentration stabilizer.
  • a substance consisting of fructose-1,6-diphosphate (FBP) and a blood concentration capable of stabilizing FBP (collectively referred to as FBP blood medicine)
  • FBP blood medicine a substance consisting of fructose-1,6-diphosphate
  • FBP blood medicine a blood concentration capable of stabilizing FBP
  • the medicament comprises a therapeutically effective amount of fructose-1,6-diphosphate and an effective amount of a blood concentration stabilizer and a pharmaceutically acceptable excipient or carrier.
  • the ratio of FBP to stabilizer in the drug is determined by the concentration of the stable FBP that the stabilizer can exert, and thus the ratio of different stabilizers to FBP can be different; the drug is in preventing and treating metabolic diseases and metabolism
  • the effective dose is determined by the specific disease, and specifically, the dose of the tumor FBP is 1 to 5 times higher than that of the other diseases.
  • the pharmaceutical form of the FBP includes a fructose-1,6-diphosphate prototype and fructose-1,6-diphosphate and a prodrug or derivative thereof.
  • the acceptable salts include, but are not limited to, ammonium, sodium, potassium, calcium, magnesium, manganese, copper, methylamine, dimethylamine, trimethylamine, butyric acid, acetic acid, dichloroacetic acid, hydrochloric acid, hydrogen formed by the compound. Salts and hydrates of the acid groups of bromic acid, sulfuric acid, trifluoroacetic acid, citric acid or maleic acid.
  • the 8-molecular hydrate of fructose-1,6-diphosphate trisodium salt is in a pharmaceutically acceptable form.
  • the blood drug concentration stabilizer refers to a diabetes drug or substance capable of slowing the rapid degradation of fructose-1,6-diphosphate in the drug preparation, including dipeptidyl peptidase represented by sitagliptin. 4 (DPP-4) inhibitors, glucagon-like peptide 1 (GLP-1) receptor agonists, biguanides represented by metformin, insulins and glitazones are also known as thiazolidinediones. Class and fructose-1,6-bisphosphatase inhibitors.
  • the ratio of fructose-1,6-diphosphate to stabilizer when fructose-1,6-diphosphate is combined with any of the stabilizers is: 8-molecular hydration of fructose-1,6-diphosphate trisodium salt
  • the ratio of the substance (g) to metformin (g) is 1:0.1 to 1:1, preferably in a ratio of 1:0.2 to 1:1; the 8-molecular hydrate of fructose-1,6-diphosphate trisodium salt (g)
  • the ratio with sitagliptin (g) is 1:0.001 to 1:0.5, preferably 1:0.01 to 1:0.1; the 8 molecule hydrate of fructose-1,6-diphosphate trisodium salt (g) and
  • the ratio of insulin (unit, IU) is 1:0.02 to 1:0.002, and the preferred ratio is 1:0.006 to 1:0.008.
  • the metabolic diseases and metabolic dysfunction-related diseases specifically include: indications of existing fructose-1,6-diphosphate preparations (mainly: angina pectoris for improving coronary heart disease, acute myocardial infarction, arrhythmia and heart failure) Myocardial ischemia and adjuvant treatment of viral myocarditis), cerebral hypoxia caused by cerebral infarction, cerebral hemorrhage, blood system cancer and various solid tumors, diabetes and its complications, fatty liver, epilepsy, neurodegenerative diseases (including senile dementia, Parkinson's disease, multiple sclerosis) and mental behavior disorders.
  • indications of existing fructose-1,6-diphosphate preparations mainly: angina pectoris for improving coronary heart disease, acute myocardial infarction, arrhythmia and heart failure
  • Myocardial ischemia and adjuvant treatment of viral myocarditis cerebral hypoxia caused by cerebral infarction, cerebral hemorrhage, blood system cancer and various solid tumors
  • diabetes and its complications fatty liver
  • the invention discloses the extensive regulation of exogenous fructose-1,6-diphosphate (FBP) on metabolic activities, in particular, the protective regulation of normal cells and the reversal of tumor metabolic characteristics, thereby protecting normal cells for FBP. It also provides a scientific basis for killing various cancer cells and supports the medical use of FBP to prevent and treat various metabolic diseases and metabolic disorders or disorders.
  • the diseases include diabetes and its complications, cardiovascular Diseases, neurological disorders (epilepsy, schizophrenia, depression, etc.) and neurodegenerative diseases (senile dementia, vascular dementia, Parkinson's disease, multiple sclerosis, etc.) and tumors.
  • the present invention discloses a mechanism by which exogenous FBP is rapidly degraded and finds a group of substances which can slow down the degradation rate of FBP in vivo and thereby increase the peak blood concentration of FBP and prolong its half-life (hereinafter referred to as FBP blood concentration). Stabilizer), and significantly enhance the efficacy of FBP, which supports the medical use of FBP in combination with its blood concentration stabilizer, FBP and its blood concentration stabilizer, in the preparation of a novel FBP drug with FBP as a pharmaceutically active ingredient. .
  • the present invention not only provides a novel FBP drug that prevents FBP from being rapidly degraded in vivo, but also expands the pharmaceutical range of FBP.
  • the novel FBP drug comprises FBP and a FBP blood concentration stabilizer which inhibits the rapid degradation of FBP in vivo, and the FBP blood concentration stabilizer can slow down the acute degradation of FBP in the body and block the prolongation with the treatment time course.
  • the degradation of FBP is accelerating, so that the pharmaceutical formulation of this composition can produce a higher peak of FBP plasma concentration (either once or after multiple treatments) and prolong its half-life. Therefore, the composition preparation can not only improve various pharmacological effects of FBP, but also expand the dose range of FBP, and can also alleviate the phosphoric acid poisoning phenomenon of the existing FBP preparation caused by rapid increase of phosphoric acid level caused by rapid degradation of FBP.
  • FBP composition preparations are effective for long-term administration, and thus the medicinal value of FBP for treating chronic diseases including tumors, epilepsy, diabetes, and neurodegenerative diseases can be fully exerted.
  • the FBP blood concentration stabilizer overcomes the defect that FBP can be rapidly degraded in vivo as a metabolic intermediate itself, thereby radically improving the medicinal value of FBP.
  • the FBP blood concentration stabilizer refers to an active substance capable of alleviating the degradation of exogenous FBP in vivo, including existing hypoglycemic drugs and hypoglycemic drugs continuously developed in the future, and indirect or direct inhibition of newly discovered or directed synthesis.
  • FBPase Fructose-1,6-bisphosphatase
  • the existing hypoglycemic agents include dipeptidyl peptidase-4 (DPP-4) inhibitors, glucagon-like peptide 1 (GLP-1) receptor agonists represented by sitagliptin, Metformin is represented by biguanides, insulins, glitazones (also known as thiazolidinediones) and fructose-1,6-bisphosphatase inhibitors (if sugar-2,6-diphosphate).
  • DPP-4 dipeptidyl peptidase-4
  • GLP-1 glucagon-like peptide 1
  • Metformin is represented by biguanides
  • insulins glitazones (also known as thiazolidinediones)
  • fructose-1,6-bisphosphatase inhibitors if sugar-2,6-diphosphate.
  • the FBP may constitute a medicinal component of the drug in an appropriate ratio to one or more of the above FBP blood concentration stabilizers.
  • Exogenous fructose-1,6-diphosphate may regulate cell metabolic activity extensively after entering the body, which may prevent various metabolic abnormal diseases and metabolic abnormalities; meanwhile, as an intermediate of glucose metabolism, exogenous After entering the body, FBP may act as an energy metabolism substrate through the glycolysis pathway and then enter the tricarboxylic acid cycle and eventually be oxidatively phosphorylated to produce energy (ATP). It may also be dephosphorylated to produce glucose by the FBPase-initiated gluconeogenesis pathway. The final product glycogen.
  • FBP Exogenous fructose-1,6-diphosphate
  • the present invention confirms this scientific hypothesis and finds a solution to the problem.
  • FBP exogenous FBP
  • FBP exhibits different metabolic regulation effects on normal cells and tumor cells, thus providing a scientific basis for FBP to protect normal cells and their functions as well as to kill various cancer cells.
  • FBP can promote mitochondrial oxidative phosphorylation activity, thereby increasing ATP levels; 2) promoting normal cell pentose phosphate metabolic bypass (PPP), increasing endogenous antioxidant
  • PPP pentose phosphate metabolic bypass
  • GSH reduced glutathione
  • FBP inhibits PPP in cancer cells, decreases NADPH and GSH levels, increases ROS levels and leads to mitochondrial damage and induces cancer cell senescence and apoptosis.
  • FBP can down-regulate multiple key metabolic enzymes in the tumor metabolic network, block the flow of glycolysis intermediates and tricarboxylic acid cycle intermediates to biosynthesis and reverse the epigenetic features of the tumor.
  • gluconeogenesis pathway inhibiting FBP is essential for maintaining the blood concentration of exogenous FBP, but also for stabilizing the blood concentration of exogenous FBP, especially the blood concentration of FBP preparations requiring long-term treatment.
  • Clue and molecular target Standard different types of hypoglycemic agents may inhibit different pathways of gluconeogenesis pathway through different mechanisms, which may indirectly or directly inhibit the gluconeogenesis of FBP, thereby protecting exogenous FBP from in vivo. Rapid degradation and inhibition of repeated repeated treatment of FBP-induced chronic activation of the gluconeogenesis pathway, increased peak and half-life of exogenous FBP plasma concentrations, and thus may significantly improve the efficacy of FBP formulations.
  • the applicant explored the stabilizing effect of different types of hypoglycemic agents on the blood concentration of FBP, and tried to find the defects that can overcome the rapid degradation of FBP in vivo, which is not conducive to its anticancer effect or other pharmacodynamic effects.
  • the present inventors have found that sitagliptin phosphate, metformin or insulin given a clinically used hypoglycemic dose 0.5 hours before intragastric administration of FBP can improve the blood after FBP or after repeated administration of FBP.
  • the peak concentration of FBP in the blood prolong the half-life of FBP in the blood, block the repetition of FBP-induced FBPase protein level up-regulation and the corresponding FBP blood concentration down-regulation, and significantly increase the overall anti-cancer efficacy of FBP.
  • Metformin is a classic drug for the treatment of type 2 diabetes, which can inhibit excessive gluconeogenesis in liver and kidney, because dephosphorylation of fructose-1,6-diphosphate under the catalysis of FBPase is a rate limit in the process of gluconeogenesis.
  • Link therefore, metformin inhibits gluconeogenesis and indirectly inhibits fructose-1,6-diphosphate as a process of dephosphorylation of gluconeogenesis, thereby increasing the peak plasma concentration of exogenous FBP and prolonging the half-life of FBP.
  • the inhibitory effect of sitagliptin on gluconeogenesis can also indirectly or directly protect FBP from degradation by dephosphorylation of FBPase.
  • Sitagliptin exerts a hypoglycemic effect by inhibiting dipeptidyl peptidase-4 (DPP-4).
  • DPP-4 dipeptidyl peptidase-4
  • GLP-1 activity is negatively regulated by DPP-4, so the inhibition of DPP-4 by sitagliptin restores GLP-1 activity and inhibits gluconeogenesis and upstream gluconeogenesis pathways.
  • insulin can play a hypoglycemic effect by inhibiting glycogen synthesis, and can also protect FBP from dephosphorylation and degradation.
  • the glitazone troglitazone can directly inhibit FBPase, so it can also increase the peak plasma concentration of exogenous FBP and prolong its half-life, thereby improving the clinical use of FBP as a pharmacophore. value.
  • Fructose-2,6-diphosphate is the isomer of fructose-1,6-diphosphate. It is the most active endogenous FBPase inhibitor known to date. It can also be combined with FBP to prepare a compound with higher bioavailability of FBP. preparation.
  • the present invention also found that no significant anticancer activity was observed in the cell culture system of sitagliptin; in the whole animal tumor model, the clinical hypoglycemic dose of sitagliptin showed certain anticancer activity in some models, which The overall anticancer activity may be the result of its regulation of glucose metabolism, or it may be the result of other effects such as improving the body's immunity.
  • the combination of FBP and the hypoglycemic dose of sitagliptin produced stronger anticancer effects than the individual treatments alone, which strongly supported the combination of FBP and sitagliptin in the preparation of novel anticancer drugs. Value.
  • FBP combined with sitagliptin can also significantly increase the body weight caused by high-fat diet feeding, and the individual treatment alone has not seen this effect; FBP can reduce fat accumulation caused by high-fat diet feeding, FBP and The efficacy of sitagliptin combination therapy is enhanced, while sitagliptin alone does not.
  • the results of the study not only prove that FBP can promote fat metabolism, but also support the application value of the novel FBP drug of the present invention in weight loss and prevention and treatment of diabetes, especially type 2 diabetes.
  • the present invention also finds that FBP can significantly counteract peripheral neuralgia caused by cancer chemotherapeutic drugs, which further supports the application value of the novel FBP drug of the present invention for treating cancer.
  • traditional chemotherapeutic drugs are still the mainstream drugs against cancer in clinical practice, but their side effects include peripheral nerve pain, which not only seriously reduces the quality of life of patients, but also often leads patients to abandon chemotherapy. Therefore, finding a drug that can reduce the side effects of chemotherapy drugs without reducing its anticancer effect is of great significance for cancer treatment.
  • the FBP anticancer preparation produced by the invention can also be used in combination with a traditional chemotherapeutic drug in the clinic, thereby further enhancing the anticancer effect and overcoming the neurotoxic side reaction.
  • Metabolic dysfunction especially mitochondrial oxidative phosphorylation, and its malignant chain reaction, including insufficient energy substance ATP, increased ROS production, and reduction of endogenous antioxidants, and inflammatory reactions are peripheral nerves caused by anticancer drugs.
  • the common pathological mechanism of pain and other causes of neuralgia, the pharmacological activity of FBP against peripheral chemotherapy caused by chemotherapeutic drugs is highly consistent with its role in mediating normal cell metabolism, and also supports FBP for the prevention of neuropathic pain caused by other causes. use.
  • novel FBP drugs of the present invention are also applicable to the various indications of the disclosed FBP, including myocardial ischemia for improving angina pectoris, acute myocardial infarction, arrhythmia and heart failure in coronary heart disease, and Adjuvant therapy for viral myocarditis, used to improve cerebral hypoxia caused by cerebral infarction, cerebral hemorrhage, prevention of blood system cancer and various solid cancers, prevention and treatment of diabetes and its complications, epilepsy and neurodegenerative diseases (including senile Dementia, Parkinson's disease, multiple sclerosis).
  • the medicinal dose of the 8-molecular hydrate of fructose-1,6-diphosphate trisodium salt is 100-5000 mg/kg body weight/day, preferably 300-2000 mg/kg body weight/ Day;
  • the pharmaceutical dosage of metformin is 1 to 1000 mg/kg body weight/day, preferably 50 to 300 mg/kg body weight/day;
  • the pharmaceutical dosage of sitagliptin is 0.1 to 500 mg/kg body weight/day, preferably 1 to 100 mg/kg body weight/day;
  • the pharmaceutical dose of insulin varies from 10 to 100 U/kg body weight/day depending on the type thereof.
  • the treatment of the novel FBP drug takes a single or multiple treatments, which are processed 2-4 times a day.
  • the "medicinal dose” means to achieve the purpose of preventing, effectively controlling or treating diseases.
  • the doctor follows the principle of individualized treatment, and adjusts the dosage of the drug of the individual according to the patient's disease condition. Accordingly, the dosages and ratios of the compositions provided in the present invention are understood not to limit the dosages and ratios of the pharmaceutical compositions used in the present invention, but are preferred to the present invention.
  • the dose and range of human pharmaceuticals can be converted to an appropriate dose of the drug and the animal, especially a mammal, such as a rat, a mouse, a dog, etc. range.
  • the dosage form of the novel FBP medicine prepared by the composition includes an injection, a common tablet, a granule, a capsule, a double-layer tablet, a controlled release double-layer tablet, a sustained-release tablet, a single-chamber controlled-release tablet, a dispersion tablet, Enteric-coated tablets, enteric-coated capsules, fixed-point release tablets, Sustained release capsules, sustained release pellets, capsules containing pellets or tablets, targeted preparations, but are not limited to these dosage forms.
  • the preferred dosage form is a controlled release solid preparation which can release the stabilizer first by releasing the FBP for 15 minutes to 60 minutes; the stabilizer can also be made into the A oral preparation or the injection preparation, and the FBP is made into the B oral preparation or the injection preparation for clinical use.
  • the A preparation was first used for 15 minutes to 60 minutes, and then the B preparation was used.
  • excipients used in the two-layer sheet are selected from, but not limited to, the following excipients: methyl cellulose, hydroxyethyl cellulose, hydroxyethyl methyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxyl Propylmethylcellulose, hydroxymethylcellulose, sodium carboxymethylcellulose, glucose, chitin, chitosan, galactomannan, beeswax, hydrogenated vegetable oil, synthetic wax, butyl stearate, stearic acid Acid, carnauba wax, glyceryl stearate, propylene glycol-stearate and stearyl alcohol, polyvinyl alcohol and polyhydroxyethylene 934; stabilizer selected from sodium citrate, citric acid; lubricant selected from hard fat Magnesium, stearic acid, colloidal silica, talc.
  • the preparation method includes a powder direct compression method, a wet granulation tablet, a dry granulation tablet, a heavy pressing method, and the like.
  • the wet granulation tableting method not only has a simple process, is time-saving, and can protect the stability of the drug
  • the specific preparation method comprises: mixing the active ingredient, the filler and the binder according to the prescription of the layer A and the layer B, respectively. Drying and granulating after wet granulation; mixing the dry granules of layer A and B with the disintegrant and the lubricant respectively, and then pressing, the fructose-1,6-diphosphate 3 sodium salt contains 8 molecules of hydrate-sig Lenin double sheet.
  • the composition is prepared into an 8-molecular hydrate containing fructose-1,6-diphosphate trisodium salt and a sustained-release pellet of sitagliptin by a blank pellet.
  • the drug immediate release layer and the sustained release layer are composed of three parts.
  • fructose-1,6-diphosphate is used as a coated sustained-release pellet, and the sitagliptin is encapsulated as a common film coating component.
  • excipients used in the sustained release pellets are selected from, but not limited to, the following excipients:
  • the blank pellet the filler is selected from the group consisting of lactose, starch, microcrystalline cellulose, etc.; the binder is selected from the group consisting of sucrose, methylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, etc.; the lubricant is hard Magnesium citrate, stearic acid, colloidal silica, talc, and the like.
  • the immediate release pellet the selected polymer film material is polyvinylpyrrolidone, hypromellose, polyethylene glycol and the like.
  • the sustained release pellet the selected retarder is acrylic resin, ethyl cellulose, and cerium wax; the porogen is lactose, hypromellose, polyethylene pyrrolidone, talc; plasticizing The agent is triethyl citrate, diethyl phthalate, polyethylene glycol 6000, tributyl citrate, dibutyl glutamate; anti-adhesive agent is talc, magnesium stearate, mono-hard fat Acid glyceride.
  • the preparation method comprises the following steps: preparing a fructose-1,6-bisphosphate pellet by a spheron extrusion method, and then coating the fluidized bed, and then directly applying metformin as a film coating component to fructose-1,6-diphosphate sustained-release micro Pill outer layer.
  • the specific preparation method is as follows:
  • Sustained-release pellet coating a coating liquid was prepared by adding an appropriate amount of Eudragit Ne30d (polymer concentration: 5%), talc (corresponding to a polymer amount of 60%), deionized water, and then fluidized bed coating.
  • the present invention is also applicable to the clinical use of a separate preparation of FBP and a stabilizer, which is taken simultaneously and sequentially according to the actual situation, preferably with the stabilizer taken in advance for 30 minutes.
  • Exogenous fructose-1,6-diphosphate is rapidly degraded into the body, and this rapid degradation is exacerbated with the prolonged treatment time.
  • the higher the dose the more serious the degradation is. Therefore, existing FBP preparations are difficult to produce and maintain effective blood concentration, which greatly limits the medicinal value of FBP, especially for the prevention and treatment of chronic diseases.
  • the present invention firstly discloses that the gluconeogenesis pathway is involved in the rapid degradation of exogenous FBP, and in particular, the activation of this metabolic pathway leads to the gradual disappearance of the drug with prolonged treatment time.
  • hypoglycemic agents can inhibit the rapid degradation of exogenous FBP in vivo and greatly improve the overall efficacy of FBP including anticancer efficacy.
  • the results support the application of hypoglycemic agents as a complex of FBP in vivo and FBP to prepare drugs for the prevention and treatment of metabolic diseases and metabolic related diseases.
  • the inventiveness and scientific nature of the present invention are also reflected in a common pathological mechanism for a variety of different diseases including neurodegenerative diseases, neurological disorders, obesity and diabetes, and tumors, namely metabolic dysfunction (metabolic disorders of differentiated cells).
  • metabolic dysfunction metabolic disorders of differentiated cells.
  • metabolic reprogramming of tumor cells including glycolysis activity and de novo biosynthesis and attenuation of mitochondrial oxidative phosphorylation, and inhibition of excessive sugar by FBP
  • the beneficial effect a kind of FBP compound preparation containing fructose-1,6-diphosphate (FBP) and its blood concentration stabilizer as the main active ingredient, which is the existing active ingredient with FBP as the only active ingredient.
  • FBP fructose-1,6-diphosphate
  • the FBP formulation has several advantages.
  • the key problem of limiting the medicinal value of the existing FBP preparations is that the problem that the exogenous fructose-1,6-diphosphate (FBP) enters the body is rapidly degraded, so the FBP composite preparation provided by the present invention can produce more High peak FBP plasma concentrations and more stable blood levels, and thus more significant pharmacological effects, can also reduce the FBP dose and reduce the toxicity of large amounts of inorganic phosphorus entering the systemic circulation after hydrolysis of large doses of FBP.
  • the FBP composite preparation overcomes the problem that the existing FBP preparation accelerates the metabolism of FBP in the body with the treatment time course, and thus the FBP composite preparation has significant advantages in treating various metabolic chronic diseases and metabolic-related chronic diseases.
  • stabilizers in the FBP complex formulation pair can improve metabolic status through a mechanism different from the mechanism of action of FBP, so both can produce synergistic pharmacokines mediated by different mechanisms of action.
  • the slow and controlled release preparations and the targeted nano preparations and the preparations of different content specifications are prepared by selecting suitable excipients, auxiliary ratios and preparation methods, thereby improving the clinical compliance of the pharmaceutical composition.
  • Figure 1 shows the regulation of fructose-1,6-diphosphate on the metabolism of human astrocytes in normal cells.
  • FBP fructose-1,6-diphosphate.
  • Figure 2 is a diagram showing fructose-1,6-diphosphate inhibiting glycolysis of glioma cells.
  • FBP fructose-1,6-diphosphate.
  • Figure 3 is a graph of fructose 1,6-diphosphate blocking the flow of glycolysis intermediates to biosynthesis.
  • FBP fructose 1,6-diphosphate
  • GAP glyceraldehyde 3-phosphate
  • PEP phosphoenolpyruvate
  • Pyr pyruvic acid
  • G6P glucose-6-phosphate
  • PGA triglyceride
  • La lactic acid
  • Ser serine
  • Gly glycine
  • R5P 5-phosphate ribose
  • ATP adenosine triphosphate
  • UTP uridine triphosphate
  • A adenosine
  • C cytidine
  • U uridine
  • T thymidine
  • the drug-administered group was compared with the control group by
  • Figure 4 is a graph showing the effect of fructose-1,6-diphosphate and its combination with metformin or sitagliptin on the level of fructose-1,6-diphosphosidase 1 protein.
  • FBP fructose-1,6-diphosphate
  • FBPase1 fructose-1,6-bisphosphatase 1
  • Met metformin
  • STG sitagliptin.
  • Figure 5 is a graph showing the effects of metformin, sitagliptin, and insulin on the peak concentration of fructose 1,6-diphosphate and stabilizing the blood concentration of FBP.
  • FBP fructose 1,6-diphosphate
  • Met metformin
  • STG sitagliptin
  • Ins insulin.
  • Experimental data were analyzed using the least significant difference method. ***P ⁇ 0.001, *P ⁇ 0.05 vs. 0 hours (before administration), #P ⁇ 0.05 vs. FBP single use group.
  • HA Human normal astrocytes
  • medium containing different concentrations of fructose-1,6-diphosphate trisodium salt (0 mM, 0.25 mM, 0.5 mM, 1 mM), and cultured at 12 h and 24 h, respectively.
  • HA Human normal astrocytes
  • PFKFB3 fructokinase 3
  • LDH5 lactate dehydrogenase
  • Cyto C cytochrome C
  • HA Human normal astrocytes
  • medium containing different concentrations of fructose-1,6-diphosphate trisodium salt (0 mM, 0.25 mM, 0.5 mM, 1 mM, 2 mM, 2.5 mM) for 36 h.
  • the ratio of intracellular reduced glutathione GSH level to fructose-1,6-diphosphate trisodium salt 1.6 mM group NADPH/NADP+.
  • the results showed that compared with the control group, the GSH in the treated group was significantly increased (the treatment group compared with the control group ***P ⁇ 0.001), and the proportion of NADPH / NADP + was significantly increased (the treatment group compared with the control group * **P ⁇ 0.001).
  • fructose-1,6-diphosphate trisodium salt can inhibit the glycolysis of human normal astrocyte HA, promote the tricarboxylic acid cycle and oxidative phosphorylation, and enhance its ability to resist oxidative stress. .
  • Astrocytes are the most abundant and versatile cell types in the brain, especially the astrocytes are extremely active in metabolic activities, their metabolic activities and multiple functions (including providing metabolic support for neurons and maintaining neurotransmitters). Dynamic equilibrium and redox dynamic equilibrium are closely related. Astrocyte metabolic disorders (including excessive glycolysis and decreased mitochondrial oxidative phosphorylation activity) are closely related to neurodegenerative and degenerative diseases. FBP inhibits excessive glycolysis of astrocytes from reducing lactic acid accumulation; FBP promotes oxidative phosphorylation and increases ATP levels; FBP increases endogenous antioxidants NADPH and glutathione (GSH) levels Improve the ability of antioxidant damage. It can be seen that the above findings strongly support the medical use of FBP for the prevention and treatment of neurodegenerative diseases.
  • the medium of fructose-1,6-diphosphate trisodium salt was cultured in LC-MS/MS for the determination of glucose-6-phosphate G6P and fructose-1,6-diphosphate FBP in each cell for 36 h.
  • the experimental results show that the tumor cells do not consume exogenous FBP, and some of the FBP entering the cell can undergo the first degradation reaction along the glycolytic pathway (generating is GAP and DHAP), and stop at this step, resulting in GAP. And the accumulation of DHAP.
  • elevated levels of intracellular F6P by exogenous FBP suggest that FBP may also be degraded by fructose-1,6-bisphosphatase (FBPase) in tumor cells.
  • G6P glucose-6-phosphate
  • FBP fructose-1,6-diphosphate
  • GAP glyceraldehyde 3-phosphate
  • DHAP dihydroxyacetone phosphate
  • PGA 3-phosphoglycerate.
  • Human glioma cell line (U87-MG, KNS-89, SHG-44) in a medium containing 0.8 mM and 1.6 mM fructose-1,6-diphosphate trisodium salt, 1.6 mM 2-deoxyglucose, respectively
  • the content of lactic acid released from the cells in the medium was determined at 12h, 24h, 36h and 48h.
  • the lactic acid level in the treatment group was significantly lower than that in the non-drug control group (CON). ***P ⁇ 0.001) (Schedule 2a-c).
  • Human glioma cell line (U87-MG) was cultured in medium containing 0.8 mM 1,6-diphosphate fructose for 1 h, 3 h, 6 h, 12 h, 24 h, 36 h, 48 h.
  • Western blotting was used to analyze cells at various time points. Changes in the levels of key metabolic enzymes in the glycolytic pathway indicate that hexokinase 2 (HK2), 6-phosphate fructokinase 2 (PFKFB3), pyruvate kinase 2 (PKM2), and lactate dehydrogenase 5 (LDH5) are both fast, Continued downward adjustment (Figure 2).
  • the experimental results show that fructose 1,6-diphosphate can inhibit the glycolysis of various glioma cells.
  • Example 4.1 fructose 6-diphosphate promotes mitochondrial oxidative phosphorylation in glioma cells
  • Rat glioma cell line (C6) and human glioma cell line (KNS-89, SHG-44) were cultured in medium containing 0.8 mM or 1.6 mM fructose-1,6-diphosphate trisodium salt, respectively.
  • the ATP/ADP ratio of each cell line was observed (the ratio of fructose in the 1,6-diphosphate to the control group was ***P ⁇ 0.001), and the ratio of NADH/NAD+ was significantly increased (1,6-diphosphate fructose group)
  • the control group had a significantly higher ATP level than ***P ⁇ 0.001) (the ratio of fructose 1,6-diphosphate to control group was ***P ⁇ 0.001) (Schedule 3a-b).
  • the experimental results show that fructose 1,6-diphosphate promotes mitochondrial oxidative phosphorylation in glioma cells.
  • Human glioma cell line (U87MG) was cultured in medium containing 13 C-labeled glucose (U- 13 C-Glc) and treated with 1.6 mM fructose-1,6-diphosphate trisodium salt for 36 h, using liquid-mass association
  • the intermediates of the intracellular glycolysis pathway, the pentose phosphate pathway, the "one carbon unit” metabolic pathway, and the de novo synthesis pathway of nucleic acids were determined by techniques (LC-MS/MS).
  • fructose-1,6-diphosphate trisodium salt can accumulate glycolytic intermediates in the glycolysis pathway, reducing its metabolism through the pentose phosphate pathway, serine biosynthesis and "one carbon unit" metabolism. Reduce de novo synthesis of nucleic acids.
  • F6P fructose 6-phosphate
  • FBP fructose-1,6-diphosphate
  • GAP glyceraldehyde 3-phosphate
  • PEP phosphoenolpyruvate
  • Lac lactic acid
  • ATP adenosine triphosphate
  • UTP uridine triphosphate
  • A adenosine
  • C cytidine
  • U uridine.
  • Human glioma cell line (U87MG) was cultured in 13 C-labeled glucose (U- 13 C-Glc) and treated with 1.6 mM 1,6-diphosphate fructose trisodium salt for 36 h using liquid chromatography-mass spectrometry (LC) - MS/MS) Determination of intracellular tricarboxylic acid cycle intermediates, amino acid-derived amino acid-derived amino acid and nucleotide de novo synthesis pathway intermediates. The results showed that: (1) The levels of ⁇ -ketoglutaric acid ( ⁇ -KG) and oxaloacetate (OAA) in the treatment group were significantly higher than those in the control group (compared with the control group).
  • ⁇ -ketoglutaric acid ⁇ -KG
  • OAA oxaloacetate
  • Fructose-1,6-diphosphate trisodium salt blocks the excretion of tricarboxylic acid intermediates into mitochondria, destroys epigenetic features of tumors, and extensively down-regulates protein levels of tumor metabolizing enzymes.
  • Human glioma cell line (U87MG) was isolated from medium containing 1.6 mM fructose-1,6-diphosphate trisodium salt for 36 h, and the cytoplasm and mitochondria were separated. LC-MS/MS The levels of the tricarboxylic acid cycle intermediates in the cytosol and mitochondria were determined separately. It can be seen that the levels of the carboxylic acid cycle intermediates acetyl-CoA, citrate (Cit), ⁇ -ketoglutarate ( ⁇ -KG) and oxaloacetate (OAA) in the treatment group were significantly reduced in the cytosol.
  • U87MG Human glioma cell line
  • Human glioma cell line (U87MG) was cultured for 0, 1, 3, 6, 12, 24, 36 and 48 hours in medium containing 1.6 mM fructose-1,6-diphosphate trisodium salt.
  • the method (WB) examines changes in the levels of enzyme proteins associated with fatty acid and nucleic acid biosynthetic pathways. It can be seen that the protein levels of the fatty acid and nucleic acid biosynthesis-related enzymes (CAD, TS, ACL, FASN) of the treatment group were significantly reduced with time (Fig. 3b).
  • the experimental results show that fructose 1,6-diphosphate can significantly down-regulate the metabolic enzymes of tumor cells.
  • Human glioma cell line (U87MG) was cultured in medium containing 1.6 mM fructose-1,6-diphosphate trisodium salt for 24 h and 36 h, respectively, and 5-hydroxymethylcytosine was examined by immunocytochemistry (5- At the level of hmC), the 5-hmC of the treatment group was significantly increased.
  • the epigenetic related proteins (Ac-Foxo, H3K9ac, H3K9me2) of the tumor cells were rapidly down-regulated.
  • Experimental results indicate that fructose 1,6-diphosphate can alter the epigenetic characteristics of tumor cells (Fig. 3c).
  • FBP fructose-1,6-diphosphate trisodium salt
  • Ac-CoA acetyl-CoA
  • Cit citric acid
  • ⁇ -KG ⁇ -ketoglutaric acid
  • OAA oxaloacetate
  • Cyto cytosol
  • Rat glioma cell line (C6), human glioma cell line (KNS-89) was cultured in a medium containing 0.8 mM fructose-1,6-diphosphate trisodium salt, and the cells were prolonged with treatment time.
  • the level of internal reactive oxygen species (ROS) is gradually increased (Schedule 7a), while the mitochondrial membrane potential (MMP) is gradually reduced (Schedule 7b).
  • Rat glioma cell line (C6) human glioma cell line (KNS-89, SHG-44) was cultured in a medium containing 1.6 mM fructose-1,6-diphosphate trisodium salt for 36 h, liquid- Mass spectrometry (LC-MS/MS) is used to determine important antioxidants in cells.
  • the results of the experiment showed that the level of glutathione (GSH, GSSG) decreased sharply (Schedule 7c), while the proportion of NADPH/NADP+ decreased drastically (Schedule 7d).
  • fructose-1,6-diphosphate trisodium salt increased the production of reactive oxygen species, inhibited the synthesis of antioxidant component glutathione, and blocked the transformation of NADP+ to NADPH, thereby destroying the oxidative reduction of glioma cells from multiple levels. balance.
  • FBP fructose-1,6-diphosphate trisodium salt
  • GSH reduced glutathione
  • GSSG oxidized glutathione
  • Tumor cells are reprogrammed by metabolism, in particular, can produce a large number of glycolysis intermediates and tricarboxylic acid cycle intermediates and use these intermediates for biosynthesis, thereby providing a prerequisite for rapid division and proliferation and growth of tumor cells.
  • acetyl-CoA, fumaric acid and succinic acid derived from the intermediate of the tricarboxylic acid cycle support the epigenetic features of the tumor, and thus participate in the regulation of the expression of the oncogenic protein and the down-regulation of the expression of the tumor suppressor protein.
  • FBP can reverse the characteristics of tumor metabolism, destroy the tumor metabolic network, and have significant anti-cancer activity in vitro and in vivo.
  • Example 9 Long-term treatment of fructose-1,6-diphosphate trisodium salt leads to increased stress level of fructose-1,6-bisphosphatase protein and decreased blood concentration of fructose-1,6-diphosphate, metformin and phosphoric acid Sitagliptin is able to combat this fructose-1,6-diphosphate metabolism change
  • 180-200 g SD rats were divided into 4 groups: saline control group, fructose-1,6-diphosphate trisodium hydrate group (500 mg/kg, ig), metformin group (150 mg/kg, ig) or west.
  • Gliptin group (20 mg/kg, ig), fructose-1,6-diphosphate combined with metformin or sitagliptin group, 5 rats in each group, all groups were treated with gavage, metformin or sitagliptin
  • fructose-1,6-diphosphate It was still as high as 99.23 ⁇ g/ml after 3 hours of treatment, compared with the control group **P ⁇ 0.01; compared with metformin group *P ⁇ 0.05 ( Table 4). Therefore, long-term treatment with fructose-1,6-diphosphate leads to a significant up-regulation of fructose-1,6-bisphosphatase 1 protein level, making fructose-1,6-diphosphate more susceptible to degradation in vivo, unable to maintain high and stable
  • the blood concentration affects the anti-tumor effect of fructose-1,6-diphosphate; metformin and sitagliptin do not affect the normal expression of FBPase1 in tissues, but can effectively fight against fructose-1,6-diphosphate.
  • Fructose-1,6-bisphosphatase 1 stress increases, which restores it to normal levels, which is conducive to the stability of FBP in vivo, thus exerting a stronger
  • Example 10 Stabilization of blood concentration of fructose-1,6-diphosphate by metformin, sitagliptin and insulin
  • mice Six-week-old ICR mice were divided into 4 groups: saline control group, fructose-1,6-diphosphate trisodium hydrate group (500 mg/kg, ig), fructose-1,6-diphosphate combined with metformin ( 150mg/kg, ig) group, fructose-1,6-diphosphate combined with sitagliptin (20mg/kg) group, fructose-1,6-diphosphate combined with insulin (4U/kg) group, each group 7 Only mice, all groups were treated with gavage, metformin, sitagliptin and insulin were treated in advance with fructose 1,6-diphosphate for 0.5 hours.
  • the plasma concentrations of 6-diphosphate were 82.5, 89.2, and 91.7 ⁇ g/ml, respectively, which were higher than the control group (compared with 1.5, 3 hours and 0 hours in the combined treatment group *P ⁇ 0.001), and compared with fructose-1,6 -
  • the diphosphoric acid single-use group the combined treatment group was greatly improved in peak concentration and blood drug concentration maintenance time (1.5 hours combined treatment group and fructose-1,6-diphosphate group comparison #P ⁇ 0.05, 3 hours combined with West Gliptin and insulin group were compared with FBP group #P ⁇ 0.05).
  • the above results indicate that FBP combined with metformin, insulin or sitagliptin can effectively increase the peak concentration of FBP and stabilize the maintenance time of FBP in vivo, which can effectively improve the anti-tumor effect of FBP in vivo.
  • FBPase1 1,6-diphosphate fructase 1
  • the present invention further explores the stabilizing effects of hypoglycemic agents including metformin, sitagliptin and insulin on the blood concentration of FBP, and attempts to find a defect that overcomes the rapid degradation of FBP in vivo and is not conducive to its anticancer effect.
  • hypoglycemic agents can increase and maintain the blood concentration of FBP, and prevent the acceleration of FBP metabolism with prolonged treatment time.
  • different hypoglycemic agents including metformin, sitagliptin, and insulin, have different mechanisms of action, but all have the effect of inhibiting gluconeogenesis. Therefore, based on the key role of the gluconeogenesis enzyme 1,6-diphosphate fructase (FBPase1) in the degradation of exogenous FBP, the above results indicate that the fructose-1,6-bisphosphatase inhibitor and the existing different mechanisms of action decline.
  • Sugar drugs and new hypoglycemic agents that continue to appear in the future can inhibit the degradation of exogenous FBP in the body, thereby increasing the medicinal value of exogenous FBP.
  • Example 11 Metformin and sitagliptin do not significantly affect the effect of fructose-1,6-diphosphate trisodium salt on human intestinal cancer cells in vitro
  • the human intestinal cancer cell lines SW620 and HCT-8 cultured for 24 hours contained 0.8 mM fructose-1,6-diphosphate trisodium salt, 0.2 mM metformin/100 ⁇ M sitagliptin or 0.8 mM fructose-1,6, respectively.
  • the medium was further cultured for 72 hours in a medium of trisodium diphosphate and 0.2 mM metformin/100 ⁇ M sitagliptin.
  • there is no drug treatment group also called control group, Con.
  • Cell viability was determined by Sulforhodamine B (SRB) staining assay.
  • fructose-1,6-diphosphate trisodium salt Compared with the control group, ***P ⁇ 0.001; the combined treatment group compared with the control group ***P ⁇ 0.001; the combined treatment group had no significant difference compared with the fructose-1,6-diphosphate trisodium salt group) (Table 5) .
  • the results showed that fructose-1,6-diphosphate trisodium salt combined with metformin or sitagliptin did not antagonize the anti-intestinal effect of fructose-1,6-diphosphate trisodium salt in vitro. Produce significant synergy.
  • Example 12 Metformin and sitagliptin do not significantly affect the effect of fructose-1,6-diphosphate trisodium salt on human hepatoma cells in vitro
  • the human hepatoma cell line Bel-7402 and huh-7 cultured for 24 hours contained 1.6 or 0.8 mM fructose-1,6-diphosphate trisodium salt, 0.2 mM metformin or 25 ⁇ M sitagliptin or 0.8 mM fructose, respectively.
  • the medium was neutralized with trisodium 1,6-diphosphate and 0.2 mM metformin/25 ⁇ M sitagliptin for 72 hours.
  • there is no drug treatment group also called control group, Con.
  • Cell viability was determined by Sulforhodamine B (SRB) staining assay.
  • fructose-1,6-diphosphate trisodium salt group compared with the control group ***P ⁇ 0.001;
  • the combined treatment group compared with the control group ***P ⁇ 0.001; the combined treatment group had no significant difference compared with the fructose-1,6-diphosphate trisodium salt group) (Table 6).
  • the experimental results show that fructose-1,6-diphosphate trisodium salt combined with metformin or sitagliptin does not antagonize the anti-hepatocarcin effect of fructose-1,6-diphosphate trisodium salt in vitro. Produce significant synergy.
  • the mouse melanoma B16 cells cultured for 24 hours contained 0.8 mM fructose-1,6-diphosphate trisodium salt, 20 ⁇ M sitagliptin or 0.8 mM fructose-1,6-diphosphate trisodium salt and 20 ⁇ M West.
  • the gliclastatin was cultured for an additional 72 hours.
  • there is no drug treatment group also called control group, Con).
  • Cell viability was determined by Sulforhodamine B (SRB) staining assay.
  • fructose-1,6-diphosphate trisodium salt inhibited B16 cell viability at 22 mM at 22 mM, and sitagliptin at 16 ⁇ M inhibited B16 cell viability by 16%.
  • Effect on the efficacy of fructose-1,6-diphosphate trisodium salt (sitagliptin group compared with control group ***P ⁇ 0.001; fructose-1,6-diphosphate trisodium salt group compared with control group ***P ⁇ 0.001; the combined treatment group compared with the control group ***P ⁇ 0.001; there was no significant difference between the combined treatment group and the fructose-1,6-diphosphate trisodium salt group) (Table 7).
  • fructose-1,6-diphosphate trisodium salt combined with sitagliptin will not antagonize the anti-melanoma effect of fructose-1,6-diphosphate trisodium salt in vitro, nor Produce significant synergy.
  • Mouse hepatoma cells H22 were inoculated into the right flank of adult male ICR mice according to a conventional method. After 24 hours of inoculation, they were randomly divided into the following experimental groups: saline control group, fructose-1,6-diphosphate trisodium salt.
  • FBP Sodium group
  • FBP metformin
  • F+M pharmaceutical composition
  • metformin or sitagliptin was treated with fructose-1,6-diphosphate trisodium salt for 0.5 hours for 7 consecutive days, and the animals were observed during the experiment. The animals were sacrificed 24 hours after the last treatment. Tumor masses were weighed and the average tumor weight of each group of animals was used as an indicator of efficacy.
  • the inhibition rate of tumor growth by fructose-1,6-diphosphate trisodium salt was 54.39% (fructose-1,6-diphosphate trisodium salt group compared with the control group ***P ⁇ 0.001
  • the inhibitory rate of tumor growth inhibition was 46.12% (the sitagliptin group compared with the control group ***P ⁇ 0.001).
  • the anti-tumor effect was not shown in the experiment, and the overall experiment showed a certain anti-tumor effect, indicating that sitagliptin may exert anti-tumor effect by stimulating the body immunity; after FBP is combined with metformin or sitagliptin, The overall anti-tumor effect was greatly improved, and the inhibition rates reached 74.2% and 75.3%, respectively (compared with the control group and ***P ⁇ 0.001); metformin combined with fructose-1,6-diphosphate trisodium salt and metformin group ###P ⁇ 0.001, compared with fructose-1,6-diphosphate trisodium salt group#P ⁇ 0.05; sitagliptin combined with fructose-1,6-diphosphate trisodium salt group and sitagliptin group, Fructose-1,6-diphosphate trisodium salt group comparison #P ⁇ 0.05).
  • Group Con Met FBP Met+FBP Tumor weight (g) 1.66 ⁇ 0.29 1.46 ⁇ 0.23 0.91 ⁇ 0.23 *** 0.43 ⁇ 0.20 ***;###;& Group Con STG FBP STG+FBP Tumor weight (g) 1.48 ⁇ 0.34 0.78 ⁇ 0.31 ** 0.67 ⁇ 0.28 *** 0.38 ⁇ 0.14 ***;#;&
  • the present inventors have found that the combination of FBP in the treatment of diabetic doses of metformin, sitagliptin or insulin can increase and stabilize the blood concentration of FBP, thereby significantly increasing the overall anticancer efficacy of FBP.
  • metformin exceeding the hypoglycemic dose has certain anticancer activity, this high dose can not improve the anticancer effect of FBP, indicating that metformin enhances the anticancer effect of FBP, which is to improve and stabilize the blood concentration of FBP.
  • the result of direct anticancer efficacy rather than metformin.
  • Sitagliptin showed no obvious anti-cancer activity in the same concentration of FBP, but in the mouse liver cancer H22 model, its clinical hypoglycemic dose showed certain anti-cancer activity.
  • This overall anticancer activity may be the result of sitagliptin regulating overall glucose metabolism. Therefore, the results support the use of FBP in combination with hypoglycemic agents, especially sitagliptin, in the preparation of novel anticancer drugs.
  • mice Forty-nine 17-19 g ICR male mice were first divided into two groups. Eight normal animals were given basic diet and drinking pure water; the other 32 obese animals were fed with high fat diet (high fat diet containing 45% of basic materials including crude protein 18%, fat 4%, fiber 8%, calcium 1.5%, Amino acid 8%, and 55% additives include refined lard 13%, soybean oil 3%, sugar 8% and peanuts, soybeans, eggs, bone meal, sesame, corn, buckwheat, salt, multivitamins) After 4 weeks of feeding, obese animals began to be grouped after 8% of normal animals. The normal group (Naive) animals were still fed with the basic diet. The model animals were divided into 4 groups on average, and the high-fat diet was continued.
  • high fat diet high fat diet containing 45% of basic materials including crude protein 18%, fat 4%, fiber 8%, calcium 1.5%, Amino acid 8%, and 55% additives include refined lard 13%, soybean oil 3%, sugar 8% and peanuts,
  • FBP can significantly counter the elevation of Lee'S INDEX caused by high-quality feed, and sitagliptin can not further enhance the efficacy of FBP.
  • the Lee's index of the model group was 353.28 ⁇ 9.64, which was significantly different from the normal group of 337.05 ⁇ 9.96 (**P ⁇ 0.01), indicating that the model group mice were obese.
  • the Lee's index of the sitagliptin single-use group was 346.34 ⁇ 9.36, which was not significantly different from the model group.
  • FBP can significantly increase the fat coefficient caused by high-fat diet feeding.
  • Sitagliptin has no such effect, but FBP is more effective when combined with sitagliptin.
  • the fat coefficient of obese mice in the model group was 3.96 ⁇ 0.83%, which was significantly higher than that in the normal group (1.58 ⁇ 0.68%) (compared with the normal group ***P ⁇ 0.001).
  • the fat coefficient of the animals in the treatment group decreased, and the sitagliptin single-use group was 3.28 ⁇ 1.14%, which did not form a significant difference with the model group.
  • fructose-1,6-diphosphate trisodium salt group (2.81 ⁇ 0.81%) and sitagliptin combined with fructose-1,6-diphosphate trisodium salt group (2.50 ⁇ 0.98%) were associated with the model group.
  • Significant difference (fructose-1,6-diphosphate trisodium salt group compared with model group *P ⁇ 0.05, sitagliptin combined with fructose-1,6-diphosphate trisodium salt group compared with model group**P ⁇ 0.01 ), wherein the combined group has the best anti-obesity effect.
  • mice had an adaptive feeding to the high-fat diet.
  • the initial intake was slightly lower than that of the normal feed.
  • the high-fat diet group had a feed intake of 6.68 g/day/day, and the normal group was 6.71 g.
  • the food intake in the treatment group was slightly lower than that in the model group, which was 5.99 g/day/day for the sitagliptin group and 6.46 g/fructose for the fructose-1,6-diphosphate trisodium salt group.
  • sitagliptin combined with fructose-1,6-diphosphate trisodium salt group 6.37 / day / day, but did not significantly affect the appetite of the mice.
  • the blood glucose level at the 15th minute was normal group (12.91 ⁇ 2.57mmol/L), model group (13.26 ⁇ 3.63mmol/L), and sitagliptin group (13.74). ⁇ 4.27mmol/L), fructose-1,6-diphosphate trisodium salt group (14.28 ⁇ 2.23mmol/L), sitagliptin combined with fructose-1,6-diphosphate trisodium salt group (13.72 ⁇ 3.83mmol) /L), at the 30th minute, the blood glucose of each group began to fall, and at 120 minutes, the blood glucose of each group returned to the initial level. It can be seen that FBP and sitagliptin and their combination do not affect normal blood glucose levels and glucose tolerance.
  • FBP can promote fat metabolism
  • sitagliptin can further enhance the efficacy of FBP in promoting fat metabolism, which supports the medical use of FBP combined with sitagliptin for weight loss and prevention and treatment of type 2 diabetes.
  • Obesity not only directly affects the quality of life, but also indicates metabolic disorders and subsequent diabetes. Therefore, preventing obesity is an important measure to prevent diabetes.
  • FBP and sitagliptin have a significant antagonistic effect.
  • FBP alone can reduce the degree of obesity but not lose weight, while sitagliptin alone has no significant effect. This strongly supports the use of FBP in combination with sitagliptin in the preparation of diet pills and drugs for the prevention of fatty liver and type 2 diabetes.
  • Example 16 Fructose-1,6-diphosphate trisodium for the treatment of tumor chemotherapy drug paclitaxel-induced peripheral neuralgia
  • paclitaxel (2.8 mg/kg, 10 ml/kg) was administered intraperitoneally four times a day (days 1, 3, 5, 7) to induce a model of peripheral neuropathic pain in ICR female mice weighing 20-24 g.
  • the model was used to observe the preventive effect of fructose-1,6-diphosphate on peripheral nerve pain caused by chemotherapy drugs.
  • Paclitaxel antineoplastic drugs have become the first line of drugs for humans to fight malignant tumors.
  • the dose-limiting toxicity of paclitaxel is mainly neurotoxicity and myelosuppression.
  • paclitaxel-induced peripheral nerve pain is representative of pain after cancer treatment, and an animal model of pain caused by paclitaxel violet is also representative.
  • the hot plate method was used to screen mice with relatively homogeneous heat sensitive reactions for experiments.
  • 21 eligible mice were divided into blank control group (normal saline group, ip), paclitaxel model group and fructose-1,6-diphosphate trisodium salt hydrate (400 mg/kg, 10 ml/kg, ig) for prophylaxis Group, 7 in each group.
  • Fructose-1,6-diphosphate was treated once a day, and the fructose-1,6-diphosphate was pretreated for 2 hours each time. Continue to give fructose-1,6-diphosphate trisodium after paclitaxel withdrawal until the end of the experiment.
  • thermosensitive reaction of the hind paw of the mouse was measured by hot plate test (52 ° C ⁇ 0.3) at 2-4 pm each time. Place the bilateral hind paws of the mouse on the hot plate of the hot plate. When the animal feels the pain caused by the heat stimulation, the animal will have a hind paw or retract and grasp, record the hind paw or retract and grasp the incubation period. The shorter the incubation period, the lower the pain threshold, and the prolongation of the pain threshold for paclitaxel animals indicates an antagonistic effect on the neuropathic pain induced by the chemotherapeutic drug.
  • the fructose-1,6-diphosphate was continued after the end of paclitaxel injection and the thermosensitive response of each group of animals was continued.
  • the experimental results show that fructose-1,6-diphosphate has a significant inhibitory effect on paclitaxel-induced peripheral nerve pain in mice.
  • the hind paw withdrawal latency of the three groups of animals was comparable, and on the 7th, 9th, 11th, and 13th days after paclitaxel administration, the hind paw withdrawal latency of the model group.
  • Significantly shorter than the blank control group indicating that paclitaxel induced significant peripheral nerve pain (**P ⁇ 0.01, ***P ⁇ 0.001); on the 7th day and subsequent time points of the fructose-1,6-diphosphate group
  • the incubation period was significantly longer than the model group (**P ⁇ 0.01, ***P ⁇ 0.001).
  • the above experimental results support the clinical use value of fructose-1,6-diphosphate in the prevention and treatment of pain after cancer treatment, especially chemotherapy-induced peripheral nerve pain.
  • Peripheral nerve pain and the commonly used chemotherapy drug paclitaxel are widely used in many malignant tumors such as ovarian cancer, breast cancer, lung cancer and head and neck cancer, but the toxicity and side effects are relatively large, and neurotoxicity is the main dose-limiting toxicity. Consistent with clinical neurotoxic side effects, the anticancer dose of paclitaxel can rapidly induce a model of peripheral nerve pain in mice; an appropriate dose of fructose-1,6-diphosphate and paclitaxel can significantly counteract paclitaxel-induced peripheral nerve pain in mice.
  • low-dose FBP 200 mg/kg
  • high-dose FBP 400 mg/kg
  • the results not only support the potential of FBP in the prevention and treatment of neurotoxic side effects of chemotherapeutic drugs, especially in combination with commonly used chemotherapeutic drugs (including taxanes, vinblastines, platinum and proteasome inhibitors), and further indicate that they cannot pass.
  • the novel FBP drug of the present invention is characterized in that its medicinal ingredients include fructose-1,6-diphosphate (FBP) and one or more components which can slow down the degradation of FBP in vivo (also called FBP blood concentration stable) Agent).
  • FBP fructose-1,6-diphosphate
  • the stabilizer is used as a medicinal ingredient, and its technical characteristics mainly achieve the drug effect by increasing and stabilizing the blood concentration of FBP, but it does not exclude the pharmacodynamic effect produced by the stabilizer itself on the metabolic regulation and the pharmacodynamic effect produced by FBP. Plus or synergy.
  • the novel FBP drug of the present invention is applicable to the prevention of obesity and chemotherapeutic drugs for treating peripheral nerve pain caused by cancer, and is also applicable to the disclosed various indications of FBP including for improving coronary heart disease.
  • Analgesia, acute myocardial infarction, arrhythmia and myocardial ischemia of heart failure and adjuvant treatment of viral myocarditis, used to improve cerebral hypoxia caused by cerebral infarction, cerebral hemorrhage, etc., for prevention and treatment of hematological cancer and various entities Cancer (Chinese invention patent: ZL201110066413.6) for the prevention and treatment of diabetic complications (Chinese invention patent: CN00112023.9), for epilepsy (Chinese invention patent: ZL201310498212.2) and neurodegenerative diseases (Chinese invention patent: CN01107519 .8).
  • the pharmacodynamic essence of the novel FBP drug provided by the present invention is to regulate metabolism to improve metabolic function and correct abnormal metabolic state, so professionals can understand other metabolic diseases not involved here and diseases closely related to metabolic disorders (such as Mental disorders - schizophrenia, depression, etc.) are also included in the indications of the novel FBP drugs described. In the same way, those skilled in the art should understand that the idea of improving and stabilizing the blood concentration of FBP by any means to improve the efficacy of FBP is within the scope of this patent.
  • the A layer and the B layer were separately mixed with the active ingredient, the filler and the binder, and wet granulated by a wet granulator (I agitating II shearing, 5 minutes), and dried at 60 ° C in a box drying oven.
  • the two-layer tablet press was pressed to obtain a fructose-1,6-diphosphate trisodium salt-sitagliptin bilayer tablet.
  • the obtained double-layer sheet was complete, smooth and friable, and the friability was ⁇ 0.9%. There was no significant difference in tablet weight, and the disintegration time limit was ⁇ 7 minutes.
  • the obtained two-layer tablets were of a specification of 0.5 g/tablet, each containing 0.125 g of fructose-1,6-diphosphate trisodium, and clinically used a single oral administration of 20 tablets three times a day.
  • the fructose-spinning mechanism produces fructose-1,6-diphosphate trisodium salt pellets.
  • the obtained pellets were dried at 50 ° C for 6 hours, and the obtained pellets of 18-24 mesh sieves were used.
  • Sustained-release pellet coating A coating solution was prepared by using Eudragit Ne30d (polymer concentration 5%), talc powder (corresponding to 60% of the polymer amount) and deionized water, and then coated with a fluidized bed.
  • Preparation of compound sustained-release pellets accurately weigh a certain amount of sitagliptin, dissolved in deionized water, and spray the sitagliptin aqueous solution to the fructose-1,6-diphosphate trisodium salt solution using a fluidized bed device. The surface of the pellet was released, and the compound pellets were prepared.
  • the pharmaceutical forms of the FBP include fructose-1,6-diphosphate proto form and fructose-1,6-diphosphate and prodrugs or derivatives thereof in pharmaceutically acceptable salts including but not Limited to ammonium, sodium, potassium, calcium, magnesium, manganese, copper, methylamine, dimethylamine, trimethylamine, butyric acid, acetic acid, dichloroacetic acid, hydrochloric acid, hydrobromic acid, sulfuric acid, trifluoroacetic acid, a salt and a hydrate of citric acid or the acid radical of maleic acid.
  • the FBP stabilizer includes DPP-4 inhibitors represented by sitagliptin, GLP-1 receptor agonists, biguanides represented by metformin, insulins and glitazones.
  • DPP-4 inhibitors represented by sitagliptin
  • GLP-1 receptor agonists represented by metformin
  • biguanides represented by metformin
  • insulins and glitazones Existing hypoglycemic substances of the class and fructose-1,6-bisphosphatase inhibitors, which may be in a pharmaceutically acceptable form, in the form of an existing pharmaceutical form, a protoplast, and a respective prodrug or derivative thereof.
  • Salts include, but are not limited to, ammonium, sodium, potassium, calcium, magnesium, manganese, copper, methylamine, dimethylamine, trimethylamine, butyric acid, acetic acid, dichloroacetic acid, hydrochloric acid, hydrobromic acid, phosphoric acid, a salt and a hydrate of sulfuric acid, trifluoroacetic acid, citric acid or an acid radical of maleic acid.
  • FBP may constitute a medicinal ingredient of the drug in an appropriate ratio with one or more of the above FBP blood concentration stabilizers, each of which is prepared with a pharmaceutically acceptable excipient or carrier.
  • Common pharmaceutical preparations including oral and injection-treated preparations), suppositories, films and various new preparations prepared by applying new materials and new technologies (including but not limited to controlled-release bilayer tablets, controlled-release nano-preparations, microcapsules) , microspheres, enteric preparations and various long-acting preparations).
  • the novel FBP drug is prepared as a bilayer tablet that achieves sequential release or a long-acting sustained release formulation with sustained release characteristics.
  • the two-layer sheet which can be successively released is technically characterized in that the stabilizer is first released for 15 minutes to 60 minutes, preferably for 30 minutes. Since one of the stabilizers is combined with FBP to achieve the purpose of stabilizing the blood concentration of FBP, in practical application, FBP is preferentially combined with a stabilizer in the preparation of a novel FBP drug, and preferably with West. Glettine combination.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Epidemiology (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Diabetes (AREA)
  • Hematology (AREA)
  • Obesity (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Endocrinology (AREA)
  • Emergency Medicine (AREA)
  • Cardiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Child & Adolescent Psychology (AREA)
  • Psychiatry (AREA)
  • Hospice & Palliative Care (AREA)
  • Vascular Medicine (AREA)
  • Urology & Nephrology (AREA)
  • Zoology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Oncology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

果糖-1,6-二磷酸（FBP）与其血药浓度稳定剂组合物在制备预防和治疗代谢性疾病和代谢功能失常相关性疾病的药物中的应用。所述果-1,6-二磷酸还可以为其原形、其前药或其衍生物的药学上可接受的盐或水合物。所述血药浓度稳定剂是指能减缓药物制剂中FBP在体内快速降解的治疗糖尿病药物或物质。所述组合物能产生更高的FBP血药浓度峰值和更稳定的血药浓度，可降低FBP剂量和减少大剂量FBP水解后造成大量无机磷进入体循环所致毒性。

Description

果糖-1,6-二磷酸与其血药浓度稳定剂组合物的医药用途 技术领域
本发明属于制药领域，涉及果糖-1,6-二磷酸(又名果糖-1,6-二磷酸和果糖二磷酸)与其血药浓度稳定剂组合物在制备防治代谢性疾病和代谢紊乱相关疾病的药物中的应用,所述疾病包括肿瘤、脂肪肝，糖尿病、高脂血症、心血管疾病、外周神经性疾病和中枢疾病。
背景技术
果糖-1,6-二磷酸(fructose-1,6-bisphosphate,FBP)，是存在于体内的糖代谢中间产物，外源性FBP通过调节糖代谢中若干酶的活性产生药理作用(果糖二磷酸钠片说明书，国家药品监督管理局2002年公布的第二批化学药品说明书)。外源性FBP可提高细胞内三磷酸腺苷和磷酸肌酸的浓度、促进钾离子内流、增加红细胞内二磷酸甘油酸的含量、抑制氧自由基和组织胺释放等多种药理作用，能减轻机体因缺血、缺氧造成的损害，尤其是对缺血性心脏病更显出良好的保护作用。我国已有多种FBP制剂用于临床用于休克、冠状粥样硬化性心脏病(冠心病)、心绞痛、急性心肌梗死、心力衰竭及心律失常等辅助治疗(注射用果糖二磷酸钠说明书，果糖二磷酸钠片说明书，国家药品监督管理局2002年公布的第二批化学药品说明书)。FBP具有治疗糖尿病或糖尿病性及合并的心脑血管疾病的作用(中国发明专利：CN00112023.9)。申请人也公开了FBP抗癫痫的作用(中国发明专利：ZL201310498212.2)和抗肿瘤作用(中国发明专利：ZL201110066413.6)。特别是，FBP的抗癫痫药效显著优于现有抗癫痫药物，突出表现为在控制癫痫发作用的同时可以修复癫痫脑的功能，显著提高癫痫动物的认知能力和产生停药后可持续的抗癫痫作用，具有广谱而显著的抗癌作用同时对正常细胞高度安全。可见，FBP具有巨大潜在药用价值。
然而，外源性FBP在体内的代谢特点限制了现有FBP制剂的药用价值。现有果糖-1,6-二磷酸制剂使用剂量大(推荐口服制剂：每次1g，一天3-4次；静脉处理：每日10g,分2次静脉输注)。有研究证明，目前推荐口服剂量未能显著提高血液中FBP水平，因此建议提高临床使用剂量(Acta Pharm.65(2015)147–157)。外源性FBP在体内被快速降解。给健康志愿者静脉输注本品(250mg/kg)，5分钟内其血浓度可达770mg/L，半衰期约10～15分钟，经过水解形成无机磷及果糖从血浆中消除，极少部分从尿中排除(注射用果糖二磷酸钠说明书，国家药品监督管理局2002年公布的第二批化学药品说明书)。申请人公开的研究进一步证明，随着处理时程的延长，FBP的血药浓度逐步降低；而且剂量越大，这种现象越严重(中国发明专利：ZL201310498212.2)。与此一致的是，FBP抗癫痫作用和抗癌作用的剂量范围小，对大鼠慢性癫痫长期有效剂量灌胃100-200mg/kg/天,对小鼠肿瘤模型有效剂量腹腔注射400-450mg/kg/次。可见，无法通过提高FBP的剂量来提高其血药浓度，这就限制了FBP的医药用途。因此，阐明外源性FBP在体内的代谢机制并寻找稳定外源性FBP血药浓度的方法和物质对发挥FBP的广泛医药用途具有十分重要的意义。
现有对FBP作用机制的认识远不能解释FBP具有如此广泛的药理学活性。是否有一种共同的生物活性支持着FBP广泛的药理活性，从而可产生广泛的临床治疗作用？越来越多的研究证明，代谢功能紊乱或失调是多种疾病的共同病理机制。这些疾病包括多种重大疾病，如 糖尿病及其并发症、心血管疾病、神经功能紊乱性疾病(癫痫、精神分裂症、抑郁症等)和神经退化性疾病(老年性痴呆、血管性痴呆、帕金森氏病、多发性硬化等)和肿瘤等。其中老年性痴呆现也被称为3型糖尿病(Biochem Pharmacol.2014Apr 15；88(4):548-59.Eur Neuropsychopharmacol.2014Dec；24(12):1954-60.Neurol Sci.2015Oct；36(10):1763-9.)，而肿瘤在上世纪60年代就被德国生物化学家也是诺贝尔奖获得者Otto Warburg称为是代谢性疾病。线粒体功能障碍或失常是上述疾病的一种共同代谢特征。比如，线粒体功能障碍发生在不同原因引起的各种神经疼痛中，包括化疗诱导的神经病变、糖尿病神经病变和外伤性神经病变等(Mol Pain.2015；11:58.)，而且线粒粒功能障碍诱发的恶性连锁反应包括氧化磷酸化功能减弱引起的能量物质(ATP)不足、氧自由基(ROS)产生增加和清除减少引起的氧化应激损伤以及炎症反应等是抗癌药和其它原因诱发的神经痛的共同病理机制(Pain.2013Nov；154(11):2432-40.Neurosci Lett.2015Jun 2；596:90-107.Curr Neuropharmacol.2016；14(6):593-609.)，也是上述其它疾病的共同病理事件(Nature.2006Oct 19；443(7113):787-95；Neurobiol Dis.2013Mar；51:27-34；Biomed Pharmacother.2015 Aug；74:101-10.Biochim Biophys Acta.2017May；1863(5):1037-1045；Biochim Biophys Acta.2017 May；1863(5):1132-1146)。特别是，近十多年来的研究结果揭示了更多的肿瘤代谢特点，确定了肿瘤代谢重编程是癌症的核心特征，与肿瘤发生和发展以及癌症治疗耐药密切相关。通过代谢重编程，癌细胞可以使用普通营养物质特别是利用葡萄糖和谷氨酰胺可同时满足能量需求、氧化还原平衡和高度活跃的生物合成，从而保证了肿瘤细胞快速分裂和永生的前提条件。肿瘤表观遗传学异常与抗癌基因静止和促癌基因过度表达密切相关，新近研究证明肿瘤特征性代谢还维持着肿瘤的表观遗传学特征。可见，调控代谢和/或逆转病理代谢模式回到正常代谢模式对防治以上代谢性疾病和代谢相关性疾病可能具有广泛的应用前景，果糖-1,6-二磷酸作为一种糖分解代谢和糖异生的中间产物可能具有广泛的代谢调控作用和/或逆转病理代谢模式回到正常代谢模式的作用，从而对防治代谢疾病和代谢相关疾病可能具有广泛的医药用途。显然，系统揭示外源性果糖-1，6-二磷酸(FBP)对细胞代谢包括肿瘤细胞代谢的调控作用及其作用机制有利于更好地利用其药用价值。
发明内容
本发明的目的是提供一种果糖-1,6-二磷酸与其血药浓度稳定剂组合物的医药用途，是一种由果糖-1,6-二磷酸与血药浓度稳定剂组成的组合物在制备预防和治疗代谢性疾病和代谢功能失常相关性疾病的药物中的应用，是一种由果糖-1,6-二磷酸(FBP)与能稳定FBP血药浓度的物质(统称FBP血药浓度稳定剂)组成的组合物在制备预防和治疗代谢性疾病和代谢功能失常相关性疾病的药物中的应用。所述药物包含治疗有效量的果糖-1,6-二磷酸和有效剂量的血药浓度稳定剂以及药学上可以接受的赋形剂或者载体。所述药物中FBP与稳定剂的比例的确定以稳定剂可发挥其稳定FBP的血药浓度为准，因此不同稳定剂与FBP的比例可以不同；所述药物在预防和治疗代谢性疾病和代谢功能失常相关性疾病时，其有效剂量是依具体的疾病而定，具体地说治疗肿瘤FBP的剂量要高于治疗其它所述疾病的1至5倍。
所述FBP的药用形式包括果糖-1,6-二磷酸原形和果糖-1,6-二磷酸及其前药或衍生物在药 学上可接受的盐包括但不限于化合物所形成的铵、钠、钾、钙、镁、锰、铜、甲胺、二甲胺、三甲胺、丁酸、乙酸、二氯乙酸、盐酸、氢溴酸、硫酸、三氟乙酸、柠檬酸或者马来酸的酸根所成的盐及水合物。优选地，以果糖-1,6-二磷酸三钠盐的8分子水合物为药用形式。
所述的血药浓度稳定剂是指能减缓所述药物制剂中果糖-1,6-二磷酸在体内快速降解的治疗糖尿病药物或物质，包括以西格列汀为代表的二肽基肽酶-4(DPP-4)抑制剂类、胰高血糖素样肽1(GLP-1)受体激动剂类、以二甲双胍为代表的双胍类、胰岛素类和格列酮类也称为噻唑烷二酮类和果糖-1,6-二磷酸酶抑制剂。果糖-1,6-二磷酸与所述任何一种稳定剂构成组合物时果糖-1,6-二磷酸与稳定剂的比例为：果糖-1,6-二磷酸三钠盐的8分子水合物(克)与二甲双胍(克)的比例为1:0.1～1:1，优选比例为1:0.2～1:1；果糖-1,6-二磷酸三钠盐的8分子水合物(克)与西格列汀(克)的比例为1:0.001～1:0.5，优选比例为1:0.01～1:0.1；果糖-1,6-二磷酸三钠盐的8分子水合物(克)与胰岛素(单位，IU)的比例为1：0.02～1：0.002，优选比例为1:0.006～1:0.008。
所述代谢性疾病和代谢功能失常相关性疾病具体包括：现有果糖-1,6-二磷酸制剂的适应症(主要有：用于改善冠心病的心绞痛、急性心肌梗死、心律失常和心力衰竭的心肌缺血以及病毒性心肌炎的辅助治疗)，脑梗死，脑出血等引起的脑缺氧症状，血液系统癌症和各种实体瘤，糖尿病及其并发症，脂肪肝，癫痫、神经退化性疾病(包括老年性痴呆、帕金森氏病、多发性硬化)和精神行为障碍性疾病。
本发明揭示了外源性果糖-1,6-二磷酸(FBP)对代谢活动的广泛调节作用，特别是对正常细胞的保护性调节作用以及逆转肿瘤代谢特征，从而为FBP既可保护正常细胞及其功能又能杀灭各种不同的癌细胞提供了科学依据，支持了FBP防治各种代谢性疾病和代谢紊乱或失常相关性疾病的医药用途，所属疾病包括糖尿病及其并发症、心血管疾病、神经功能紊乱性疾病(癫痫、精神分裂症、抑郁症等)和神经退化性疾病(老年性痴呆、血管性痴呆、帕金森氏病、多发性硬化等)和肿瘤。更重要的是，本发明揭示了外源性FBP被快速降解的机制并发现了一组可减缓FBP体内降解速度从而提高FBP血药浓度峰值和延长其半衰期的物质(后统称为FBP血药浓度稳定剂)，并显著增强FBP药效，这就支持了FBP与其血药浓度稳定剂联合即FBP与其血药浓度稳定剂的组合物在制备以FBP为药效活性成分的新型FBP药物的医药用途。总之，本发明不仅提供了一组可以避免FBP在体内被快速降解的新型FBP药物，而且扩大了FBP的药用范围。
所述新型FBP药物包括FBP和抑制FBP在体内被快速降解的成分即FBP血药浓度稳定剂，所述FBP血药浓度稳定剂可减慢FBP在体内急性降解并阻断随处理时程延长出现的FBP降解不断加速的现象，从而使这种组合物的药物制剂可产生更高FBP血药浓度峰值(无论是一次性处理还是多次处理后)并延长其半衰期。因此，这种组合物制剂不仅可提高FBP的各种药效，而且扩大了FBP的剂量范围，还可减轻FBP快速降解导致的磷酸水平迅速升高引起的现有FBP制剂磷酸中毒现象。特别是，这种FBP组合物制剂长期用药有效，因此可充分发挥FBP治疗慢性疾病(包括肿瘤、癫痫、糖尿病和神经退化性疾病)的药用价值。综上所述， 所述FBP血药浓度稳定剂克服了FBP作为代谢中间产物自身无法克服的在体内被快速降解的缺陷，从而突破性地提高了FBP的药用价值。
所述FBP血药浓度稳定剂是指凡能减缓外源性FBP体内降解的活性物质，包括现有降血糖药物和今后不断研发出的降血糖药物以及新发现或定向合成的可间接或直接抑制果糖-1,6-二磷酸酶(FBPase)物质。所述现有降糖药物包括以西格列汀为代表的二肽基肽酶-4(DPP-4)抑制剂类、胰高血糖素样肽1(GLP-1)受体激动剂类、以二甲双胍为代表的双胍类、胰岛素类、格列酮类(也称为噻唑烷二酮类)和果糖-1,6-二磷酸酶抑制剂(如果糖-2,6-二磷酸)。在实际应用中，FBP可与上述FBP血药浓度稳定剂中的一种或多种按合适比例构成药物的药效成分。
外源性果糖-1,6-二磷酸(FBP)进入体内后可能广泛调控细胞代谢活动，从而可能防治各种代谢异常性疾病和代谢异常相关疾病；同时，作为糖代谢中间产物，外源性FBP进入体内后既可能作为能量代谢底物通过糖酵解通路而后进入三羧酸循环最终被氧化磷酸化而产生能量(ATP)，也可能通过FBPase启动的糖异生通路被脱磷酸生成葡萄糖和终产物糖原。可见，这两种可能的FBP代谢降解可直接导致了外源性FBP进入体内后被快速消耗因而难于产生足以发挥药效的FBP血药浓度和维持足够长的半衰期，从而使外源性FBP无法发挥其调控代谢的药理活性和相应的药效。
本发明证实了这个科学假设，并找到解决问题的方法。
首先，本发明发现在用细胞培养系统外源性FBP不作为能量代谢底物被消耗而是发挥对代谢的广泛调节作用。特别是，FBP对正常细胞和肿瘤细胞表现出不同的代谢调节作用，从而为FBP既可保护正常细胞及其功能又能杀灭各种不同的癌细胞提供了科学依据。具体地说，1)无论何种细胞类型，FBP可促进线粒体氧化磷酸化活动，从而升高ATP水平；2)促进正常细胞的磷酸戊糖代谢旁路(PPP)，升高内源性抗氧化物质NADPH和还原性谷胱甘肽(GSH)的水平，因此对正常细胞具有抗氧化损伤的作用。相反，FBP抑制癌细胞的PPP，降低NADPH和GSH水平、升高ROS水平并导致线粒体损伤和诱导癌细胞衰老和凋亡。此外，FBP可下调肿瘤代谢网络中的多个关键代谢酶，阻断糖酵解中间产物和三羧酸循环中间产物流向生物合成并逆转肿瘤的表观遗传学特征。
而后，本发明发现对整体肿瘤模型动物重复性给予外源性FBP数天后显著升高了体内FBPase的蛋白水平，而且FBP剂量越高FBPase上调的出现就越早，并伴随相应的FBP血药浓度的降低。这些研究结果不仅揭示了FBP药效剂量范围小的科学道理，而且从机制上进一步指出无法通过提高FBP制剂的剂量达到升高FBP血药浓度的目的。基于FBPase对FBP在体内快速脱磷酸降解的关键作用，有理由认为FBPase水平上调加速了外源性FBP在体内的快速降解并导致了FBP药效随处理时程延长而逐渐降低和当FBP剂量高到一定水平时剂量越高反而药效越不好的现象。显然，克服外源性FBP在体内的快速降解是提高FBP血药浓度和延长其半衰期的关键，也是充分发挥外源性FBP药效的关键。
以上发现不仅指出抑制FBP的糖异生通路对维持外源性FBP的血药浓度至关重要，也为稳定外源性FBP的血药浓度特别是需要长期处理的FBP制剂的血药浓度提供了线索和分子靶 标。从理论上讲，不同种类的降糖药可能通过不同的机制抑制糖异生通路的不同环节，因而可能间接或直接地抑制FBP的糖异生代谢，从而可保护外源性FBP避免在体内的快速降解并抑制多次重复处理FBP诱导的糖异生通路的慢性激活，提高外源性FBP血药浓度的峰值和半衰期，因此可能显著提高FBP制剂的药效。
所以，申请人探索了不同类型的降糖药对FBP血药浓度的稳定作用，试图找到能克服FBP在体内被快速降解从而不利于其抗癌药效或其它药效发挥的缺陷。本发明发现，在灌胃给以FBP前0.5小时，给以临床使用的降血糖剂量的磷酸西格列汀、二甲双胍或胰岛素均能提高一次性灌胃FBP后或多次重复给以FBP后血液中FBP浓度峰值、延长FBP在血液中的半衰期、阻断重复给以FBP诱导的FBPase蛋白水平上调和相应的FBP血药浓度下调，同时显著升高FBP的整体抗癌药效。
二甲双胍是经典的治疗Ⅱ型糖尿病的药物，可抑制肝、肾的过度糖原异生，因为果糖-1,6-二磷酸在FBPase的催化下脱磷酸是糖原异生过程中的一个限速环节，因此二甲双胍抑制糖原异生可间接抑制果糖-1,6-二磷酸作为糖异生的底物被脱磷酸降解的过程，从而起到提高外源性FBP血药浓度峰值和延长FBP半衰期的作用。西格列汀抑制糖原异生的作用也可间接或直接保护FBP免受FBPase对其脱磷酸化而被降解。西格列汀通过抑制二肽基肽酶-4(DPP-4)发挥降糖作用。体内胰高血糖素样肽-1(GLP-1)可通过多种机制发挥降低血糖的作用，其中机制之一是减少糖原异生。GLP-1活性受到DPP-4的负向调控，因此西格列汀抑制DPP-4可恢复GLP-1的活性从而抑制糖原异生和上游的糖异生通路。同理，胰岛素通过抑制糖原合成来发挥降糖的作用，也可以保护FBP免受脱磷酸化而被降解。格列酮类药物曲格列酮(troglitazone)则可直接抑制FBPase，因此也可升高外源性FBP的血药浓度峰值和延长其半衰期，从而提高以FBP为药效活性成分制剂的临床使用价值。果糖-2,6-二磷酸是果糖-1，6-二磷酸的异构体是至今已知的活性最强的内源性FBPase抑制剂也可FBP联合制备出FBP生物利用度更高的复合制剂。
在此需要特别指出的是，国内外有不少研究报道了二甲双胍的抗肿瘤活性，而且二甲双胍的抗癌活性引起国际抗肿瘤领域的广泛关注，但是动物实验结果证明二甲双胍的抗肿瘤有效剂量要远远高于治疗糖尿病时所需剂量，这就说明其抗癌作用不是通过调节血糖水平来实现的。本发明发现FBP与临床降血糖剂量的二甲双胍联合时，FBP抑制肿瘤生长的药效被增强。相反，与本身可产生一定的抑制肿瘤生长的高剂量二甲双胍联合，FBP的抗癌药效反而被减弱。这就说明，本发明使用二甲双胍治疗糖尿病的剂量与FBP联合，利用了其稳定FBP的血药浓度的作用而不是其抗癌作用。
本发明还发现：在细胞培养体系西格列汀未见明显的抗癌活性；在整体动物肿瘤模型上，临床降糖剂量的西格列汀在某些模型显示了一定的抗癌活性，这种整体抗癌活性可能是其调节糖代谢的结果，也可能是其它作用如提高机体免疫力的结果。特别是，FBP与降糖剂量的西格列汀联合产生了比各自单独处理更强的抗癌药效，这就有力地支持了FBP与西格列汀组合物在制备新型抗癌药物中的应用价值。
本发明还发现，FBP与西格列汀联合还可显著对高脂饲料喂养引起的体重升高，而各自单独治疗仅未见此作用；FBP可减少高脂食料喂养引起的脂肪堆积，FBP与西格列汀联合治疗这一药效被加强，而西格列汀单独使用无此药效。所述研究结果不仅证明FBP也可以促进脂肪代谢，也支持了本发明所述的新型FBP药物在减肥和防治糖尿病特别是二型糖尿病的应用价值。
本发明还发现FBP可显著对抗癌症化疗药引起的外周神经痛，这就进一步支持了本发明所述的新型FBP药物对治疗癌症的应用价值。特别是，至今传统化疗药在临床上还是抗癌的主流药物,但是其毒副作用包括外周神经疼痛不仅严重降低了患者的生活质量,而且常导致患者放弃化疗。因此，寻找一种可减轻化疗药毒副作用又不减弱其抗癌药效的药物对癌症治疗具有重要意义。可见，本发明产生的FBP抗癌制剂在临床上也可与传统化疗药物联合使用，从而可进一步加强抗癌药效同时克服神经毒副反应。基于代谢功能失常特别是线粒体氧化磷酸化功能减弱和其引起的恶性连锁反应包括能量物质ATP的不足、ROS产生增加和内源性抗氧化物质的降低以及炎症反应等是抗癌药引起的外周神经痛和其它原因诱发的神经痛的共同病理机制，FBP对抗癌化疗药引起的外周神经痛的药理活性与其调解正常细胞代谢的作用高度一致，也支持了FBP防治其它原因引起的神经疼痛的医药用途。
本发明所述的新型FBP药物其要点是通过联合其血药浓度稳定剂可产生更高的FBP血药浓度峰值和更长的半衰期，从而更好地发挥FBP的药效。因此，专业人员可以理解，本发明所述的新型FBP药物也适用于已公开的FBP各种适应症，包括用于改善冠心病的心绞痛、急性心肌梗死、心律失常和心力衰竭的心肌缺血以及病毒性心肌炎的辅助治疗，用于改善脑梗死、脑出血等引起的脑缺氧症状，防治血液系统癌症和各种实体癌症，防治糖尿病及其并发症，癫痫和神经退化性疾病(包括老年性痴呆、帕金森氏病、多发性硬化)。
所述组合物制备的新型FBP药物中，果糖-1,6-二磷酸三钠盐的8分子水合物的药用剂量为100～5000mg/kg体重/天，优选为300～2000mg/kg体重/天；二甲双胍的药用剂量为1～1000mg/kg体重/天，优选为50～300mg/kg体重/天；西格列汀的药用剂量为0.1～500mg/kg体重/天，优选为1～100mg/kg体重/天；胰岛素的药用剂量根据其种类不同在10-100U/kg体重/天范围内。所述新型FBP药物的处理方式采取单次或多次处理，所述多次处理方式为每日处理2-4次。
所述“药用剂量”意为达到预防、有效控制或治疗疾病的目的，临床使用时医生遵循个体化治疗原则，依据患者疾病情况调整患病个体药物的剂量。因此，本发明中所提供的组合物剂量及比例应当理解不是对本发明中的药物组合物使用剂量及比例进行限制，而是对本发明的优选。
本发明中“患病个体”尤指人类。但应当理解为，在现有药理学理解范围内，人类药用剂量与范围可与动物，尤其是哺乳动物，例如大鼠、小鼠、狗等，进行换算得出相适应的药用剂量与范围。
所述组合物制备的新型FBP药物的剂型包括注射剂、普通片剂、颗粒剂、胶囊剂、双层片剂、控释双层片、缓释片剂、单室控释片剂、分散片、肠溶片、肠溶胶囊、定点释药片剂、 缓控释胶囊剂、缓释微丸、含微丸或小片的胶囊、靶向制剂，但不限于这些剂型。优先剂型是可实现稳定剂先释放15分钟至60分钟而后释放FBP的控释固体制剂；也可将稳定剂制成A口服制剂或注射制剂，FBP制成B口服制剂或注射制剂，在临床使用是先使用A制剂15分钟至60分钟，而后使用B制剂。
所述双层片所用的辅料选自但不限于以下辅料：甲基纤维素、羟乙基纤维素、羟乙基甲基纤维素、羟丙基纤维素、羟丙基甲基纤维素、羟丙甲纤维素、羟甲基纤维素、羟甲基纤维素钠、葡萄糖、壳多糖、脱乙酰壳多糖、半乳糖甘露聚糖、蜂蜡、氢化植物油、合成蜡、硬脂酸丁酯、硬脂酸、巴西棕榈蜡、甘油硬脂酸酯、丙二醇－硬脂酸酯和十八烷醇、聚乙烯醇和聚羟乙烯934；稳定剂选自柠檬酸钠、枸橼酸；润滑剂选自硬脂酸镁、硬脂酸、胶态二氧化硅、滑石粉。
制备方法包括粉末直接压片法、湿法制粒压片、干法制粒压片、重压法等。
优选地，采用湿法制粒压片法，不仅工艺简单、省时，且可以保护药物稳定性，具体制备方法包括：分别将A层与B层按处方将活性成分、填充剂与粘合剂混合，湿法制粒后干燥并整粒；将A、B层干燥颗粒分别与崩解剂和润滑剂混合后压制，即得果糖-1,6-二磷酸3钠盐含8分子水合物-西格列汀双层片。
为提高临床用药顺应性，减少服药次数，将所述组合物制备成为含有果糖-1,6-二磷酸三钠盐的8分子水合物及西格列汀的缓释微丸，由空白微丸、药物速释层及缓释层三部分组成，根据缓释剂型的需要，将果糖-1,6-二磷酸制成包衣缓释微丸，将西格列汀作为普通薄膜衣成分包裹于果糖-1,6-二磷酸缓释微丸的外层。
所述缓释微丸所用的辅料选自但不限于以下辅料：
1.所述空白微丸：填充剂选自乳糖、淀粉、微晶纤维素等；粘合剂选自蔗糖、甲基纤维素、羟丙基甲纤维素、聚乙烯吡咯烷酮等；润滑剂为硬脂酸镁、硬脂酸、胶态二氧化硅、滑石粉等。
2.所述速释微丸：所选择高分子膜衣材料为聚乙烯吡咯烷酮、羟丙甲纤维素、聚乙二醇等。
3.所述缓释微丸：所选阻滞剂为丙烯酸树脂、乙基纤维素、川蜡；致孔剂为乳糖、羟丙甲纤维素、聚乙烯比咯烷酮、滑石粉；增塑剂为柠檬酸三乙酯、邻苯二甲酸二乙酯、聚乙二醇6000、柠檬酸三丁酯、葵二酸二丁酯；抗黏剂为滑石粉、硬脂酸镁、单硬脂酸甘油酯。
4.制备方法为采用滚圆挤出法制备果糖-1,6-二磷酸素丸，而后流化床包衣，再将二甲双胍直接作为薄膜衣成分包在果糖-1,6-二磷酸缓释微丸外层。
优选的，具体制备方法如下：
(1)素丸的制备：称取药物及辅料，辅料过筛后混匀，加水制成软材，经挤出滚圆机制得果糖-1,6-二磷酸微丸。所得微丸干燥后过筛备用。
(2)缓释微丸包衣：以Eudragit Ne30d(聚合物浓度5％)、滑石粉(相当于聚合物用量60％)加去离子水适量制成包衣液，而后流化床包衣。
(3)复方缓释微丸的制备：精密称取一定量的西格列汀，溶于去离子水中，采用流化床装置 将西格列汀水溶液喷至果糖-1,6-二磷酸缓释微丸表面，制得复方微丸。
发明人认为，因为果糖-1,6-二磷酸与西格列汀各自药代动力学代谢特点及两药联合抗肿瘤的特殊机制，所以有关其制剂研究的关键点在于同时或顺序释放两药物，因此，包含两药物的普通剂型，或缓控释制剂，包括缓释微丸、双层骨架片剂、膜控释片等复方缓释制剂，均有良好的开发及应用前景。
本领域内人员可以理解，本发明也适用于FBP和稳定剂的独立制剂在临床上的联合使用，根据实际情况采取同时服用和先后服用，优选地提前服用所述稳定剂30分钟。
本发明的要点及产生的有益效果在于：
外源性果糖-1,6-二磷酸(FBP)进入体内被快速降解，而且随着处理时程的延长这种快速降解被加剧，特别是剂量越大这种降解加速的现象就越严重，因此现有FBP制剂难以产生和维持有效血药浓度，这就极大地限制了FBP的药用价值特别是对防治慢性疾病的价值。本发明首先揭示了糖异生通路参与了外源性FBP的快速降解，特别是此代谢通路的激活导致了随处理时程延长的药效逐渐消失。而后，发现降血糖药可以抑制外源性FBP在体内的快速降解并极大地提高了FBP整体药效包括抗癌药效。研究结果支持了降糖药作为FBP体内稳定剂与FBP构成复合物在制备防治代谢性疾病和代谢相关疾病的药物中的应用。
本发明的创造性和科学性还体现在，针对多种不同疾病(包括神经退化性疾病、神经功能紊乱性疾病、肥胖和糖尿病以及肿瘤)的共同病理机制，即代谢功能失常(分化细胞的代谢障碍包括糖酵解活动增强和线粒体氧化磷酸化活动减弱以及相关联的氧化损伤，肿瘤细胞的代谢重编程包括糖酵解活动和从头生物合成增强以及线粒体氧化磷酸化减弱)，利用FBP可抑制过度糖酵解并促进线粒体氧化磷酸化的药理作用来治疗所述多种不同疾病。
产生的有益效果：提供了一类由果糖-1,6-二磷酸(FBP)和其血药浓度稳定剂为主药成分的FBP复合制剂，这类药物与以FBP为唯一活性成分的现有FBP制剂比较具有多种优势。首先，解决了限制现有FBP制剂药用价值的关键问题，即外源性果糖-1，6-二磷酸(FBP)进入体内被快速降解的问题，因此本发明提供的FBP复合制剂能产生更高的FBP血药浓度峰值和更稳定的血药浓度以及因此产生的更显著的药效，还可降低FBP剂量和减少大剂量FBP水解后造成大量无机磷进入体循环所致毒性。特别是，所述FBP复合制剂克服了现有FBP制剂随处理时程延长FBP在体内代谢不断加快的问题，因此FBP复合制剂在治疗各种代谢性慢性疾病和代谢相关慢性疾病具有显著优势。此外，FBP复合制剂对中的稳定剂可通过不同于FBP作用机制的机制来改善代谢状态，因此二者可产生通过不同作用机制介导的协同药效。通过选择适宜的辅料、辅料配比及制备方法，制备缓、控释制剂和靶向纳米制剂以及不同含量规格的制剂，提高了所述药物组合物的临床顺应性。
附图说明
图1是果糖-1,6-二磷酸对正常细胞人星形胶质细胞代谢的调控作用。FBP：果糖-1,6-二磷酸。注：采用单因素方差分析的方法分析实验数据,用LSD方法检测各组之间显著性差异。给药组与对照组比***P<0.001。
图2是果糖-1,6-二磷酸抑制胶质瘤细胞的糖酵解。FBP：果糖-1,6-二磷酸。
图3是1,6-二磷酸果糖阻断糖酵解中间产物流向生物合成。FBP：1,6-二磷酸果糖；GAP：3-磷酸甘油醛；PEP：磷酸烯醇式丙酮酸；Pyr：丙酮酸；G6P：6-磷酸葡萄糖；PGA：三磷酸甘油酸；La：乳酸；Ser：丝氨酸；Gly：甘氨酸；R5P：5-磷酸核糖；ATP：三磷酸腺苷；UTP：三磷酸尿苷；A：腺苷；C:胞苷；U:尿苷；T：胸苷；A：腺嘌呤；G：鸟嘌呤；注：采用单因素方差分析的方法分析实验数据,用LSD方法检测各组之间显著性差异。给药组与对照组比***P<0.001。
图4是果糖-1,6-二磷酸及其联合二甲双胍或西格列汀重复处理果糖-1,6-二磷酸糖酶1蛋白水平的影响。FBP：果糖-1,6-二磷酸；FBPase1:果糖-1,6-二磷酸酶1；Met:二甲双胍；STG：西格列汀。
图5是二甲双胍、西格列汀及胰岛素提高1,6-二磷酸果糖峰浓度并稳定FBP血药浓度的作用。FBP：1,6-二磷酸果糖；Met：二甲双胍；STG：西格列汀；Ins：胰岛素。注：采用最小显著性差异法分析实验数据。***P<0.001,*P<0.05对比于0小时(给药前)，#P<0.05对比于FBP单用组。
具体实施方式
本发明结合附图和具体实施例作进一步的说明。但不应理解为本发明的范围仅限于以下实施例，应理解上述所实现的内容均属于本发明的范围，依照本发明内容进行的在任何本领域内的替换，均应属于本发明保护范围之内。
实施例1.果糖-1，6-二磷酸对正常细胞人星形胶质细胞代谢的调控作用
人正常星形胶质细胞(HA)在含不同浓度的果糖-1,6-二磷酸三钠盐(0mM、0.25mM、0.5mM、1mM)的培养基中培养，分别在12h、24h测定培养基中乳酸的水平和24h细胞内ATP的水平。研究结果显示：与对照组相比，处理组在12h和24h的乳酸水平均随着果糖-1，6-二磷酸三钠盐浓度升高而显著降低(处理组与对照组比***P<0.001)；处理组在24h的ATP水平随着果糖-1,6-二磷酸三钠盐浓度升高而显著升高(处理组与对照组比***P<0.001)(图1a,b)
人正常星形胶质细胞(HA)在含0.8mM的果糖-1,6-二磷酸三钠盐的培养基中培养，在36h用蛋白免疫印迹法(Western Blot)测定细胞内2,6-二磷酸果糖激酶3(PFKFB3)、乳酸脱氢酶(LDH5)、细胞色素C(Cyto C)的蛋白水平(β-actin作为内参)。实验结果显示：与对照组相比，处理组PFKFB3和LDH5蛋白水平显著降低，而Cyto C的蛋白水平显著升高。(图1c)
人正常星形胶质细胞(HA)在含不同浓度的果糖-1，6-二磷酸三钠盐(0mM、0.25mM、0.5mM、1mM、2mM、2.5mM)的培养基中培养，测定36h细胞内还原型谷胱甘肽GSH水平和果糖-1,6-二磷酸三钠盐1.6mM组NADPH/NADP+的比例。实验结果显示，与对照组相比，处理组细胞内GSH显著升高(处理组与对照组比***P<0.001)，并且NADPH/NADP+的比例显著升高(处理组与对照组比***P<0.001)。(图1d,e)
上述实验结果表明，果糖-1，6-二磷酸三钠盐可抑制人正常星形胶质细胞HA的糖酵解，促进三羧酸循环和氧化磷酸化，同时增强其抗氧化应激的能力。
讨论与小结：
星形胶质细胞是脑内数量最多而且功能多样的细胞类型，特别是星形胶质细胞的代谢活动极其活跃，其代谢活动与多种功能(包括为神经元提供代谢支持、维持神经递质动态平衡和氧化还原动态平衡等)密切相关，星形胶质细胞代谢失常(包括过度糖酵解和线粒体氧化磷酸化活动减弱)与神经退化和退化性疾病密切相关。FBP可抑制星形胶质细胞的过度糖酵解，从减少乳酸的堆积；FBP促进氧化磷酸化，升高ATP水平；FBP升高内源性抗氧化物质NADPH和谷胱甘肽(GSH)水平，提高抗氧化损伤能力。可见,上述研究结果强烈地支持了FBP防治神经退化性疾病的医药用途。
实施例2.肿瘤细胞不消耗外源性果糖-1,6-二磷酸，但其糖酵解中间产物被显著升高
大鼠胶质瘤细胞株(C6)，人胶质瘤细胞株(U87-MG,U-251,SHG-44)以及患者来源的胶质瘤细胞(肿瘤1,肿瘤3)在含1.6mM的果糖-1,6-二磷酸三钠盐的培养基中培养，采用LC-MS/MS测定36h各细胞内糖酵解中间产物葡萄糖-6-磷酸G6P、果糖-1,6-二磷酸FBP、3-磷酸甘油醛(GAP)、磷酸二羟丙酮(DHAP)、3-磷酸甘油酸PGA的水平；另外，测定各胶质瘤细胞株在12h和36h培养基中果糖-1,6-二磷酸的水平。实验结果显示，与对照组相比，果糖-1,6-二磷酸处理组细胞内糖酵解产物FBP、GAP和DHAP水平显著升高(处理组与对照组比***P<0.001)(附表1a)；果糖-1,6-二磷酸处理组培养基中FBP的浓度随着处理时间延长不显著降低(N.S.P>0.1)(附表1b)。实验结果表明，肿瘤细胞不消耗外源性FBP，部分进入细胞内的FBP可顺着糖酵解通路发生第一步降解反应(产生是GAP和DHAP)，并停止于此步，以此导致GAP和DHAP的堆积。此外，外源性FBP引起的胞内F6P水平升高说明FBP在肿瘤细胞内也可能被果糖-1,6-二磷酸酶(FBPase)降解。
表1a细胞内糖酵解中间产物升高的倍数(与对照组比)
细胞株 F6P FBP GAP DHAP PGA
U87-MG 47.31 149.69 1222.86 944.4 3.03
C6 19.89 92.22 132.85 66.04 2.2
KNS-89 1.89 21.97 19.75 8.74 3.28
SHG-44 3.12 10.74 21.03 12.48 2.04
Tumor 1 2.02 8.27 7.22 4.9 1.13
Tumor 3 14.12 22.94 76.22 55.58 1.86
表1b培养基中果糖-1,6-二磷酸的水平(mg/ml)
Figure PCTCN2017114371-appb-000001
G6P：葡萄糖-6-磷酸、FBP：果糖-1,6-二磷酸、GAP：3-磷酸甘油醛、DHAP：磷酸二羟丙酮、PGA：3-磷酸甘油酸。
实施例3.果糖-1,6-二磷酸抑制胶质瘤细胞的糖酵解
人胶质瘤细胞株(U87-MG,KNS-89,SHG-44)分别在含0.8mM和1.6mM的果糖-1，6-二磷酸三钠盐、含1.6mM 2-脱氧葡萄糖的培养基中培养，12h、24h、36h、48h分别测定培养基中细胞释放的糖酵解终产物乳酸的含量，处理组乳酸水平均显著低于不加药对照组(CON)(处理组与对照组比***P<0.001)(附表2a-c)。人胶质瘤细胞株(U87-MG)在含0.8mM 1,6-二磷酸果糖的培养基中培养1h、3h、6h、12h、24h、36h、48h，用蛋白质印迹法分析各时间点细胞糖酵解通路关键代谢酶的水平变化，可见己糖激酶2(HK2)、6-磷酸果糖激酶2(PFKFB3)、丙酮酸激酶2(PKM2)，和乳酸脱氢酶5(LDH5)均快速、持续下调(附图2)。实验结果表明：1,6-二磷酸果糖可以抑制多种胶质瘤细胞的糖酵解。
表2a果糖-1,6-二磷酸处理后胶质瘤细胞U87-MG乳酸的相对水平(与对照组相比)
Figure PCTCN2017114371-appb-000002
表2b果糖-1,6-二磷酸处理后胶质瘤细胞KNS-89乳酸的相对水平(与对照组相比)
Figure PCTCN2017114371-appb-000003
表2c果糖-1,6-二磷酸处理后胶质瘤细胞SHG-44乳酸的相对水平(与对照组相比)
Figure PCTCN2017114371-appb-000004
注：采用单因素方差分析的方法分析实验数据,用LSD方法检测各组之间显著性差异。(处理组与对照组比*P<0.1显著差异，***P<0.001极显著差异)。FBP：果糖-1,6-二磷酸；2-DG:2-脱氧-D-葡萄糖
实施例4.1,6-二磷酸果糖促进胶质瘤细胞线粒体氧化磷酸化
大鼠胶质瘤细胞株(C6)，人胶质瘤细胞株(KNS-89,SHG-44)分别在含0.8mM或1.6mM果糖-1,6-二磷酸三钠盐的培养基中培养36h，可观察到各细胞株ATP/ADP比例(1,6-二磷酸果糖组与对照组比***P<0.001)、NADH/NAD+比例大幅升高(1,6-二磷酸果糖组与对照组比***P<0.001)，同时ATP水平显著升高(1，6-二磷酸果糖组与对照组比***P<0.001)(附表3a-b)。实验结果表明：1,6-二磷酸果糖促进胶质瘤细胞线粒体氧化磷酸化。
表3a.果糖-1,6-二磷酸升高胶质瘤细胞ATP与ADP比例
Figure PCTCN2017114371-appb-000005
表3b.果糖-1,6-二磷酸升高胶质瘤细胞NADH与NAD+比例
Figure PCTCN2017114371-appb-000006
注：采用单因素方差分析的方法分析实验数据,用LSD方法检测各组之间显著性差异。处理组与对照组比***P<0.001极显著差异。FBP：果糖-1,6-二磷酸三钠盐
实施例5.果糖-1,6-二磷酸三钠盐阻断糖酵解中间产物流向生物合成
人胶质瘤细胞株(U87MG)在含13C标记葡萄糖(U-13C-Glc)的培养基中培养并用1.6mM果糖-1,6-二磷酸三钠盐处理36h，采用液-质联用技术(LC-MS/MS)测定细胞内糖酵解通路、磷酸戊糖通路、“一碳单位”代谢通路和核酸从头合成通路的中间产物。实验结果显示：(1)处理组糖酵解中间产物1，6-二磷酸果糖(FBP)、3-磷酸甘油醛(GAP)、磷酸烯醇式丙酮酸(PEP)的水平较对照组显著升高(与对照组相比***P<0.001)，而糖酵解产物乳酸(Lac)的水平显著降低(与对照组相比***P<0.001)(附表4a)；(2)处理组U-13C-Glc经丝氨酸生物合成通路生成的丝氨酸(Ser)(M+3)显著升高(对照组1±0.03，处理组1.57±0.04，对照组与 处理组相比***P<0.001)，而丝氨酸经“一碳单位”代谢通路生成的甘氨酸(Gly)(M+2)显著降低(对照组1±0.07，处理组0.63±0.06，对照组与处理组相比***P<0.001)；(3)磷酸戊糖通路产物5-磷酸核糖(R5P)中13C标记的比例由对照组的68.96±5.03％降低为处理组的17.32±1.23％(对照组与处理组比***P<0.001)；(4)处理组游离的核酸生物合成中间产物三磷酸腺苷(ATP)、三磷酸尿苷(UTP)、腺苷(A)、胞苷(C)、尿苷(U)、胸苷(T)中核糖带标记的比例较未处理组显著降低(与对照组相比*P<0.05；***P<0.001)(附表4b)；
实验结果表明，果糖-1,6-二磷酸三钠盐可使糖酵解中间产物在糖酵解通路中堆积，减少其通过磷酸戊糖通路，丝氨酸生物合成和“一碳单位”代谢，进而减少核酸的从头合成。
表4a.果糖-1,6-二磷酸处理后糖酵解中间产物的相对水平(与对照组相比)
  F6P FBP GAP PEP Lac
CON 1±0.01 1±0.05 1±0.18 1±0.06 1±0.01
FBP 2.06±0.04*** 5.01±0.34*** 4.27±0.07*** 8.00±0.32*** 1.06±0.02N.S.
表4b.果糖-1,6-二磷酸处理后游离核苷及核苷酸中核糖的13C标记比例
  F6P FBP GAP PEP Lac
CON 13.03±0.38 41.00±4.28 31.97±1.11 59.40±1.11 61.60±1.95
FBP 2.79±0.14*** 34.99±1.63* 11.31±1.75*** 13.71±0.54*** 30.70±2.11***
注：采用单因素方差分析的方法分析实验数据,用LSD方法检测各组之间显著性差异。给药组与对照组比(N.S.无显著性差异；*P<0.05显著性差异；***P<0.001极显著性差异)。F6P：6-磷酸果糖；FBP：果糖-1,6-二磷酸；GAP：3-磷酸甘油醛；PEP：磷酸烯醇式丙酮酸；Lac：乳酸；ATP：三磷酸腺苷；UTP：三磷酸尿苷；A：腺苷；C:胞苷；U:尿苷。
实施例6.果糖-1,6-二磷酸三钠盐阻断三羧酸循环中间产物流向生物合成
人胶质瘤细胞株(U87MG)在含13C标记的葡萄糖(U-13C-Glc)中培养并用1.6mM 1,6-二磷酸果糖三钠盐36h，采用液-质联用技术(LC-MS/MS)测定细胞内三羧酸循环中间产物、三羧酸循环中间产物衍生的氨基酸和核苷酸从头合成通路中间产物。实验结果显示：(1)处理组三羧酸循环中间产物α-酮戊二酸(α-KG)和草酰乙酸(OAA)的水平较对照组显著升高(与对照组相比***P<0.001)(附表5a)；(2)处理组三羧酸循环中间产物衍生的天冬氨酸(Asp)和谷氨酸(Glu)较对照组显著降低(与对照组相比***P<0.001)(附表5b)；(3)处理组游离核苷及核苷酸中嘌呤环和嘧啶环13C标记的比例显著降低(与对照组相比**P<0.005，***P<0.001)(附表5c)。实验结果表明，1,6-二磷酸果糖三钠盐可阻断三羧酸循环中间产物转化为氨基酸，进而阻断其参与的核酸从头合成。
表5a果糖-1,6-二磷酸处理后部分三羧酸循环中间产物的相对水平(与对照组相比)
  α-KG OAA
CON 1±0.01 1±0.14
FBP 1.61±0.01*** 1.87±0.04***
表5b果糖-1,6-二磷酸处理后部分氨基酸的相对水平(与对照组相比)
  Asp Glu
CON 1±0.02 1±0.05
FBP 0.16±0.07*** 0.35±0.04***
表5c果糖-1,6-二磷酸处理后游离核苷及核苷酸中嘌呤环和嘧啶环的13C标记比例
  ATP A G UTP U
CON 38.26±1.50 12.41±1.19 53.75±1.47 50.67±0.96 20.58±2.30
FBP 23.77±0.57*** 7.70±1.50** 41.83±4.06** 18.19±0.87*** 8.33±0.90***
注：采用单因素方差分析的方法分析实验数据,用LSD方法检测各组之间显著性差异。给药组与对照组比(**显著性差异；***P<0.001极显著性差异)FBP：果糖-1,6-二磷酸；α-KG：α-酮戊二酸；OAA：草酰乙酸；Asp天冬氨酸；Glu：谷氨酸；ATP：三磷酸腺苷；UTP：三磷酸尿苷；A：腺苷；C:胞苷；U:尿苷。
实施例7.果糖-1,6-二磷酸三钠盐阻断三羧酸中间产物流出线粒体、破坏肿瘤表观遗传学特征、广泛下调肿瘤代谢酶的蛋白水平
人胶质瘤细胞株(U87MG)在含1.6mM果糖-1,6-二磷酸三钠盐的培养基中培养36h后分离胞浆和线粒体，液-质联用技术(LC-MS/MS)分别测定胞浆和线粒体中三羧酸循环中间产物的水平。可见处理组三羧酸循环中间产物乙酰辅酶A(Ac-CoA)、柠檬酸(Cit)、α-酮戊二酸(α-KG)和草酰乙酸(OAA)在胞浆中的水平显著降低(与对照组相比**P<0.01；***P<0.001)，而在线粒体中的水平显著升高(与对照组相比***P<0.001)(附表6)；同时胞浆与线粒体间的苹果酸穿梭相关酶(ME1、MDH1、MDH2、GOT1、GOT2)的蛋白水平随时间显著降低(附图3a)。实验结果表明，1，6-二磷酸果糖三钠盐可阻断三羧酸循环代谢中间产物流出线粒体。
人胶质瘤细胞株(U87MG)在含1.6mM果糖-1,6-二磷酸三钠盐的培养基中分别培养0、1、3、6、12、24、36和48小时，蛋白免疫印迹法(WB)考察脂肪酸和核酸生物合成通路相关酶蛋白水平的变化。可见处理组脂肪酸和核酸生物合成相关酶(CAD、TS、ACL、FASN)的蛋白水平随时间显著降低(附图3b)。实验结果表明，1,6-二磷酸果糖可广泛下调肿瘤细胞的代谢酶。
人胶质瘤细胞株(U87MG)在含1.6mM果糖-1,6-二磷酸三钠盐的培养基中分别培养24h和36h后采用免疫细胞化学法考察5-羟甲基胞嘧啶(5-hmC)的水平，可见处理组5-hmC显著升高；同时，处理后肿瘤细胞表观遗传学相关蛋白(Ac-Foxo、H3K9ac、H3K9me2)快速下调。实验结果表明，1,6-二磷酸果糖可改变肿瘤细胞的表观遗传学特征(附图3c)。
表6.胞浆和线粒体中三羧酸循环中间产物的相对水平(与对照组相比)
Figure PCTCN2017114371-appb-000007
Figure PCTCN2017114371-appb-000008
注：采用单因素方差分析的方法分析实验数据,用LSD方法检测各组之间显著性差异。处理组与对照组比(*P<0.05显著性差异；***P<0.001极显著性差异)
FBP：果糖-1,6-二磷酸三钠盐；Ac-CoA：乙酰辅酶A；Cit：柠檬酸；α-KG：α-酮戊二酸；OAA：草酰乙酸；Cyto：胞浆；Mito：线粒体
实施例8.果糖-1,6-二磷酸三钠盐破坏胶质瘤细胞氧化还原平衡
大鼠胶质瘤细胞株(C6)，人胶质瘤细胞株(KNS-89)在含0.8mM果糖-1,6-二磷酸三钠盐的培养基中培养，随着处理时间的延长细胞内活性氧水平(ROS)逐步升高(附表7a)，而线粒体膜电位(MMP)则逐步降低(附表7b)。
大鼠胶质瘤细胞株(C6)，人胶质瘤细胞株(KNS-89,SHG-44)在含1.6mM果糖-1,6-二磷酸三钠盐的培养基中培养36h，液-质联用技术(LC-MS/MS)测定细胞中重要的抗氧化物质。实验结果表明：谷胱甘肽(GSH,GSSG)水平急剧降低(附表7c)，同时NADPH/NADP+比例剧烈下降(附表7d)。
实验结果表明：果糖-1,6-二磷酸三钠盐增加活性氧的生成，抑制抗氧化成分谷胱甘肽的合成，阻碍NADP+向NADPH转化，从而从多个层面破坏胶质瘤细胞氧化还原平衡。
表7a.活性氧的相对水平(与对照组相比)
  0h 12h 24h 48h 72h
C6 1±0.04 1.26±0.04*** 1.89±0.03*** / /
U251 1±0.02 1.20±0.04 1.19±0.03 1.42±0.03*** 1.82±0.03***
表7b.线粒体膜电位的相对水平(与对照组相比)
  0h 12h 24h 48h 72h
U251 1±0.03 1.08±0.03 1.18±0.01 0.6±0.06*** 0.4±0.07***
C6 1±0.08 0.8±0.14*** 0.6±0.11*** 0.1±0.09*** 0.1±0.07***
表7c GSH与GSSG的相对含量(与对照组相比)
Figure PCTCN2017114371-appb-000009
表7d.NADPH与NADP+的相对比例(与对照组相比)
    C6 KNS-89 SHG-44
NADPH/NADP+ CON 1±0.11 1±0.03 1±0.02
  FBP 0.14±0.01*** 0.44±0.04*** 0.39±0.01***
注：采用单因素方差分析的方法分析实验数据,用LSD方法检测各组之间显著性差异。(处理组与对照组比***P<0.001极显著性差异)。
FBP：果糖-1,6-二磷酸三钠盐；GSH：还原型谷胱甘肽；GSSG：氧化型谷胱甘肽
讨论与小结(实施3至实施例8)——FBP逆转肿瘤代谢特征
肿瘤细胞通过代谢重编程，特别是可产生大量的糖酵解中间产物和三羧酸循环中间产物并将这些中间产物用于生物合成，从而为肿瘤细胞的快速分裂增殖和生长提供前提条件。此外，来源于三羧酸循环中间产物的乙酰辅酶A、富马酸和琥珀酸支持着肿瘤表观遗传学特征，从而参与了促癌蛋白表达上调和抑癌蛋白表达下调的调节。FBP可逆转肿瘤代谢特征，摧毁肿瘤代谢网络，具有显著的体内外抗癌活性。突出表现为促进葡萄糖和谷氨酰胺进入三羧酸循环和氧化磷酸化，还可阻断糖酵解和三羧酸循环中间产物流向生物合成，并阻断三羧酸中间产物流出线粒体因而可破坏肿瘤表观遗传学特征，广泛下调肿瘤代谢酶的蛋白水平。研究结果强烈地支持了FBP治疗各种肿瘤的医药用途。
实施例9.果糖-1,6-二磷酸三钠盐长期处理导致果糖-1,6-二磷酸酶蛋白水平应激性增加和果糖-1,6-二磷酸血药浓度降低，二甲双胍和磷酸西格列汀能够对抗这种果糖-1,6-二磷酸代谢改变
将180-200g的SD大鼠分为4组：生理盐水对照组、果糖-1，6-二磷酸三钠水合物组(500mg/kg,i.g)、二甲双胍组(150mg/kg，i.g)或西格列汀组(20mg/kg，i.g)、果糖-1,6-二磷酸钠联合二甲双胍或西格列汀组，每组5只，所有组别均采用灌胃处理，二甲双胍或西格列汀提前于果糖-1,6-二磷酸0.5小时处理，连续处理14天，于末次果糖-1,6-二磷酸灌胃处理后3小时，取大鼠血液检测FBP血药浓度，取肾脏组织，Western-blot考察果糖-1,6-二磷酸酶1蛋白水平。结果显示，相比于对照组，果糖-1,6-二磷酸组长期处理后果糖-1,6-二磷酸酶1蛋白水平出现显著上调，二甲双胍及西格列汀组均未见显著影响，而联合二甲双胍或西格列汀后后恢复至正常水平；相应的，果糖-1,6-二磷酸组果糖-1,6-二磷酸血药浓度无法维持，处理3小时后降低至60μg/ml，与体内正常FBP水平相同，而联合二甲双胍后显著升高其血药浓度，处理3小时后仍高达99.23μg/ml，与对照组比较**P<0.01；与二甲双胍组比较*P<0.05(表4)。因此，果糖-1,6-二磷酸长期处理会导致果糖-1,6-二磷酸酶1蛋白水平的显著上调，使得果糖-1,6-二磷酸在体内更易被降解，无法维持高而稳定的血药浓度，影响了果糖-1,6-二磷酸抗肿瘤药效；二甲双胍及西格列汀不影响FBPase1在组织中的正常表达，而可以有效对抗果糖-1,6-二磷酸引起的果糖-1,6-二磷酸酶1应激性增加，使其恢复至正常水平，有利于FBP在体内血药浓度的稳定，从而发挥更强的抗肿瘤作用。
表4. 1,6-二磷酸果糖及其联合二甲双胍重复处理对1，6-二磷酸果糖血药浓度的影响
Figure PCTCN2017114371-appb-000010
注：采用单因素方差分析的方法分析实验数据,用LSD方法检测各组之间显著性差异 (Met+FBP组与对照组、FBP组相比**P<0.01；与Met组比*P<0.05)。FBP：果糖-1,6-二磷酸；Met:二甲双胍。
实施例10.二甲双胍、西格列汀及胰岛素对果糖-1,6-二磷酸血药浓度的稳定作用
将6周龄ICR小鼠分为4组：生理盐水对照组、果糖-1,6-二磷酸三钠水合物组(500mg/kg,i.g)、果糖-1,6-二磷酸钠联合二甲双胍(150mg/kg,i.g)组、果糖-1,6-二磷酸钠联合西格列汀(20mg/kg)组、果糖-1,6-二磷酸钠联合胰岛素(4U/kg)组，每组7只小鼠，所有组别均采用灌胃处理，二甲双胍、西格列汀及胰岛素提前于1，6-二磷酸果糖0.5小时处理。于果糖-1,6-二磷酸处理后1.5及3小时，取各组别小鼠血液，分离血浆，酶法测定血液中1，6-二磷酸果糖浓度。结果显示，1,6-二磷酸果糖组处理1.5小时后血液中FBP浓度从53.3μg/ml升高到77.5μg/ml，3小时后下降至70μg/ml(1,6-二磷酸果糖组1.5小时与0小时比较*P<0.001)；而联合二甲双胍、胰岛素或西格列汀后，1.5小时后FBP血药浓度分别提高至97.5、97.5及106μg/ml，3小时后各组果糖-1,6-二磷酸血药浓度分别为82.5、89.2及91.7μg/ml，均高于对照组(联合处理组1.5、3小时与0小时比较*P<0.001)，且相较于果糖-1,6-二磷酸单用组，联合处理组在达峰浓度及血药浓度维持时间上均大大提高(1.5小时联合处理组与果糖-1,6-二磷酸组比较#P<0.05，3小时联合西格列汀及胰岛素组与FBP组比较#P<0.05)。以上结果表明，FBP联合二甲双胍、胰岛素或西格列汀可以有效提高FBP峰浓度并稳定FBP在体内的血药浓度的维持时间，从而能够有效提高FBP体内抗肿瘤作用。
讨论与小结(实施例9和10)—降糖药可以升高和维持FBP血药浓度，阻止随处理时间延长产生的FBP代谢加速
对整体肿瘤模型动物给以FBP长期治疗,会引起FBP降解酶1,6-二磷酸果糖酶(FBPase1)蛋白水平的上调并加速FBP的降解,从而出现随着处理时间的延长FBP血药浓度逐渐降低的现象,阻碍了FBP抗癌药效的发挥。因此，本发明进一步探索了降糖药包括二甲双胍、西格列汀和胰岛素对FBP血药浓度的稳定作用，试图找到克服FBP在体内被快速降解从而不利于其抗癌药效发挥的缺陷。研究结果证明，这些降糖药均能升高和维持FBP血药浓度，阻止随处理时间延长产生的FBP代谢加速。特别要指出的是，包括二甲双胍、西格列汀和胰岛素的不同降糖药具有不同作用机制，但均能产生抑制糖异生的药效。因此，基于糖异生酶1,6-二磷酸果糖酶(FBPase1)对外源性FBP降解的关键作用，上述研究结果指出果糖-1,6-二磷酸酶抑制剂和现有不同作用机制的降糖药以及今后不断出现的新降糖药均能抑制外源性FBP在体内的降解，从而提高外源性FBP的药用价值。
实施例11.二甲双胍和西格列汀不显著影响果糖-1,6-二磷酸三钠盐体外抗人肠癌细胞的作用
培养24小时的人肠癌细胞株SW620、HCT-8分别在含0.8mM果糖-1,6-二磷酸三钠盐、0.2mM二甲双胍/100μM西格列汀或同时含0.8mM果糖-1,6-二磷酸三钠盐及0.2mM二甲双胍/100μM西格列汀的培养基中再培养72小时。同时设不加药处理组(也称对照组，Con)。用磺基罗丹明B(Sulforhodamine B,SRB)染色分析方法测定细胞活力。结果显示，FBP在0.8mM时对SW620细胞活力抑制率达到41％，二甲双胍在0.2mM时不会影响SW620细胞活力，两药 联合不会对果糖-1,6-二磷酸三钠盐的药效产生影响；果糖-1,6-二磷酸三钠盐在0.8mM时对HCT-8细胞活力抑制率达到41％，西格列汀在100μM时不会影响HCT-8的细胞活力，两药联合不会对果糖-1,6-二磷酸三钠盐的药效产生影响(果糖-1,6-二磷酸三钠盐组与对照组比较***P<0.001；联合处理组与对照组比较***P<0.001；联合处理组较果糖-1,6-二磷酸三钠盐组无显著性差异)(表5)。实验结果表明：果糖-1,6-二磷酸三钠盐联合二甲双胍或西格列汀在离体实验中不会对果糖-1,6-二磷酸三钠盐的抗肠癌作用产生拮抗也不产生明显的协同作用。
表5. 1,6-二磷酸果糖联合二甲双胍或西格列汀对人肠癌细胞活力的影响
  Con Met FBP Met+FBP
SW620 100±2.99 95.92±7.54 49.90±3.91*** 42.67±2.57***
  Con STG FBP STG+FBP
HCT-8 100±2.57 95.43±3.79 85.93±3.29*** 79.10±6.17***
注：采用单因素方差分析的方法分析实验数据,用LSD方法检测各组之间显著性差异(***P<0.001对比于对照组(Con))。FBP：1,6-二磷酸果糖；Met：二甲双胍；STG：西格列汀。
实施例12.二甲双胍和西格列汀不显著影响果糖-1,6-二磷酸三钠盐体外抗人肝癌细胞的作用
培养24小时的人肝癌细胞株Bel-7402及huh-7分别在含1.6或0.8mM果糖-1,6-二磷酸三钠盐、0.2mM二甲双胍或25μM西格列汀或同时含0.8mM果糖-1,6-二磷酸三钠盐及0.2mM二甲双胍/25μM西格列汀的培养基中再培养72小时。同时设不加药处理组(也称对照组，Con)。用磺基罗丹明B(Sulforhodamine B,SRB)染色分析方法测定细胞活力。结果显示，FBP在0.8mM时对Bel-7402细胞活力抑制率为41％，二甲双胍在0.2mM时对Bel-7402细胞活力抑制率为5％，两药联合不会对果糖-1,6-二磷酸三钠盐的药效产生影响；果糖-1,6-二磷酸三钠盐在0.8mM时对Huh-7细胞活力抑制率达到20％，西格列汀在25μM时细胞活力与对照组无显著性差异，两药联合不会对果糖-1,6-二磷酸三钠盐的药效产生影响(果糖-1,6-二磷酸三钠盐组与对照组比较***P<0.001；联合处理组与对照组比较***P<0.001；联合处理组较果糖-1,6-二磷酸三钠盐组无显著性差异)(表6)。实验结果表明：果糖-1,6-二磷酸三钠盐联合二甲双胍或西格列汀在离体实验中不会对果糖-1,6-二磷酸三钠盐的抗肝癌作用产生拮抗作用也不产生明显的协同作用。
表6. 1,6-二磷酸果糖联合二甲双胍或西格列汀对人肝癌细胞活力的影响
  Con Met FBP Met+FBP
Bel-7402 100±4.81 91.69±1.62 60.43±4.66*** 60.51±3.87***
  Con STG FBP STG+FBP
Huh-7 100±4.38 92.08±1.35 80.38±3.63*** 79.52±11.85***
注：采用单因素方差分析的方法分析实验数据,用LSD方法检测各组之间显著性差异(***P<0.001对比于对照组(Con))。FBP:1,6-二磷酸果糖；Met:二甲双胍；STG:西格列汀。
实施例13.二甲双胍和西格列汀不显著影响果糖-1,6-二磷酸三钠盐体外抗黑色素瘤细胞 的作用
培养24小时的小鼠黑色素瘤B16细胞在含0.8mM果糖-1,6-二磷酸三钠盐、20μM西格列汀或同时含0.8mM果糖-1,6-二磷酸三钠盐及20μM西格列汀的培养基中再培养72小时。同时设不加药处理组(也称对照组，Con)。用磺基罗丹明B(Sulforhodamine B,SRB)染色分析方法测定细胞活力。结果显示，果糖-1,6-二磷酸三钠盐在0.8mM时对B16细胞活力抑制率为22％，西格列汀在20μM时对B16细胞活力抑制率为16％，两药联合不会对果糖-1,6-二磷酸三钠盐的药效产生影响(西格列汀组与对照组比较***P<0.001；果糖-1,6-二磷酸三钠盐组与对照组比较***P<0.001；联合处理组与对照组比较***P<0.001；联合处理组与果糖-1,6-二磷酸三钠盐组比较无显著性差异)(表7)。实验结果表明：果糖-1,6-二磷酸三钠盐联合西格列汀在离体实验中不会对果糖-1,6-二磷酸三钠盐的抗黑色素瘤作用产生拮抗作用，也不产生明显的协同作用。
表7. 1,6-二磷酸果糖联合西格列汀对黑色素瘤B16细胞活力的影响
  Con Met FBP Met+FBP
  100±2.89 86.84±3.84 77.67±7.29*** 70.38±5.01***；#
注：采用单因素方差分析的方法分析实验数据,用LSD方法检测各组之间显著性差异注(***P<0.001对比于对照组(CON)；#P<0.05对比于STG组)。FBP：1,6-二磷酸果糖；STG：西格列汀。
实施例14.二甲双胍和西格列汀增强果糖-1,6-二磷酸三钠盐的整体抗肿瘤药效
按常规方法将小鼠肝癌细胞H22接种于成年雄性ICR小鼠右侧腋皮下，于接种24小时后，随机分为以下实验组：生理盐水对照组、果糖-1,6-二磷酸三钠盐钠组(FBP)组(500mg/kg,i.g)、二甲双胍(Met)组(150mg/kg,i.g)、药物组合物(F+M)组(FBP 500mg/kg+Met 150mg/kg，i.g),或者生理盐水对照组、果糖-1,6-二磷酸三钠盐钠组(FBP)组(500mg/kg,i.g)、西格列汀(STG)组(20mg/kg,i.g)、药物组合物(FBP+STG)组(FBP 500mg/kg+STG 20mg/kg，i.g)，动物数每组7只。每天处理三次，二甲双胍或西格列汀提前于果糖-1,6-二磷酸三钠盐0.5小时处理，连续7天，并观测实验过程中动物的情况，于末次处理24小时后处死动物，取肿瘤块称重，以每组动物的平均瘤重量作为疗效指标。
如表8所示：果糖-1,6-二磷酸三钠盐钠对肿瘤生长的抑制率为54.39％(果糖-1,6-二磷酸三钠盐组与对照组比较***P<0.001)，二甲双胍单独使用与对照组平均瘤重无显著性差异，西格列汀单独使用对肿瘤生长抑制率为46.12％(西格列汀组与对照组比较***P<0.001)，其细胞实验中并未显示出抗肿瘤作用，而整体实验中显示出一定的抗肿瘤作用，表明西格列汀可能通过刺激机体免疫而发挥抗肿瘤作用；FBP与二甲双胍或西格列汀联合使用后，其整体抗肿瘤作用大大提高，抑制率分别达到74.2％和75.3％(联合用药组与对照组比较***P<0.001；二甲双胍联合果糖-1,6-二磷酸三钠盐组与二甲双胍组比较###P<0.001、与果糖-1,6-二磷酸三钠盐组比较#P<0.05；西格列汀联合果糖-1,6-二磷酸三钠盐组与西格列汀组、果糖-1,6-二磷酸三钠盐组比较#P<0.05)。
表8. 1,6-二磷酸果糖联合二甲双胍或西格列汀的抗小鼠肝癌H22模型肿瘤生长药效
组别 Con Met FBP Met+FBP
肿瘤重量(克) 1.66±0.29 1.46±0.23 0.91±0.23*** 0.43±0.20***；###；&
组别 Con STG FBP STG+FBP
肿瘤重量(克) 1.48±0.34 0.78±0.31** 0.67±0.28*** 0.38±0.14***；#；&
注：采用单因素方差分析的方法分析实验数据,用LSD方法检测各组之间显著性差异注(***P<0.001，**P<0.01对比于对照组(Con)；###P<0.001，#P<0.05对比于Met或STG组；&P<0.05对比于FBP组)。FBP：1,6-二磷酸果糖；Met：二甲双胍；STG：西格列汀。
讨论与小结(实施例11至实施例14)——降糖药可显著增强FBP的体内抗癌药效，而对体外抗癌药效无明显影响
本发明发现FBP联合治疗糖尿病剂量的二甲双胍、西格列汀或胰岛素使用可升高和稳定FBP的血药浓度,从而显著提高FBP的整体抗癌药效。相反，超过降糖剂量的二甲双胍虽然其本身有一定的抗癌活性，但此高剂量反而不能提高FBP抗癌效果，说明二甲双胍加强FBP的抗癌药效是其提高和稳定FBP的血药浓度所致，而不是二甲双胍的直接抗癌药效的结果。西格列汀在FBP的同等浓度未见明显体外抗癌活性，而在小鼠肝癌H22模型中，其临床降糖剂量出现一定的抗癌活性。这种整体抗癌活性可能是西格列汀调节整体糖代谢的结果。因此，所述研究结果支持了FBP与降糖药联合，特别是西格列汀联合，在制备新型抗癌药物中的应用。
实施例15.果糖-1,6-二磷酸三钠联合西格列汀对抗高脂饲料喂养引起的体重升高和脂肪堆积
将40只17-19g的ICR雄性小鼠先分为2组。8只正常动物给予基础饲料喂养，饮用纯净水；其余32只肥胖动物给予高脂饲料喂养(高脂饲料含45％基础料包括粗蛋白18％、脂肪4％、纤维8％、钙1.5％、胺基酸8％，和55％添加物包括精炼猪油13％、豆油3％、糖8％以及花生、黄豆、鸡蛋、骨粉、芝麻、玉米、荞麦、盐、多种维生素)同样饮用纯净水；饲养4周后肥胖动物相较正常动物肥胖率达8％后开始分组处理。正常组(Naive)动物依旧基础饲料喂养，模型动物平均分为4组，继续给予高脂饲料，另外根据小鼠饮水量，将一定剂量药物溶于饮用水中处理。其中模型组(Model)动物，饮用纯净水，果糖-1,6-二磷酸组动物饮用水含0.18％的果糖-1,6-二磷酸钠盐含8分子水合物(相当于FBP 300mg/kg)，西格列汀组动物饮用水含0.012％的西格列汀磷酸盐(相当于STG 20mg/kg)，合用组(FBP 500mg/kg+STG 20mg/kg)。连续处理6周，每周测定体重和饮食饮水量，最后一周的最后一天测定体重和量身长(CM)并计算Lee's指数(Lee's INDEX＝体重(g)^(1/3)*1000/体长(cm))，以了解动物肥胖状态；而后脱颈椎处死动物并解剖小鼠，取附睾脂肪垫称重，计算脂肪系数来进一步考察小鼠肥胖程度。处死前一天测试小鼠口服糖耐量。在小鼠禁食12小时后口服灌胃10％的葡萄糖，分别在灌胃后第0min，15min，30min，60min，120min测试小鼠血糖，考察肥胖小鼠的糖代谢变化。
获得以下研究结果：
1.只有西格列汀与FBP联合处理才能对抗高脂食料喂养引起的体重升高。正常动物体重呈时间依赖性增长，6周时间从36.57±2.79g增长到42.07±3.3g。而高脂饲料喂养增长速度明显 高于正常，6周后体重高达47.89±3.34g，与正常组有显著区别(模型组与正常组比较***P<0.001)，说明这种高脂食料喂养ICR小鼠6周能显著升高体重。单独给果糖-1,6-二磷酸三钠盐组以及西格列汀组，体重分别为45.93±5.1g和46.78±6.1g，与模型组无显著区别。西格列汀联合果糖-1,6-二磷酸三钠盐组动物开始处理后显著减缓了动物增长速度，6周后联合用药动物体重为40.98±3.65g，显著低于模型组动物(联合用药组与模型组比较***P<0.001)。
2.FBP能显著对抗高质饲料饲养引起的Lee'S INDEX升高，西格列汀不能进一步加强FBP的此药效。模型组小鼠Lee's指数为353.28±9.64，与正常组动物337.05±9.96相比有显著性差异(**P<0.01)，说明模型组小鼠处于肥胖状态。西格列汀单用组动物Lee＇s指数为346.34±9.36，与模型组无明显区别。而果糖-1,6-二磷酸三钠盐组动物的Lee'S INDEX(326.14±11.10)和西格列汀联合果糖-1,6-二磷酸三钠盐组动物的Lee's INDEX(338.29±9.48)均显著低于模型组的Lee'S INDEX(***P<0.001)，但联合西格列汀未能进一步升高FBP的作用。
3.FBP可显著对抗高脂饲料喂养引起的脂肪系数升高，西格列汀无此作用，但西格列汀联合处理时FBP的药效更好。模型组肥胖小鼠脂肪系数为3.96±0.83％，显著高于正常组小鼠1.58±0.68％(模型组与正常组比较***P<0.001)。处理组动物脂肪系数均有所下降，其中西格列汀单用组为3.28±1.14％，与模型组未形成显著性差别。而果糖-1,6-二磷酸三钠盐组动物(2.81±0.81％)和西格列汀联合果糖-1,6-二磷酸三钠盐组动物(2.50±0.98％)均与模型组有显著差异(果糖-1,6-二磷酸三钠盐组与模型组比较*P<0.05，西格列汀联合果糖-1,6-二磷酸三钠盐组与模型组比较**P<0.01)，其中合用组抗肥胖效果最佳。
4.FBP和西格列汀及其联合不影响正常饮食和正常血糖水平
本实验小鼠一开始对高脂饲料有一个适应性的进食，一开始进食量略低于普通饲料，一周后高脂饲料组小鼠进食量为6.68g/只/天，与正常组6.71g/只/天没有显著区别，处理组进食量相较与模型组略低，分别为西格列汀组5.99g/只/天，果糖-1,6-二磷酸三钠盐组6.46g/只/天，西格列汀联合果糖-1,6-二磷酸三钠盐组6.37/只/天，但们没有显著影响小鼠食欲。
本实验中高脂饲料诱导并没有引起肥胖小鼠的糖耐量变化，FBP和西格列汀及其二者联用同样不影响小鼠的正常血糖水平。禁食12h后，各组血糖水平分别为正常组(4.50±0.66mmol/L)，模型组(4.09±1.06mmol/L)，西格列汀组(5.21±1.22mmol/L)，果糖-1,6-二磷酸三钠盐组(4.24±1,12mmol/L)，西格列汀联合果糖-1,6-二磷酸三钠盐组(4.66±1.60mmol/L)，组间均无显著性区别。灌胃葡萄糖后，小鼠血糖有一个急剧升高，第15min时血糖水平分别为正常组(12.91±2.57mmol/L)，模型组(13.26±3.63mmol/L)，西格列汀组(13.74±4.27mmol/L)，果糖-1,6-二磷酸三钠盐组(14.28±2.23mmol/L)，西格列汀联合果糖-1,6-二磷酸三钠盐组(13.72±3.83mmol/L)，第30min时各组血糖开始下落，120min时各组血糖均回到初始水平。可见FBP和西格列汀及其联合不影响正常血糖水平和糖耐量。
综上述结果，证明FBP可促进脂肪代谢，西格列汀可进一步加强FBP促进脂肪代谢的药效，这就支持了FBP联合西格列汀用于减肥和防治2型糖尿病的医药用途。
讨论与小结——FBP联合西格列汀对抗高脂饲料喂养引起的体重升高和脂肪堆积,因此FBP联合西格列汀制备的复方制剂可用于减肥和预防脂肪肝以及2型糖尿病
肥胖不仅直接影响了生活质量，更指示着代谢障碍和后续糖尿病，因此防止肥胖是预防糖尿病的重要措施。对高脂食料喂养产生的肥胖，FBP和西格列汀联合具有显著的对抗作用，FBP单用能降低肥胖程度但不能减轻体重，而西格列汀单用均未见显著药效。这就强烈的支持了FBP联合西格列汀在制备减肥药和预防脂肪肝以及2型糖尿病的药物中的用途。
实施例16.果糖-1，6-二磷酸三钠对抗治疗肿瘤化疗药紫杉醇诱导的外周神经痛
本研究采用紫杉醇(2.8mg/kg,10ml/kg)隔日(第1,3,5,7天)腹腔注射共4次的方法诱导体重20-24g的ICR雌性小鼠外周神经痛模型，并用此模型观测果糖-1，6-二磷酸对化疗药物引起的外周神经痛的预防作用。紫杉醇类抗肿瘤药已成为人类抗击恶性肿瘤的一线药品。紫杉醇的剂量限制性毒性主要为神经毒性和骨髓抑制，后者已成功通过应用粒细胞集落刺激因子予以克服，但表现为神经病理性疼痛的神经毒性至今仍然世界性难题，因为这种化疗所致的疼痛对目前任何临床上所使用的镇痛药物都不敏感，常导致一部分患者被迫减量直至停药，从而严重影响化疗效果甚至使化疗归于失败，部分紫杉醇化疗痛并不会因为停药而迅速终止，常常迁延数月甚至数年，严重影响肿瘤患者的生存质量。可见，紫杉醇引起的外周神经疼痛对癌症治疗后的疼痛具有代表性，用紫杉醇紫引起的疼痛动物模型也具有代表性。
用热板法筛选热敏感反应相对均匀的小鼠进行实验。将合格的21只小鼠分为空白对照组(生理盐水组，ip)、紫杉醇模型组和果糖-1,6-二磷酸三钠盐水合物(400mg/kg,10ml/kg,ig)预防治疗组，每组7只。果糖-1,6-二磷酸每天处理一次，每逢给紫杉醇那天果糖-1,6-二磷酸预先处理2小时。在紫杉醇停药后继续给果糖-1,6-二磷酸三钠，直到实验结束。
每次于下午2-4点用热板实验(52℃±0.3)测定小鼠后爪的热敏反应。将小鼠双侧后爪置于热板仪的热板上，当动物感觉热刺激引起的疼痛时，动物则会出现舔后爪或缩回后抓，记录舔后爪或缩回后抓潜伏期，潜伏期越短说明痛阈值越低，延长给紫杉醇动物的疼阈值说明对其化疗药诱导的神经疼痛有对抗作用。在结束紫杉醇注射后继续给果糖-1,6-二磷酸并继续测定各组动物的热敏反应。实验结果证明，果糖-1,6-二磷酸对紫杉醇引起的小鼠外周神经疼痛具有显著的抑制作用。如图所示，在给紫杉醇前，三组动物的后爪回缩潜伏期相当，在给紫杉醇后的第7天、第9天、第11天和第13天，模型组的后爪回缩潜伏期明显短于空白对照组，说明紫杉醇诱导了显著的外周神经疼痛(**P<0.01，***P<0.001)；果糖-1,6-二磷酸组的第7天和以后各时间点的潜伏期均明显长于模型组(**P<0.01，***P<0.001)。以上实验结果支持了果糖-1,6-二磷酸防治癌症治疗后疼痛特别是化疗药引起的外周神经疼痛的临床使用价值。
讨论与小结——FBP对抗抗癌药紫杉醇诱导的外周神经痛，但有效剂量范围窄小
外周神经疼痛与常用化疗药紫杉醇广泛应用于卵巢癌、乳腺癌、肺癌及头颈部癌等多种恶性肿瘤，但毒副反应较大，其中神经毒性是主要的剂量限制性毒性。与临床神经毒副作用一致，抗癌剂量的紫杉醇可快速诱导小鼠外周神经疼痛模型；适当剂量的果糖-1,6-二磷酸与紫杉醇同时处理可显著对抗紫杉醇引起的小鼠外周神经疼痛。特别要指出的是，低剂量FBP(200mg/kg)和高剂量FBP(400mg/kg)呈现出相反的药效动态变化，即高剂量FBP在造模的早期也是FBP处理的早期显示出对抗外周神经痛的作用趋势，但随着治疗时程的延长这种 药效趋势逐渐消失，而低剂量出现随处理时程延长药效逐渐增强的变化趋势。研究结果不仅支持了FBP在防治化疗药神经毒副作用的潜力，特别是与常用化疗药(包括紫杉烷类、长春花碱类、铂类和蛋白酶体抑制剂)联合应用，而且进一步指出无法通过提高FBP的剂量来提高FBP药效。综上所述，本发明前述发现的随处理时程延长出现的FBP降解酶FBPase水平上调解释了高剂量药效反而无效的现象，抗糖尿病药物与FBP联合制成复合制剂可克服此FBP缺陷。
研究结果的总结(实施例1至实施例16)
本发明所述新型FBP药物，其技术特征在于其药效成分包括果糖-1,6-二磷酸(FBP)和一种或多种能减缓FBP在体内降解的成分(也称FBP血药浓度稳定剂)。所述稳定剂作为药效成分，其技术特征主要通过提高和稳定FBP的血药浓度来实现药效，但也不排除稳定剂本身对代谢调节作用所产生的药效与FBP产生的药效相加或协同。因此，专业人员可以理解，本发明所述的新型FBP药物除适用于防治肥胖和化疗药治疗癌症引起的外周神经痛之外，还适用于已公开的FBP各种适应症包括用于改善冠心病的心绞痛、急性心肌梗死、心律失常和心力衰竭的心肌缺血以及病毒性心肌炎的辅助治疗，用于改善脑梗死、脑出血等引起的脑缺氧症状，用于防治血液系统癌症和各种实体癌症(中国发明专利：ZL201110066413.6)，用于防治糖尿病并发症(中国发明专利：CN00112023.9)，用于癫痫(中国发明专利：ZL201310498212.2)和神经退化性疾病(中国发明专利：CN01107519.8)。基于本发明提供的新型FBP药物的药效本质是调控代谢从而改善代谢功能和纠正失常的代谢状态，因此专业人员可以理解其它在此未涉及的代谢性疾病和与代谢紊乱密切相关的疾病(如精神障碍性疾病-精神分裂症、抑郁症等)也包括在所述的新型FBP药物的适应症内。同理，本专业领域人员应当理解，通过任何手段来提高和稳定FBP血药浓度以提高FBP药效的思路均在本专利保护范围内。
实施例17.果糖-1,6-二磷酸-西格列汀双层片的制备
9.双层片处方：
Figure PCTCN2017114371-appb-000011
分别将A层与B层按处方将活性成分、填充剂与粘合剂混合，采用湿法制粒机湿法制粒(I搅拌II剪切，5分钟)，在箱式干燥箱中60℃干燥并整粒；将A、B层干燥颗粒按照处方分别与 崩解剂和润滑剂在混合机中混合约40分钟后，双层片压片机压制，即得果糖-1,6-二磷酸三钠盐-西格列汀双层片。所得双层片外光完整、光洁，脆碎度均≤0.9％，片重无显著性差异，崩解时限均≤7分钟。所得双层片规格为0.5克/片，每片含果糖-1,6-二磷酸三钠0.125g，临床使用单次口服20片，每日三次。
10a.溶出度检验：
Figure PCTCN2017114371-appb-000012
10b.溶出度检验：
Figure PCTCN2017114371-appb-000013
11.稳定性考察：
Figure PCTCN2017114371-appb-000014
实施例18.果糖-1,6-二磷酸-西格列汀复方缓释微丸的制备
素丸的制备：按照果糖-1,6-二磷酸三钠盐:微晶纤维素:乳糖＝6:2.5:1.5称取药物及辅料，辅料过筛后混匀，加水制成软材，经挤出滚圆机制得果糖-1,6-二磷酸三钠盐丸。所得微丸于50℃干燥6小时后取18-24目筛所得微丸备用。
缓释微丸包衣：以Eudragit Ne30d(聚合物浓度5％)、滑石粉(相当于聚合物用量60％)加去离子水适量制成包衣液，而后采用流化床包衣。
复方缓释微丸的制备：精密称取一定量的西格列汀，溶于去离子水中，采用流化床装置将西格列汀水溶液喷至果糖-1,6-二磷酸三钠盐缓释微丸表面，制得复方微丸。
本领域内人员可以理解，所述FBP的药用形式包括果糖-1,6-二磷酸原形和果糖-1,6-二磷酸及其前药或衍生物在药学上可接受的盐包括但不限于化合物所形成的铵、钠、钾、钙、镁、锰、铜、甲胺、二甲胺、三甲胺、丁酸、乙酸、二氯乙酸、盐酸、氢溴酸、硫酸、三氟乙酸、柠檬酸或者马来酸的酸根所成的盐及水合物。优选地，以果糖-1,6-二磷酸三钠盐的8分子水 合物为药用形式；所述FBP稳定剂包括以西格列汀为代表的DPP-4抑制剂类、GLP-1受体激动剂类、以二甲双胍为代表的双胍类、胰岛素类和格列酮类以及果糖-1,6-二磷酸酶抑制剂的现有降糖物质，它们的药用形式可以是现有的药用形式、原形及其各自的前药或衍生物在药学上可接受的盐包括但不限于化合物所形成的铵、钠、钾、钙、镁、锰、铜、甲胺、二甲胺、三甲胺、丁酸、乙酸、二氯乙酸、盐酸、氢溴酸、磷酸、硫酸、三氟乙酸、柠檬酸或者马来酸的酸根所成的盐及水合物。
在实际应用中，FBP可与上述FBP血药浓度稳定剂中的一种或多种按合适比例构成药物的药效成分，所述药效成分与药学上可接受的赋形剂或载体制备各种普通药物制剂(包括经口服和注射处理的制剂)、栓剂、膜剂和应用新材料和新技术制备的各种新型制剂(包括但不限于控释双层片，控释纳米制剂、微囊、微球、肠溶制剂和各种长效制剂)。优选地，将所述新型FBP药物制备成为可实现按顺序释放的双层片或具有缓控释特点的长效缓释制剂。所述可实现先后释放的双层片，其技术特征在于所述稳定剂先释放15分钟至60分钟，优选地先释放30分钟。由于所述稳定剂中的其中一种与FBP联合就可达到稳定FBP血药浓度的目的，因此在实际应用中FBP优先与一种稳定剂联合在制备新型FBP药物中的应用，再优选与西格列汀联合。

Claims (7)

  1. 一种由果糖-1,6-二磷酸与血药浓度稳定剂组成的组合物在制备预防和治疗代谢性疾病和代谢功能失常相关性疾病的药物中的应用，所述药物包含果糖-1,6-二磷酸和其血药浓度稳定剂以及药学上可以接受的赋形剂或者载体。
  2. 根据权利要求1所述的一种由果糖-1,6-二磷酸与其血药浓度稳定剂组成的组合物在制备预防和治疗代谢性疾病和代谢功能失常相关性疾病的药物中的应用，其特征在于，所述的血药浓度稳定剂包括以西格列汀为代表的二肽基肽酶-4(DPP-4)抑制剂类、胰高血糖素样肽1(GLP-1)受体激动剂类、以二甲双胍为代表的双胍类、胰岛素类和格列酮类也称为噻唑烷二酮类和果糖-1,6-二磷酸酶抑制剂。
  3. 根据权利要求1所述的一种由果糖-1,6-二磷酸与其血药浓度稳定剂组成的组合物在制备预防和治疗代谢性疾病和代谢功能失常相关性疾病的药物中的应用，其特征在于，所述果糖-1,6-二磷酸原形和果糖-1,6-二磷酸及其前药或衍生物在药学上可接受的盐包括但不限于化合物所形成的铵、钠、钾、钙、镁、锰、铜、甲胺、二甲胺、三甲胺、丁酸、乙酸、二氯乙酸、盐酸、氢溴酸、硫酸、三氟乙酸、柠檬酸或者马来酸的酸根所成的盐及水合物。
  4. 根据权利要求1所述的一种由果糖-1,6-二磷酸与其血药浓度稳定剂组成的组合物在制备预防和治疗代谢性疾病和代谢功能失常相关性疾病的药物中的应用，其特征在于，以果糖-1,6-二磷酸三钠盐的8分子水合物为药用形式。
  5. 根据权利要求1所述的一种由果糖-1,6-二磷酸与其血药浓度稳定剂组成的组合物在制备预防和治疗代谢性疾病和代谢功能失常相关性疾病的药物中的应用，其特征在于，所述代谢性疾病和代谢功能失常相关性疾病具体包括：冠心病引起的心绞痛、急性心肌梗死、心律失常和心力衰竭引起的心肌缺血以及病毒性心肌炎，脑梗死，脑出血等引起的脑缺氧症状，血液系统癌症和各种实体瘤，糖尿病及其并发症，脂肪肝，癫痫、神经退化性疾病和精神行为障碍性疾病。
  6. 根据权利要求1所述的一种由果糖-1,6-二磷酸与其血药浓度稳定剂组成的组合物在制备预防和治疗代谢性疾病和代谢功能失常相关性疾病的药物中的应用，其特征在于，所述药物中，果糖-1,6-二磷酸三钠盐的8分子水合物与二甲双胍的质量比例为1:0.1～1:1；果糖-1,6-二磷酸三钠盐的8分子水合物与西格列汀的质量比例为1:0.001～1:0.5；果糖-1,6-二磷酸三钠盐的8分子水合物与胰岛素的比例为1：0.02～1：0.002。
  7. 根据权利要求1-6任一所述的应用，其特征在于，所述药物的制剂形式包括注射剂、普通片剂、颗粒剂、胶囊剂、双层片剂、控释双层片、缓释片剂、单室控释片剂、分散片、肠溶片、肠溶胶囊、定点释药片剂、缓控释胶囊剂、缓释微丸、含微丸或小片的胶囊、靶向制剂。
PCT/CN2017/114371 2017-08-10 2017-12-04 果糖-1,6-二磷酸与其血药浓度稳定剂组合物的医药用途 WO2019029064A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US16/637,766 US20200246364A1 (en) 2017-08-10 2017-12-04 Medical application of composition of fructose-1,6-bisphosphate and blood concentration stabilizer thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201710678435.5 2017-08-10
CN201710678435.5A CN107362363B (zh) 2017-08-10 2017-08-10 果糖-1,6-二磷酸与其血药浓度稳定剂组合物的医药用途

Publications (1)

Publication Number Publication Date
WO2019029064A1 true WO2019029064A1 (zh) 2019-02-14

Family

ID=60309266

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/114371 WO2019029064A1 (zh) 2017-08-10 2017-12-04 果糖-1,6-二磷酸与其血药浓度稳定剂组合物的医药用途

Country Status (3)

Country Link
US (1) US20200246364A1 (zh)
CN (1) CN107362363B (zh)
WO (1) WO2019029064A1 (zh)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107362363B (zh) * 2017-08-10 2020-06-16 浙江大学 果糖-1,6-二磷酸与其血药浓度稳定剂组合物的医药用途
CN111657861B (zh) * 2020-06-04 2022-02-25 浙江大学 基于双光子显微镜技术的溶栓药效评价方法
CN112245442B (zh) * 2020-09-29 2022-04-29 北京华靳制药有限公司 果糖二磷酸钠反渗透浓缩液及其制备方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1265308A (zh) * 2000-01-17 2000-09-06 欧阳平凯 1,6－二磷酸果糖镁盐在医药中的应用
CN102125569A (zh) * 2011-03-18 2011-07-20 浙江大学 一种1,6-二磷酸果糖在制备抗癌药物中的应用
CN102755292A (zh) * 2011-04-26 2012-10-31 沈阳药科大学 一种含有二甲双胍囊泡类给药系统及其应用
CN103599115A (zh) * 2013-10-22 2014-02-26 浙江大学 一种组合物在制备抗癫痫药物中的应用
CN107362363A (zh) * 2017-08-10 2017-11-21 浙江大学 果糖‑1,6‑二磷酸与其血药浓度稳定剂组合物的医药用途
CN107375934A (zh) * 2017-08-10 2017-11-24 浙江大学 含有果糖‑1，6‑二磷酸的组合物在制备抗肿瘤药物中的应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101721419A (zh) * 2009-11-06 2010-06-09 连晓媛 1,6-二磷酸果糖防治帕金森氏综合症的应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1265308A (zh) * 2000-01-17 2000-09-06 欧阳平凯 1,6－二磷酸果糖镁盐在医药中的应用
CN102125569A (zh) * 2011-03-18 2011-07-20 浙江大学 一种1,6-二磷酸果糖在制备抗癌药物中的应用
CN102755292A (zh) * 2011-04-26 2012-10-31 沈阳药科大学 一种含有二甲双胍囊泡类给药系统及其应用
CN103599115A (zh) * 2013-10-22 2014-02-26 浙江大学 一种组合物在制备抗癫痫药物中的应用
CN107362363A (zh) * 2017-08-10 2017-11-21 浙江大学 果糖‑1,6‑二磷酸与其血药浓度稳定剂组合物的医药用途
CN107375934A (zh) * 2017-08-10 2017-11-24 浙江大学 含有果糖‑1，6‑二磷酸的组合物在制备抗肿瘤药物中的应用

Also Published As

Publication number Publication date
CN107362363B (zh) 2020-06-16
US20200246364A1 (en) 2020-08-06
CN107362363A (zh) 2017-11-21

Similar Documents

Publication Publication Date Title
CN107375934B (zh) 含有果糖-1，6-二磷酸的组合物在制备抗肿瘤药物中的应用
JP2013505897A (ja) Ｓ−アデノシルメチオニン製剤の改善された薬物動態
JP2008505960A (ja) 多剤癌治療
WO2019029064A1 (zh) 果糖-1,6-二磷酸与其血药浓度稳定剂组合物的医药用途
US20140235558A1 (en) Pharmaceutical composition having activity of anticancer
EP2838517B1 (en) Orally available pharmaceutical formulation suitable for improved management of movement disorders
CN103764144A (zh) Pi3k抑制剂与mek抑制剂的协同组合
KR102193989B1 (ko) 약학 조성물 및 이의 제조방법
JP2022500390A (ja) キナーゼ阻害剤としてのアルキニルニコチンアミド化合物
Nagao et al. From medications to surgery: advances in the treatment of motor complications in Parkinson’s disease
Geng et al. Extensive therapeutic effects, underlying molecular mechanisms and disease treatment prediction of Metformin: a systematic review
KR20110007255A (ko) 고형 의약 제제
KR20190046675A (ko) 간질환 예방 또는 치료용 의약 조성물
CN112294820B (zh) 一种减少高血压患者血管内皮损伤的组合物
JP2023544981A (ja) 癌の代謝療法
CN112691107B (zh) 一种药物组合物及其应用
CN111053780A (zh) 奥西替尼的药物组合物及其应用
WO2008050483A1 (fr) Préparation cardiovasculaire
CN112618533A (zh) 一种左旋多巴/甜菜碱药物组合物以及应用
CN102125569A (zh) 一种1,6-二磷酸果糖在制备抗癌药物中的应用
US20030069203A1 (en) Method for increasing human performance by reducing muscle fatigue and recovery time through oral administration of adenosine triphosphate
CN103127121A (zh) 雷沙吉兰/叶酸类化合物的药物组合物及其用途
RU2709502C1 (ru) Фармацевтическая композиция для парентерального капельного введения
CN104840480A (zh) 二甲双胍/叶酸/维生素b12药物组合物的新用途
EP1933823A1 (en) Pharmaceutical composition on basis of reverse transcriptase inhibitor and meldonium

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17920979

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17920979

Country of ref document: EP

Kind code of ref document: A1