WO2015101208A1 - 维生素d及其组合物的抗菌用途 - Google Patents

维生素d及其组合物的抗菌用途 Download PDF

Info

Publication number
WO2015101208A1
WO2015101208A1 PCT/CN2014/094893 CN2014094893W WO2015101208A1 WO 2015101208 A1 WO2015101208 A1 WO 2015101208A1 CN 2014094893 W CN2014094893 W CN 2014094893W WO 2015101208 A1 WO2015101208 A1 WO 2015101208A1
Authority
WO
WIPO (PCT)
Prior art keywords
vitamin
compound
medium
urethritis
cystitis
Prior art date
Application number
PCT/CN2014/094893
Other languages
English (en)
French (fr)
Inventor
周明东
Original Assignee
上海泽生科技开发有限公司
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 上海泽生科技开发有限公司 filed Critical 上海泽生科技开发有限公司
Priority to JP2016544430A priority Critical patent/JP2017501203A/ja
Priority to AU2014375577A priority patent/AU2014375577A1/en
Priority to EP14876219.8A priority patent/EP3111939A4/en
Priority to RU2016131454A priority patent/RU2016131454A/ru
Priority to CN201480071325.7A priority patent/CN105916510A/zh
Priority to US15/109,110 priority patent/US20160324876A1/en
Priority to CA2935597A priority patent/CA2935597A1/en
Publication of WO2015101208A1 publication Critical patent/WO2015101208A1/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
    • A61K31/59—Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene ring systems
    • A61K31/593—9,10-Secocholestane derivatives, e.g. cholecalciferol, i.e. vitamin D3
    • 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
    • A61K31/59—Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene ring systems
    • A61K31/592—9,10-Secoergostane derivatives, e.g. ergocalciferol, i.e. vitamin D2
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system

Definitions

  • the present invention relates to the use of a vitamin D compound for the preparation of a medicament for the prevention or treatment of cystitis, urethritis and/or urinary tract infection. More particularly, it relates to pharmaceutical compositions and pharmaceutical preparations for the prevention or treatment of cystitis, urethritis and/or urinary tract infections.
  • Urinary tract infection refers to the inflammation caused by pathogens in the urinary tract and invading the mucosa or tissues of the urinary tract. Bacterial urinary tract infections are the most common. Urinary tract infection is a common clinical disease. According to the US outpatient medical investigation and hospital outpatient medical investigation, there are nearly 7 million outpatient cases and 1 million emergency cases every year, and 100,000 hospitalizations, especially female patients. A large number of reports have reported a high recurrence rate of urinary tract infections, and about 25-30% of female patients have recurrent infections. More studies have shown that up to 44.1% of female patients relapse within one year. The high recurrence rate of urinary tract infections has caused significant economic losses, and some scholars estimate that the annual cost of urinary tract infection treatment in the United States is as high as $1.6 billion.
  • Urethritis is a common disease, which refers to inflammation of the urethra mucosa, which is good for women. Clinically, it can be divided into acute and chronic, non-specific urethritis and gonococcal urethritis. Usually caused by retrograde invasion of the urethra by pathogenic bacteria. When bacteria enter the bladder or the kidneys multiply in the urine, it causes urethritis.
  • cystitis an inflammation that occurs in the bladder.
  • Bacterial cystitis is the most common type of cystitis. The cause of the disease is that the bacteria on the surface of the body invade from the urethra and proliferate in the bladder. It often has frequent urination, urgency, urinary burning or pain, hematuria and other symptoms. The process of seizures is mostly rapid, causing severe discomfort within half a day, even spreading to the kidneys within a few days and becoming acute pyelonephritis.
  • E. coli is one of the most common bacteria causing cystitis, especially in the case of insufficient water and urine, it is easier for bacteria to have a chance to breed in the bladder.
  • pathogenic bacteria are not easy to stay and multiply in the urinary tract, so they will not cause infection. This is due to the physiological defense function of the urinary and reproductive systems, the most important of which is the secretion of antimicrobial peptides. Once a pathogen invades, the antimicrobial peptide can act as a bactericidal agent, and the reaction process is quite rapid, so that the pathogen has not been propagated in the body and has been eliminated. However, once this anti-infective defense function is weakened, the pathogenic bacteria will become ill and cause infection.
  • Antibacterial peptides are mostly peptides of 20 to 60 amino acids, which are generally positively charged and amphiphilic, which enables them to reach higher concentrations in both water-soluble and hydrophobic environments (eg membranes). Antimicrobial peptides are rapidly adsorbed on the membrane of invading microorganisms and killed.
  • the most studied human antimicrobial peptides are defensins and cathelicidin (also known as LL-37). Under normal conditions, low levels of cathelicidin can be detected in the human urinary tract. Once microbial invasion occurs, the mRNA expression of cathelicidin is rapidly increased. Chromek et al. infected cathelicidin knockout and wild-type mice with E. coli, respectively, and the number of bacteria in the lower urinary tract of the knockout mice was significantly higher than that of the wild type.
  • Vitamin D plays an important role in human nutrition. It is a fat-soluble vitamin.
  • the human body mainly exists in the form of vitamin D 2 (ergocalciferol) and vitamin D 3 (cholecalciferol).
  • the human body mainly relies on food intake and skin synthesis to obtain vitamin D.
  • the 7-dehydrocholesterol in the skin is synthesized by ultraviolet light having a wavelength of 290 to 320 mm to synthesize a vitamin D precursor.
  • Vitamin D from both sources cannot be circulated in the blood for a long time, but is immediately taken up by adipose tissue or liver for storage or activation.
  • Activated vitamin D can bind to its receptor VDR and enter the nucleus to regulate the expression of various genes.
  • VDR is widely present in various cells of the human body.
  • vitamin D has a wide range of biological effects, such as the promotion of calcium and phosphorus.
  • Vitamin D deficiency or deficiency has an extremely adverse effect on the body's immunity, reducing the resistance to recurrent urinary tract infections. Although there is no direct evidence from epidemiological studies that vitamin D deficiency leads to a decline in immunity, epidemiological studies have shown a decrease in serum 25(OH)D concentration and an increase in upper respiratory tract infection rate. Epidemiological studies have shown that vitamin D deficiency in mothers and infants is associated with a high incidence of acute lower respiratory tract infections in infants. Gibney et al. have shown that vitamin D deficiency is associated with the incidence of tuberculosis, and these studies have revealed vitamin D deficiency or deficiency. Systemic immunity may have a negative impact.
  • Urinary tract infection can be divided into urethritis, cystitis, and pyelonephritis according to the infection site of the pathogen; renal function damage or even failure is caused by severe pyelonephritis, and Gram-negative bacilli sepsis is caused by urinary tract infection, and a small number of sepsis patients can occur.
  • Significant shock accompanied by clinical manifestations of heart, brain, and kidney ischemia.
  • antibiotic therapy is mainly used for the treatment of cystitis, urethritis and/or urinary tract infection.
  • the commonly used antibiotics are: TMP-SMX, nitrofurantoin, ciprofloxacin, levofloxacin or its chemical analogues.
  • Some penicillin such as amoxicillin.
  • antibiotics can reduce cystitis, urethritis, and/or urinary tract infections to a certain extent, the cost of widespread use of antibiotics is also high: the increasing number of resistant bacteria and the number of normal flora in the patient's body.
  • Escherichia coli isolated from patients with cystitis, urethritis, and/or urinary tract infections was significantly resistant to ciprofloxacin, amoxicillin-clavulanic acid, and TMP-SMX between 1998 and 2003. increase.
  • Antibiotic abuse will lead to an increase in drug-resistant bacteria.
  • the resistance of bacteria to drugs makes the use of antibiotics increase or constantly seeks to sterilize more antibiotics, resulting in economic waste; and its blind bactericidal effect also leads to the reduction of beneficial bacteria in the body. , causing unnecessary harm to the human body.
  • Antibiotics can quickly inhibit the proliferation of bacteria, but can not improve the body's own innate immunity.
  • a major cause of cystitis, urethritis and/or urinary tract infections is the decline in the body's inherent immunity, and enhancing its innate immunity is one way to reduce the recurrence rate of cystitis, urethritis and/or urinary tract infections.
  • Active vitamin D and VDR vitamin d receptor
  • the present application provides a pharmaceutical preparation for effectively reducing the recurrence of cystitis, urethritis and/or urinary tract infection, improving the quality of life of patients and reducing antibiotics by reducing the recurrence of cystitis, urethritis and/or urinary tract infections.
  • the application of the current clinical antibiotic abuse situation The use of vitamin D compounds in the preparation of a medicament for the prevention or treatment of cystitis, urethritis and/or urinary tract infections will greatly change the prevention and treatment of cystitis, urethritis and/or urinary tract infections.
  • the present invention relates to the use of a vitamin D compound for the preparation of a medicament for the prevention or treatment of cystitis, urethritis and/or urinary tract infection.
  • the present invention relates to the use of a vitamin D compound for the preparation of a medicament for the prevention or treatment of cystitis.
  • the present invention relates to the use of a vitamin D compound for the preparation of a medicament for the prevention or treatment of urethritis.
  • the present invention relates to the use of a vitamin D compound for the preparation of a medicament for the prevention or treatment of a urinary tract infection.
  • the urinary tract infection includes an upper urinary tract infection and a lower urinary tract infection, and may be classified into urethritis, cystitis, and pyelonephritis according to the infection site of the pathogen.
  • the vitamin D compound is a vitamin D receptor ligand (VDR ligand).
  • the vitamin D compound is an agonist of the vitamin D receptor.
  • the vitamin D compound comprises a vitamin D 2 compound, a vitamin D 3 compound, an isomer thereof, an activated form or the like.
  • the vitamin D compound comprises vitamin D 2 , D 3 and analogs thereof.
  • the vitamin D compound is an activated form of vitamin D, e.g. calcifediol (25-OH D 3) or calcitriol (1,25- (OH) 2 D 3 ).
  • the present invention relates to a pharmaceutical composition for preventing or treating cystitis, urethritis and/or urinary tract infection, the composition comprising a vitamin D compound and others for preventing or treating cystitis, urethritis and/or A drug for urinary tract infection.
  • the present invention relates to a pharmaceutical composition for preventing or treating cystitis, which comprises a vitamin D compound and other drugs for preventing or treating cystitis.
  • the present invention relates to a pharmaceutical composition for preventing or treating urethritis, which comprises a vitamin D compound and other drugs for preventing or treating urethritis.
  • the present invention relates to a pharmaceutical composition for preventing or treating urinary tract infections, which comprises a vitamin D compound and other drugs for preventing or treating urinary tract infections.
  • the vitamin D compound is a vitamin D receptor ligand (VDR ligand).
  • the vitamin D compound is an agonist of the vitamin D receptor.
  • the vitamin D compound comprises a vitamin D 2 compound, a vitamin D 3 compound, an isomer thereof, an activated form or the like.
  • the vitamin D compound comprises vitamin D 2 , D 3 and analogs thereof.
  • the vitamin D compound is an activated form of vitamin D, e.g. calcifediol (25-OH D 3) or calcitriol (1,25- (OH) 2 D 3 ).
  • other drugs for preventing or treating cystitis, urethritis and/or urinary tract infections are certain antibiotics such as TMP-SMX, nitrofurantoin, ciprofloxacin, levofloxacin. (levofloxacin) or a chemical analogue thereof, penicillin, amoxicillin, and the like.
  • the pharmaceutical compositions of the invention are suitable for topical or oral administration to a subject.
  • the pharmaceutical compositions of the invention may be specially formulated for administration in solid or liquid form, such as oral administration, parenteral administration, topical administration, and the like.
  • the present invention provides a pharmaceutical preparation for preventing or treating cystitis, urethritis and/or urinary tract infection, the pharmaceutical preparation comprising a vitamin D compound and a pharmaceutically acceptable carrier.
  • the present invention provides a pharmaceutical preparation for preventing or treating cystitis, which comprises a vitamin D compound and a pharmaceutically acceptable carrier.
  • the present invention provides a pharmaceutical preparation for preventing or treating urethritis, which comprises a vitamin D compound and a pharmaceutically acceptable carrier.
  • the present invention provides a pharmaceutical preparation for preventing or treating a urinary tract infection, which comprises a vitamin D compound and a pharmaceutically acceptable carrier.
  • the vitamin D compound is a vitamin D receptor ligand (VDR ligand).
  • the vitamin D compound is an agonist of the vitamin D receptor.
  • the vitamin D compound comprises a vitamin D 2 compound, a vitamin D 3 compound, an isomer thereof, an activated form or the like.
  • the vitamin D compound comprises vitamin D 2 , D 3 and analogs thereof.
  • the vitamin D compound is an activated form of vitamin D, e.g. calcifediol (25-OH D 3) or calcitriol (1,25- (OH) 2 D 3 ).
  • the pharmaceutically acceptable carrier is one such as: sugars, starches, celluloses and derivatives thereof, gelatin, talc, excipients, oils, polyols, Other non-toxic compatible substances used in esters, buffers, alginic acid or pharmaceutical preparations.
  • the composition of the present invention can be added to various conventional excipients required for preparing different dosage forms, such as a disintegrator, a lubricant, a binder, an antioxidant, a complexing agent and the like, and can be made into any conventional preparation method.
  • a commonly used oral dosage form such as dispersible tablets, granules, capsules, oral liquids and the like.
  • the pharmaceutical preparation of the present invention may be in the form of a tablet, a capsule, an injection, an infusion, an inhalation ointment or a suppository, but is not limited thereto.
  • the present invention relates to a kit for preventing or treating cystitis, urethritis and/or urinary tract infection, comprising a pharmaceutical composition or a pharmaceutical preparation as described above and how to use the composition or Instructions for pharmaceutical preparations.
  • the present invention is directed to a kit for preventing or treating cystitis, which comprises a pharmaceutical composition or a pharmaceutical preparation as described above and instructions on how to use the composition or pharmaceutical preparation.
  • the present invention is directed to a kit for preventing or treating urethritis, which comprises a pharmaceutical composition or a pharmaceutical preparation as described above and instructions on how to use the composition or pharmaceutical preparation.
  • the present invention is directed to a kit for preventing or treating a urinary tract infection, comprising a pharmaceutical composition or a pharmaceutical preparation as described above and instructions on how to use the composition or pharmaceutical preparation.
  • vitamin D compound includes any vitamin D compound capable of preventing or treating cystitis, urethritis and/or urinary tract infection, and in general, is a vitamin D receptor ligand (VDR ligand) and can be prevented. Or a vitamin D compound for treating cystitis, urethritis and/or urinary tract infection is considered to be within the scope of the invention, and the vitamin D compound is preferably an agonist of the vitamin D receptor.
  • vitamin D compounds are intended to include ring-opening steroids, and examples of specific vitamin D compounds suitable for use in the methods of the invention are further described herein.
  • the vitamin D compound includes a vitamin D 2 compound, a vitamin D 3 compound, an isomer thereof, an activated form or the like.
  • vitamin D 3 compounds form vitamin D 3 compounds, which are ligands of the vitamin D receptor (more preferably an agonist).
  • the vitamin D 2 compound and the vitamin D 3 compound include vitamin D 2 , D 3 and the like, respectively.
  • the vitamin D compound may be a steroid, a steroid such as an open loop, e.g. calciferol (VD 3), calcifediol (25-OH D 3) or calcitriol (1,25 -(OH) 2 D 3 ).
  • antibiotics such as TMP-SMX, nitrofurantoin, ciprofloxacin, levofloxacin or its chemical analogues, penicillin, amoxicillin and the like.
  • pharmaceutically acceptable means suitable for use in contact with human and animal tissues within the scope of sound medical judgment without undue toxicity, irritation, allergies or other problems or complications.
  • pharmaceutically acceptable carrier includes pharmaceutically acceptable materials, compositions or vehicles, such as liquid or solid fillers, diluents, excipients, solvents or encapsulating materials which are involved in the subject matter An organ or part of the body is carried or transported to another organ or part of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the patient.
  • administering includes the introduction of a vitamin D compound into a subject to exert their intended function.
  • routes of administration include injection (subcutaneous, intravenous, parenteral, intraperitoneal), oral, inhalation, rectal, transdermal.
  • Oral administration is preferred if the skin is instilled via the bladder.
  • the formulations of the invention may be administered orally, or parenterally, intravenously, subcutaneously, intramuscularly, transdermally, intranasally, or rectally, or by inhalation.
  • Dosage forms for oral administration include tablets, pills, powders, granules, liquids, suspensions, syrups, capsules and the like.
  • Figure 1 is an antibacterial experiment in which VD3 stimulates bladder epithelial cells. 10 ⁇ L of different concentrations of the test solution or vehicle are added to each well of a six-well plate.
  • Figure 2 is a colony forming unit (CFU) of 5637 cell lysate in each dose group in an antibacterial experiment in which VD3 stimulates bladder epithelial cells.
  • CFU colony forming unit
  • Fig. 3 is an antibacterial experiment in which VD3 stimulates bladder epithelial cells.
  • the CFU in the lysate of each dose group of 5637 cells was counted, and the ratio of each group to the CFU of the ethanol solvent control group was calculated, and the relative CFU was obtained, and the statistical results of repeated experiments were repeated.
  • the statistical test method is One-Way ANOVA LSD Test. * indicates t ⁇ 0.05 compared with the ethanol control group, and ** indicates t ⁇ 0.01 compared with the ethanol control group.
  • Figure 4 shows the ELISA detection of LL-37 in the lysate of each dose group in the antibacterial experiment of VD3 stimulating bladder epithelial cells. the result of.
  • Figure 5 is a CFU of SV-HUC1 cell lysate in each dose group in an antibacterial experiment in which VD3 stimulates urothelial cells.
  • Drug name supplier Item number lot number Storage Conditions 1,25(OH) 2 D 3 Sigma D1530 023M4010V -20 ° C
  • the 1,25(OH) 2 D 3 was dissolved in absolute ethanol to prepare a stock solution having a concentration of 200 ⁇ 10 ⁇ 6 M, and after storage, it was stored in a refrigerator at -20° C. for use.
  • RPMI 1640 basal medium 1.5 g of sodium bicarbonate, 2.5 g of glucose and 0.11 g of sodium pyruvate were added per 10.4 g of lyophilized powder.
  • RPMI 1640 Complete Medium with 10% FBS 50 mL fetal bovine serum (FBS) was added per 450 mL of basal medium.
  • RPMI 1640 incomplete medium containing 1% FBS 1 mL fetal calf serum (FBS) was added per 49 mL of basal medium.
  • LB liquid medium accurately weighed 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and dissolved in 900 mL of deionized water. After adjusting the pH to 7.0 with 5N NaOH, the volume was adjusted to 1 L with deionized water and autoclaved at 121 ° C for 15 min.
  • 1.5% agar plate 15 g of agar powder was added per 1000 mL of LB liquid medium, and autoclaved at 121 ° C for 15 min. 6-well plates were added with 2 mL of LB medium containing 1.5% agar. After the medium was solidified, 1.5% agar plates were placed in a 2-8 ° C refrigerator.
  • Upper agar medium 0.8 g of agar powder was added per 100 mL of LB liquid medium to prepare an upper agar medium containing 0.8% agar, and autoclaved at 121 ° C for 15 min. After the medium was cooled, the upper medium was placed in 2 Store in a -8 ° C refrigerator.
  • the cell supernatant was discarded, and the cell pellet was resuspended by adding an appropriate amount of RPMI1640 incomplete medium, and the cells were uniformly dispersed;
  • the concentration of the obtained cell suspension was adjusted to 1 ⁇ 10 5 /mL with RPMI 1640 incomplete medium;
  • Each well of a 6-well plate was inoculated with 3 mL of cell suspension, and the total number of cells per well was 3 ⁇ 10 5 ;
  • 6-well plates were cultured in a 37 ° C, 5% CO 2 incubator for 24 hours and 48 hours, respectively.
  • the 1.5% agar plate (lower layer medium) prepared in advance was taken out and allowed to stand at room temperature for at least one hour;
  • U-937 cells 1000 rpm, 5 min were centrifuged, and after centrifugation, the cells were aspirated for use;
  • the cell supernatant/bacterial mixture was incubated at 37 ° C in a shaker (200 rpm) for 90 minutes, and after the completion of the incubation, the following steps were performed;
  • Plating Mix 0.8 mL of cell supernatant/bacterial solution and 1.6 mL of the upper agar medium, and then spread the mixture on a 1.5% agar plate at 600 uL per well, repeating 3 wells.
  • Sterility test After mixing with 800 uL of PBS and 1.6 mL of the upper agar medium, plate as above.
  • Antibiotic control experiment Take 700uL PBS, 50uL bacterial solution, 2uL ampicillin, 1.6mL upper agar medium for mixing, mix and paste as above.
  • vitamin D 3 In order to investigate the role of vitamin D 3 in the prevention and treatment of cystitis, urethritis and/or urinary tract infection, this experiment used vitamin D 3 to stimulate human lymphoid U937 cells, collected supernatants for antibacterial experiments, and examined cell supernatants. The antibacterial effect of the liquid.
  • the experimental system is stable, the experimental results are controlled within a reasonable error range, and the data is credible.
  • the experimental results showed that after the U937 cells were stimulated by VD 3 , the supernatant solution showed obvious antibacterial effect.
  • CFT073 was a bacteriostatic object. Cells treated with 1,25(OH) 2 D 3 were co-incubated with E. coli CFT073, and colony forming units (CFU) of E. coli CFT073 were used to examine 1,25 (OH). 2 D 3 can stimulate human-derived cell lines to secrete antibacterial peptides in vitro to achieve antibacterial effects.
  • the 1,25(OH) 2 D 3 was dissolved in absolute ethanol to prepare a stock solution having a concentration of 200 ⁇ 10 -6 M, and after storage, it was stored in a refrigerator at -20 ° C for use.
  • RPMI 1640 basal medium 1.5 g of sodium bicarbonate, 2.5 g of glucose and 0.11 g of sodium pyruvate were added per 10.4 g of lyophilized powder.
  • RPMI 1640 Complete Medium with 10% FBS 50 mL fetal bovine serum (FBS) was added per 450 mL of basal medium.
  • 5XLBE liquid medium LB medium supplemented with Medium E. Accurately weighed 5g of tryptone, 2.5g of yeast extract, 5g of sodium chloride, 0.0986g of MgSO4 ⁇ 7H 2 O, Citric Acid ⁇ H 2 O 1.0087g, K 2 HPO 4 6.55g, NaNH 4 HPO 4 ⁇ 4H 2 O 1.6726 g was dissolved in 90 mL of deionized water. After adjusting the pH to 7.0 with 5N NaOH, the solution was made up to 100 mL with deionized water and autoclaved at 121 ° C for 15 min.
  • 0.8% agar plate 8 g of agar powder was added per 1000 mL of LB liquid medium, and autoclaved at 121 ° C for 15 min. Add 6 mL of LB medium containing 0.8% agar to each well of the 6-well plate. After the medium is solidified, place the 0.8% agar plate in a 2-8 ° C refrigerator (or it can be used on the day of the experiment).
  • 0.8% upper agar medium 0.8g agar powder was added per 100mL LB liquid medium to prepare an upper agar medium containing 0.8% agar, and autoclaved at 121 °C for 15 minutes. After the medium was cooled, the upper medium was placed. Store in a 2-8 ° C refrigerator.
  • the concentration of the obtained cell suspension was adjusted to 4 ⁇ 10 5 /mL with RPMI 1640 incomplete medium;
  • Each well of a 6-well plate was inoculated with 2 mL of cell suspension, and the total number of cells per well was 8 ⁇ 10 5 ;
  • the 6-well plates were incubated at 37 ° C in a 5% CO 2 incubator.
  • the cultured cells to which the bacteria were added were incubated in a 37 ° C incubator for 2 hours.
  • the supernatant was aspirated in a sterile EP tube for use.
  • the cells were washed three times with sterile PBS to remove bacteria that did not adhere to the cell surface.
  • the cells were lysed with 500 ⁇ L of PBS solution containing 1% Triton X-100 and protease inhibitor.
  • the cell lysate was diluted stepwise with PBS at a dilution factor of 10,000 fold. 100 ⁇ L of the diluted cell lysate was taken, placed in a 1.5 mL EP tube, 900 ⁇ L of 0.8% LB medium was added, and uniformly blown, and added to a 6-well plate in which a layer of LB medium was just laid. Take care to ensure a uniform layup. After the medium was solidified, a 1 mL/well LB medium was further applied to the upper layer, taking care to ensure uniform plating.
  • the cell lysate was obtained by the method of 3.1.4, centrifuged at 13,000 rpm for 5 min to remove bacteria and cell debris, and the supernatant of the lysate was taken, and LL-37 was quantified using a Human LL-37 ELISA Kit (Hycult, Cat. No. HK321). content.
  • CFU Colony forming units
  • the statistical test method is One-Way ANOVA LSD Test. * indicates t ⁇ 0.05 compared with the ethanol control group, and ** indicates t ⁇ 0.01 compared with the ethanol control group.
  • vitamin D 3 used vitamin D 3 to stimulate human bladder cell line 5637 and co-incubated with E. coli to investigate the antibacterial effect of its cell lysate.
  • the experimental system is stable, the experimental results are controlled within a reasonable error range, and the data is credible.
  • the experimental results showed that the 5637 cells were stimulated by VD 3 and co-incubated with E. coli, and the cell lysate showed obvious antibacterial effect.
  • Example 3 Antibacterial experiment of VD3 stimulation of urothelial cells
  • E.coli CFT073 Bacillus licheniformis E.coli CFT073 is a bacteriostatic object. Cells treated with 1,25(OH) 2 D 3 are incubated with E. coli CFT073 and examined by colony forming units (CFU) of E. coli CFT073. Whether 25(OH) 2 D 3 can stimulate the secretion of antimicrobial peptides from human-derived cell lines in vitro to achieve an antibacterial effect.
  • the 1,25(OH) 2 D 3 was dissolved in absolute ethanol to prepare a stock solution having a concentration of 200 ⁇ 10 ⁇ 6 M, and after storage, it was stored in a refrigerator at -20° C. for use.
  • F12 basal medium purchased from Hyclone, article number SH30026.01B, 500 mL per bottle, stored in a refrigerator at 2-8 ° C for use.
  • F12 complete medium containing 10% FBS 50 mL fetal bovine serum (FBS) was added per 450 mL of basal medium.
  • the F12 basal medium without FBS is incomplete medium.
  • 5XLBE liquid medium LB medium supplemented with Medium E. Accurately weighed 5g of tryptone, 2.5g of yeast extract, 5g of sodium chloride, 0.0986g of MgSO4 ⁇ 7H 2 O, Citric Acid ⁇ H 2 O 1.0087g, K 2 HPO 4 6.55g, NaNH 4 HPO 4 ⁇ 4H 2 O 1.6726 g was dissolved in 90 mL of deionized water. After adjusting the pH to 7.0 with 5N NaOH, the solution was made up to 100 mL with deionized water and autoclaved at 121 ° C for 15 min.
  • 0.8% agar plate 8 g of agar powder was added per 1000 mL of LB liquid medium, and autoclaved at 121 ° C for 15 min. Add 6 mL of LB medium containing 0.8% agar to each well of the 6-well plate. After the medium is solidified, place the 0.8% agar plate in a 2-8 ° C refrigerator (or it can be used on the day of the experiment).
  • 0.8% upper agar medium 0.8g agar powder was added per 100mL LB liquid medium to prepare an upper agar medium containing 0.8% agar, and autoclaved at 121 °C for 15 minutes. After the medium was cooled, the upper medium was placed. Store in a 2-8 ° C refrigerator.
  • SV-HUC1 cells in the exponential growth phase with a confluency of about 90% were digested with trypsin (GIBCO, Cat. No. 25200-056) for 5 min. After most of the cells were detached from the culture plate, complete medium was added. The digestion was terminated, and the cells that had not fallen off were pipetted from the culture plate with a pipette and transferred to a 15 ml centrifuge tube. After centrifugation for three minutes at 1500 rpm, the cells were discarded and the supernatant was discarded. Resuspend the cell pellet in complete medium and ensure that the cells are evenly dispersed;
  • Each well of a 6-well plate was inoculated with 2 mL of cell suspension, and the total number of cells per well was 8 ⁇ 10 5 ;
  • the 6-well plates were incubated at 37 ° C in a 5% CO 2 incubator.
  • the cultured cells to which the bacteria were added were incubated in a 37 ° C incubator for 2 hours.
  • the supernatant was aspirated in a sterile EP tube for use.
  • the cells were washed three times with sterile PBS to remove bacteria that did not adhere to the cell surface.
  • the cells were lysed with 500 ⁇ L of PBS solution containing 1% Triton X-100 and protease inhibitor.
  • Plating Cell lysate and supernatant were diluted stepwise with PBS at dilutions of 10,000 and 100,000, respectively. 100 ⁇ L of the diluted cell lysate or supernatant was taken, placed in a 1.5 mL EP tube, 900 ⁇ L of 0.8% LB medium was added, and uniformly blown, and added to a 6-well plate in which a layer of LB medium was just laid. Take care to ensure a uniform layup. After the medium was solidified, a 1 mL/well LB medium was further applied to the upper layer, taking care to ensure uniform plating.
  • vitamin D 3 In order to examine the role of vitamin D 3 in the prevention and treatment of urethritis, this experiment used vitamin D 3 to stimulate human SV-HUC1 cells and co-incubated with E. coli to investigate the antibacterial effect of the cell lysate.
  • the experimental system is stable, the experimental results are controlled within a reasonable error range, and the data is credible.
  • the experimental results showed that the SV-HUC1 cells were stimulated by VD 3 and co-incubated with E. coli, and the cell lysate showed obvious antibacterial effect.

Landscapes

  • Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Epidemiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicinal Preparation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

维生素D化合物在制备用于预防或治疗膀胱炎、尿道炎和/或尿路感染的药物中的用途、包含维生素D化合物的药物组合物、药物制剂和药盒，所述维生素D化合物包括维生素D2、维生素D3、维生素D3的活化形式或类似物，所述维生素D3的活化形式是25-羟基维生素D3或1,25-二羟基维生素D3。

Description

维生素D及其组合物的抗菌用途 技术领域
本发明涉及维生素D化合物在制备用于预防或治疗膀胱炎、尿道炎和/或尿路感染的药物中的用途。更为特别地，涉及用于预防或治疗膀胱炎、尿道炎和/或尿路感染的药物组合物和药物制剂。
背景技术
尿路感染是指病原体在尿路中生长繁殖，并侵犯泌尿道粘膜或组织而引起的炎症，以细菌性尿路感染最常见。尿路感染是临床常见病，据美国门诊医疗调查和医院门诊医疗调查，每年有近700万门诊病例和100万急诊，以及造成10万人住院治疗，尤以女性患者居多。大量文献报道尿路感染复发率高，约25-30％的女性患者发生复发感染，更有研究显示高达44.1％的女性患者在一年内复发一次。尿路感染的高复发率带来了重大的经济损失，有学者估算美国每年用于尿路感染治疗的费用高达16亿美元。
尿道炎是一种常见病，指尿道粘膜的炎症,女性好发。临床上可分为急性和慢性、非特异性尿道炎和淋菌性尿道炎。多为致病菌逆行侵入尿道引起。当细菌进入膀胱或肾脏在尿液中繁殖，便引起尿道炎。
最常见的泌尿道感染是膀胱炎，即发生在膀胱的炎症。细菌性膀胱炎是最常见的一种膀胱炎，发病原因是身体表面的细菌从尿道口入侵，在膀胱内增殖，常并有频尿、尿急、排尿灼热感或疼痛、血尿等症状。发作的过程大多很迅速，半天之内就会造成严重不适，甚至在几天内蔓延到肾脏而成为急性肾盂肾炎。大肠杆菌是最常引起膀胱炎的细菌之一，尤其在喝水量不足、憋尿的状况下，更容易让细菌有机会在膀胱滋生。
正常情况下，致病菌在尿路不易停留、繁殖，故不会引起感染。这是由于泌尿、生殖系统的生理防御功能，其中最为主要的是分泌抗菌肽。一旦有病原体入侵时，抗菌肽就能起到杀菌的作用，且这个反应过程相当迅速，使得致病菌尚未在体内繁殖就已经被清除。但是，这种抗感染的防御功能一旦减弱，致病菌就会趁虚而人，从而诱发感染。
抗菌肽多为20～60个氨基酸的肽，一般带正电且具有两亲性，后者使得它们在水溶环境和疏水环境(如膜)都能达到较高浓度。抗菌肽能快速吸附在入侵微生物的膜上并将其杀死。现研究较多的人抗菌肽是防御素和cathelicidin(也称为LL-37)。正常情况下人尿路中可检测到低水平的cathelicidin，一旦有微生物侵入，编码cathelicidin的mRNA表达量迅速上升。 Chromek等用大肠杆菌分别感染cathelicidin基因敲除和野生型鼠，结果基因敲除的鼠下尿道细菌数明显高于野生型。
维生素D在人体营养中占重要地位，它是一种脂溶性维生素，人体内主要以维生素D2(麦角钙化醇)和维生素D3(胆钙化醇)两种形式存在。人体主要依靠食物摄取和皮肤合成获得维生素D。皮肤中的7-脱氢胆固醇经波长290～320mm的紫外光照射下合成维生素D前体。两种来源的维生素D都不能在血液中长期循环，而是马上就被脂肪组织或肝脏摄取，以用来储存或活化。活化后的维生素D能够与其受体VDR结合进入细胞核内，调节多种基因的表达，VDR广泛存在于人体各种细胞中，因而维生素D具有广泛的生物学作用，比如以往所熟知的促进钙磷代谢吸收的功能，以及近年来逐渐被发现的促进细胞分化、抑制细胞增殖、促进分泌抗菌肽、增强固有免疫、抑制特异性免疫等。
通过太阳直接照射皮肤是人体产生天然维生素D的主要方式，但由于现代社会生活习惯的改变，现代人待在室内时间加长，较多地使用防晒产品，使得人们接受到足够的阳光照射机会较少，而能够提供维生素D的食物来源又非常有限(如鱼肝油、一些水栖哺乳动物的肝脏和脂肪等)，因此大部分人无法从常规饮食中补充维生素D，导致目前全球范围内维生素D缺乏呈普遍趋势，国外众多大型流行病学调查显示(包括美国、加拿大、英国、法国、德国、西班牙、俄罗斯、新西兰、以色列等)，在大龄儿童、青年和中年人中约有50％～70％的人群处于维生素D缺乏状态。而我国维生素D缺乏形势更为严峻，特别是中国人的膳食结构中缺乏富含维生素D的食物，在现代生活中接触阳光和户外活动的机会也大大减少，因此我国居民出现维生素D缺乏的几率将比西方人更高，据中科院研究发现我国中老年人群缺乏维生素D现象更为严重，其调查结果显示我国中老年人血液中含有的维生素D的水平整体较低，维生素D不足者占93.6％，维生素D充足者仅占6.4％。
维生素D缺乏或不足对人体的免疫能力具有极其不利的影响，降低对尿路感染反复发作的抵抗力。虽然暂未有流行病学研究数据证实维生素D缺乏会导致免疫能力下降的直接证据，但已有流行病学研究显示体内25(OH)D血清浓度降低而呈现上呼吸道感染率增加的关系，另有流行病学研究显示母婴体内维生素D缺乏与婴儿急性下呼吸道感染的高发病率相关，Gibney等人研究显示维生素D缺乏与肺结核发病率相关，这些研究都从侧面揭示维生素D缺乏或不足对系统免疫能力或会造成一定负面的影响。另外孕妇和高龄妇女都是维生素D缺乏或不足的高危人群，有研究显示2％～10％的孕妇及高达20％的80岁以上妇女有无症状菌尿，发病概率远远超过普通人，提示我们这两者之间可能存在某种内在联系。
尿路感染可根据病原体的感染部位分为尿道炎、膀胱炎、肾盂肾炎；肾盂肾炎严重时引发肾功能损伤甚至衰竭，而革兰氏阴性杆菌败血症多由尿路感染引起，少数败血症病人可发生明显的休克，伴有心、脑、肾缺血的临床表现。
现膀胱炎、尿道炎和/或尿路感染治疗主要采取抗生素疗法，常用的抗生素有：TMP-SMX、呋喃妥因(nitrofurantoin)、环丙沙星(ciprofloxacin)、左氧氟沙星(levofloxacin)或其化学类似物、某些盘尼西林(penicillin)如阿莫西林等。尽管抗生素一定程度上能降低膀胱炎、尿道炎和/或尿路感染，但是抗生素广泛应用的代价同样是高昂的：耐药菌的不断增加以及破坏患者体内的正常菌群数量。有一项研究表明从膀胱炎、尿道炎和/或尿路感染患者中分离的大肠杆菌在1998年至2003年5年间对环丙沙星、阿莫西林-克拉维酸、TMP-SMX抗性明显增加。抗生素滥用将导致耐药性的细菌不断增加，细菌对药的抗性使得抗生素用量加大或不断寻求杀菌更强的抗生素，造成经济上的浪费；并且其盲目杀菌作用也导致体内有益菌的减少，对人体造成不必要的伤害。抗生素可以快速抑制细菌增殖，但不能提高机体自身的固有免疫，当病原体再次侵入时，体内固有免疫仍不足以将病原体杀死，感染易复发。前文也提到膀胱炎、尿道炎和/或尿路感染的高复发率，现阶段单用抗生素疗法不能降低其高复发率，且易造成医疗资源的浪费和经济的重大损失。因此现亟需一种有效的新的防治方法，能够单独使用或同抗生素疗法联用以降低膀胱炎、尿道炎和/或尿路感染的高复发率。
膀胱炎、尿道炎和/或尿路感染的一大诱因是机体自身固有免疫力下降，增强自身固有免疫能力是降低膀胱炎、尿道炎和/或尿路感染复发率的一条途径。而活性维生素D与VDR(vitamin d receptor，维生素D受体)结合进入细胞核内，作用于多种靶基因，可增强人的固有免疫、上调抗菌肽的表达，抑制病菌粘附，杀灭或抑制病原体，减少复发率，其理论依据和实验结果使新的膀胱炎、尿道炎和/或尿路感染的防治药物成为可能。本申请提供了一种可有效降低膀胱炎、尿道炎和/或尿路感染复发的药物制剂，通过降低膀胱炎、尿道炎和/或尿路感染的复发，改善患者的生活质量，并减少抗生素的应用，缓解目前临床上抗生素滥用的状况。维生素D化合物在制备用于预防或治疗膀胱炎、尿道炎和/或尿路感染的药物中的应用必将大大改观膀胱炎、尿道炎和/或尿路感染的防治状况。
发明内容
A.发明概述
本发明涉及维生素D化合物在制备用于预防或治疗膀胱炎、尿道炎和/或尿路感染的药物 中的用途。本发明涉及维生素D化合物在制备用于预防或治疗膀胱炎的药物中的用途。本发明涉及维生素D化合物在制备用于预防或治疗尿道炎的药物中的用途。本发明涉及维生素D化合物在制备用于预防或治疗尿路感染的药物中的用途。其中所述尿路感染包括上尿路感染和下尿路感染，根据病原体的感染部位可分为尿道炎、膀胱炎、肾盂肾炎。在一个优选实施方案中，维生素D化合物是维生素D受体配体(VDR配体)。在一个更优选的实施方案中，维生素D化合物是维生素D受体的激动剂。优选地，维生素D化合物包括维生素D2化合物、维生素D3化合物、其异构体、活化形式或类似物。优选地，维生素D化合物包括维生素D2、D3和其类似物。在某些实施方案中，维生素D化合物是维生素D的活化形式，例如骨化二醇(25-OH D3)或骨化三醇(1，25-(OH)2 D3)。
另一方面，本发明涉及一种预防或治疗膀胱炎、尿道炎和/或尿路感染的药物组合物，该组合物包括维生素D化合物和其它用于预防或治疗膀胱炎、尿道炎和/或尿路感染的药物。本发明涉及一种预防或治疗膀胱炎的药物组合物，该组合物包括维生素D化合物和其它用于预防或治疗膀胱炎的药物。本发明涉及一种预防或治疗尿道炎的药物组合物，该组合物包括维生素D化合物和其它用于预防或治疗尿道炎的药物。本发明涉及一种预防或治疗尿路感染的药物组合物，该组合物包括维生素D化合物和其它用于预防或治疗尿路感染的药物。在一个优选实施方案中，维生素D化合物是维生素D受体配体(VDR配体)。在一个更优选的实施方案中，维生素D化合物是维生素D受体的激动剂。优选地，维生素D化合物包括维生素D2化合物、维生素D3化合物、其异构体、活化形式或类似物。优选地，维生素D化合物包括维生素D2、D3和其类似物。在某些实施方案中，维生素D化合物是维生素D的活化形式，例如骨化二醇(25-OH D3)或骨化三醇(1，25-(OH)2 D3)。在一个优选实施方案中，其它用于预防或治疗膀胱炎、尿道炎和/或尿路感染的药物是某一抗生素，如TMP-SMX、呋喃妥因(nitrofurantoin)、环丙沙星(ciprofloxacin)、左氧氟沙星(levofloxacin)或其化学类似物、盘尼西林(penicillin)、阿莫西林等。在某些实施方案中，本发明的药物组合物适合于局部或口服施用于受试者。在其它实施方案中，正如下文所描述的，本发明的药物组合物可以被特殊配制成以固体或液体形式施用，如口服施用、肠胃外施用、局部施用等。
另一方面，本发明提供了一种预防或治疗膀胱炎、尿道炎和/或尿路感染的药物制剂，该药物制剂含有维生素D化合物以及药学上可接受的载体。本发明提供了一种预防或治疗膀胱炎的药物制剂，该药物制剂含有维生素D化合物以及药学上可接受的载体。本发明提供了一种预防或治疗尿道炎的药物制剂，该药物制剂含有维生素D化合物以及药学上可接受的载体。本发明提供了一种预防或治疗尿路感染的药物制剂，该药物制剂含有维生素D化合物以及药 学上可接受的载体。在一个优选实施方案中，维生素D化合物是维生素D受体配体(VDR配体)。在一个更优选的实施方案中，维生素D化合物是维生素D受体的激动剂。优选地，维生素D化合物包括维生素D2化合物、维生素D3化合物、其异构体、活化形式或类似物。优选地，维生素D化合物包括维生素D2、D3和其类似物。在某些实施方案中，维生素D化合物是维生素D的活化形式，例如骨化二醇(25-OH D3)或骨化三醇(1，25-(OH)2 D3)。在某些优选实施方案中，药学上可接受的载体是下述某一物质，如：糖类、淀粉、纤维素及其衍生物、明胶、滑石粉、赋形剂、油类、多元醇、酯类、缓冲剂、海藻酸或药物制剂中采用的其它无毒的相容性物质。本发明组合物可加入制备不同剂型时所需的各种常规辅料，如崩解剂、润滑剂、粘合剂、抗氧剂、络合剂等药用载体，以常规的制剂方法制成任何一种常用的口服剂型，如分散片、颗粒剂、胶囊剂、口服液等其他剂型。本发明的药物制剂的剂型可以为片剂、胶囊、注射剂、输注剂、吸入剂软膏剂或栓剂，但不限于此。
另一方面，本发明涉及提供了一种预防或治疗膀胱炎、尿道炎和/或尿路感染的药盒，该药盒含有如上所述的药物组合物或药物制剂以及如何使用该组合物或药物制剂的说明书。本发明涉及提供了一种预防或治疗膀胱炎的药盒，该药盒含有如上所述的药物组合物或药物制剂以及如何使用该组合物或药物制剂的说明书。本发明涉及提供了一种预防或治疗尿道炎的药盒，该药盒含有如上所述的药物组合物或药物制剂以及如何使用该组合物或药物制剂的说明书。本发明涉及提供了一种预防或治疗尿路感染的药盒，该药盒含有如上所述的药物组合物或药物制剂以及如何使用该组合物或药物制剂的说明书。
B.释义
除另有定义，这里使用的所有科技术语与本发明所属技术领域的普通技术人员理解含义相同。所有专利文献、专利申请文献、公开的专利文献和其它出版物均作为参考。如本节阐述的定义与上述参考文献所述的定义不一致或相反时，以本节阐述的定义为准。
本文所用的术语“维生素D化合物”包括任何能够预防或治疗膀胱炎、尿道炎和/或尿路感染的维生素D化合物，一般而言，为维生素D受体配体(VDR配体)并且能够预防或治疗膀胱炎、尿道炎和/或尿路感染的维生素D化合物被视为本发明的范围内，维生素D化合物优选地是维生素D受体的激动剂。因此，维生素D化合物旨在包括开环甾类，适用本发明的方法的具体维生素D化合物的实例在本文中有进一步描述。维生素D化合物包括维生素D2化合物、维生素D3化合物、其异构体、活化形式或类似物。优选的维生素D化合物是维生素D3化合物，它们是维生素D受体的配体(更优选地是激动剂)。维生素D2化合物和维生 素D3化合物分别包括维生素D2、D3和其类似物。在某些实施方案中，维生素D化合物可以是甾类，例如开环甾类，例如骨化醇(VD3)、骨化二醇(25-OH D3)或骨化三醇(1，25-(OH)2D3)。
术语“其它用于预防或治疗膀胱炎、尿道炎和/或尿路感染的药物”是指已知的可用于预防或治疗膀胱炎、尿道炎和/或尿路感染的药物，包括现有的各种抗生素，如TMP-SMX、呋喃妥因(nitrofurantoin)、环丙沙星(ciprofloxacin)、左氧氟沙星(levofloxacin)或其化学类似物、盘尼西林(penicillin)，阿莫西林等。
术语“药学上可接受的”表示在合理的医学判断范围内适合于与人类和动物的组织接触使用、没有过度的毒性、刺激作用、变态反应或者其它问题或并发症。
术语“药学上可接受的载体”包括药学上可接受的材料、组合物或媒介物，例如液体或固体填充剂、稀释剂、赋形剂、溶剂或包封材料，它们参与将主题化学物质从机体的一个器官或一个部分携带或运输到机体的另一个器官或另一个部分。每种载体必须是“可接受的”，意义是与制剂其它成分相容并对患者无害。
术语“施用”包括向受试者引入维生素D化合物以发挥它们预期功能的途径，可使用的施用途径的实例包括注射(皮下、静脉内、肠胃外、腹膜内)、口服、吸入、直肠、透皮或经由膀胱滴注，口服施用是优选的。本发明的制剂可以口服、或经静脉、皮下、肌内、经皮肤、鼻内、或直肠途径的胃肠外给药，或吸入给药。用于口服给药的剂型包括片剂、丸剂、粉剂、颗粒剂、液体剂、悬浮剂、糖浆剂、胶囊剂等。
附图说明
图1是VD3刺激膀胱上皮细胞的抗菌实验中，六孔板每孔中加入10μL不同浓度的供试品溶液或溶媒。
图2是VD3刺激膀胱上皮细胞的抗菌实验中，各个剂量组5637细胞裂解液的菌落形成单位(CFU)。
图3是VD3刺激膀胱上皮细胞的抗菌实验中，计数5637细胞各剂量组裂解液中CFU，并计算各组与乙醇溶剂对照组CFU的比值，得到相对CFU，重复多次实验的统计结果。统计检验方法为One-Way ANOVA LSD Test。*表示与乙醇对照组相比t<0.05，**表示与乙醇对照组相比t<0.01。
图4为VD3刺激膀胱上皮细胞的抗菌实验中，对各剂量组裂解液中LL-37进行ELISA检测 的结果。
图5为VD3刺激尿道上皮细胞的抗菌实验中，各个剂量组SV-HUC1细胞裂解液的CFU。
具体实施方式
实施例1：VD3刺激淋巴细胞的抗菌实验
1.实验目的
本研究以维生素D的最终活化形式1，25-二羟维生素D3[1,25(OH)2D3]为供试品、以人组织细胞淋巴瘤细胞U-937细胞为实验系统，以大肠杆菌E.coli O111B4为抑菌对象，取分别经1,25(OH)2D3作用后的细胞上清液和大肠杆菌E.coli O111B4共孵育，以E.coli O111B4的菌落形成单位(CFU)来考察1,25(OH)2D3在体外是否可以刺激人来源细胞株分泌抗菌肽，以达到抗菌效果。
2.实验材料
2.1供试品1,25(OH)2D的信息
药品名 供应商 货号 批号 保存条件
1,25(OH)2D3 Sigma D1530 023M4010V -20℃
使用无水乙醇溶解1,25(OH)2D3，配制成浓度为200x l0-6M的储备液，分装后，保存于-20℃冰箱中待用。
2.2实验仪器
MODEL 3111型CO2培养箱
XDS-1B型倒置显微镜
SDG-D2型超净工作台
DK-6000型水浴锅
Biofuge Stratos型离心机
SMARTSPEC 3000型紫外分光光度计
SCS-24恒温振荡摇床
2.3细菌和细胞培养基
RPMI 1640基础培养基：每10.4g冻干粉加入1.5g碳酸氢钠、2.5g葡萄糖和0.11g丙酮酸钠。首先用800ml去离子水进行溶解，待完全溶解后，用1N HCl调pH值至7.2-7.4，并用去离子水定容至1L。基础培养基配制后，用孔径为0.22μm的滤膜过滤除菌，然后分装于 蓝盖瓶，每瓶500mL，保存于2-8℃冰箱待用。
含10％FBS的RPMI 1640完全培养基：每450mL基础培养基加入50mL胎牛血清(FBS)。
含1％FBS的RPMI 1640不完全培养基：每49mL基础培养基加入1mL胎牛血清(FBS)。
LB液体培养基：准确称取胰蛋白胨10g，酵母提取物5g，氯化钠10g，溶解于900mL去离子水中。用5N的NaOH调pH至7.0后，用去离子水定容至1L，121℃高压蒸汽灭菌15min。
1.5％琼脂平板：每1000mL LB液体培养基加入15g琼脂粉，121℃高压蒸汽灭菌15min。6孔板每孔加入2mL含1.5％琼脂的LB培养基，待培养基凝固后，将1.5％琼脂平板放入2-8℃冰箱保存。
上层琼脂培养基：每100mL LB液体培养基加入0.8g琼脂粉，制备成含0.8％琼脂的上层琼脂培养基，121℃高压蒸汽灭菌15min，待培养基冷却后，将上层培养基放入2-8℃冰箱保存。
3.实验方法
3.1接种和给药(Day 1)
将处于指数增长期U-937细胞进行离心(1000rpm，5min)；
离心结束后，弃除细胞上清，加入适量的RPMI1640不完全培养基重悬细胞沉淀，并确保细胞分散均匀；
用血细胞计数板对细胞重悬液进行计数；
用RPMI 1640不完全培养基将所得到的细胞悬液的浓度调整到1x 105/mL；
6孔板的每个孔接种3mL细胞悬液，每孔的细胞总数为3x 105；
以0.5％(v/v)的给药体积，根据标签和实验设计，每孔中加入10μL不同浓度的200x供试品溶液或溶媒；
每个剂量平行重复2孔，其中一孔24小时收获，另一孔48小时后收获；
给药后，将6孔板置于37℃、5％CO2培养箱培养，分别培养24小时和48小时。
3.2大肠杆菌感染菌液制备
Day 1下午，解冻一支E.coli O111B4甘油管，吸取10μL甘油菌接种于10mL不含任何抗生素的LB液体培养基中，37℃震荡培养过夜；
Day 2早上，吸取200μL培养过夜的菌液(按1:100比例)转接至20mL不含任何抗生素的LB液体培养基，37℃震荡培养至OD600读数0.6-1.0，进入下一步的抗菌实验(Day 3)；剩余的过夜菌液保存于2-8℃冰箱备用。
Day 2下午，吸取10μL培养过夜的菌液接种于10mL不含任何抗生素的LB培养基中，37℃震荡培养过夜；
Day 3早上，吸取200μL培养过夜的菌液(按1:100比例)转接至20mL不含任何抗生 素的LB培养基，37℃震荡培养至OD600读数0.6-1.0，进入下一步的抗菌实验(Day 3)。
3.3抗菌实验(Day 2和Day 3)
实验开始前，取出事先制备好的含1.5％琼脂平板(下层培养基)，室温至少放置一小时；
溶化上层琼脂培养基(含0.8％琼脂)，然后分装。每管分装1.6mL的上层琼脂培养基，然后将小管置于45℃水浴锅保温待用；
取10mL OD600为0.6-1.0的E.coli菌液转入50mL离心管，进行离心(1000rpm x 5min)；离心后，弃除上清，细菌沉淀加入10mL PBS进行重悬；通过倍比稀释(10倍倍距)，用PBS将上述细菌悬液稀释1x105倍；
药物作用结束后(24小时或48小时)，离心U-937细胞(1000rpm,5min)，离心结束后，吸取细胞上清备用；
每个离心管预先加入50μL稀释后的细菌悬液(约150-250个细菌)；
每份细胞上清样品取1.2mL细胞上清液加入细菌悬液中，混匀；
将细胞上清/细菌混合液置于37℃摇床中振荡(200rpm)孵育90分钟，孵育结束后如下步骤进行铺板；
铺板：取0.8mL细胞上清液/菌液的混合液和1.6mL上层琼脂培养基混合，然后将混合液铺于1.5％琼脂平板上，每孔600uL，3孔一重复。
无菌测试：取800uL PBS和1.6mL上层琼脂培养基混匀后，如上进行铺板。
抗生素对照实验：取700uL PBS、50uL菌液、2uL氨苄抗生素、1.6mL上层琼脂培养基进行混匀，混匀后如上进行铺板。
待上层琼脂培养基冷却凝固后，所有6孔板(琼脂平板)一起放入37℃培养箱培养，培养过夜。
3.4菌落计数(Day 3和Day 4)
铺板后次日，从培养箱中取出6孔板进行菌落计数。
4.实验结果
U937细胞经维生素D3诱导24h和48h后，取上清进行抗菌实验。分别计算各剂量组菌落形成单位(CFU)。实验结果如下：
Figure PCTCN2014094893-appb-000001
Figure PCTCN2014094893-appb-000002
其中，无菌实验及抗生素对照实验组均未有菌落产生。从现有数据看，不同浓度的VD3刺激U937细胞后，其细胞上清溶液显示出明显的抗菌作用。
5.讨论
为了考察维生素D3在预防和治疗膀胱炎、尿道炎和/或尿路感染方面的作用，本实验采用维生素D3刺激来源于人的淋巴U937细胞，收集上清进行抗菌实验，考察细胞上清液的抗菌效果。
该实验体系稳定，实验结果控制在合理的误差范围内，数据可信。实验结果显示，U937细胞经VD3刺激后，其上清溶液显示出明显的抗菌作用。
实施例2：VD3刺激膀胱上皮细胞的抗菌实验
1实验目的
本研究以维生素D的最终活化形式1，25-二羟维生素D3[1,25(OH)2D3]为供试品、以人膀胱癌细胞5637为实验系统，以大肠杆菌E.coli CFT073为抑菌对象，将经1,25(OH)2D3作用后的细胞与大肠杆菌E.coli CFT073共孵育，以E.coli CFT073的菌落形成单位(CFU)来考察1,25(OH)2D3在体外是否可以刺激人来源细胞株分泌抗菌肽，以达到抗菌效果。
2实验材料
2.1供试品1,25(OH)2D的信息
Figure PCTCN2014094893-appb-000003
使用无水乙醇溶解1,25(OH)2D3，配制成浓度为200x l0-6M的储备液，分装后，保存于 -20℃冰箱中待用。
2.2实验仪器
MODEL 3111型CO2培养箱
XDS-1B型倒置显微镜
SDG-D2型超净工作台
DK-6000型水浴锅
Biofuge Stratos型离心机
SMARTSPEC 3000型紫外分光光度计
SCS-24恒温振荡摇床
2.3细菌和细胞培养基
RPMI 1640基础培养基：每10.4g冻干粉加入1.5g碳酸氢钠、2.5g葡萄糖和0.11g丙酮酸钠。首先用800ml去离子水进行溶解，待完全溶解后，用1N HCl调pH值至7.2-7.4，并用去离子水定容至1L。基础培养基配制后，用孔径为0.22μm的滤膜过滤除菌，然后分装于蓝盖瓶，每瓶500mL，保存于2-8℃冰箱待用。
含10％FBS的RPMI 1640完全培养基：每450mL基础培养基加入50mL胎牛血清(FBS)。
无FBS的RPMI 1640不完全培养基。
5XLBE液体培养基：即增加了Medium E的LB培养基。准确称取胰蛋白胨5g，酵母提取物2.5g，氯化钠5g，MgSO4·7H2O 0.0986g，Citric Acid·H2O 1.0087g，K2HPO4 6.55g，NaNH4HPO4·4H2O 1.6726g溶解于90mL去离子水中。用5N的NaOH调pH至7.0后，用去离子水定容至100mL，121℃高压蒸汽灭菌15min。
0.8％琼脂平板：每1000mL LB液体培养基加入8g琼脂粉，121℃高压蒸汽灭菌15min。6孔板每孔加入1mL含0.8％琼脂的LB培养基，待培养基凝固后，将0.8％琼脂平板放入2-8℃冰箱保存(或可实验当天现配)。
0.8％上层琼脂培养基：每100mL LB液体培养基加入0.8g琼脂粉，制备成含0.8％琼脂的上层琼脂培养基，121℃高压蒸汽灭菌15min，待培养基冷却后，将上层培养基放入2-8℃冰箱保存。
3实验方法
3.1抗菌实验
3.1.1接种(Day 1)
将处于指数增长期的，汇合度90％左右的5637细胞用胰酶(GIBCO，Cat.No.25200-056)消化5min，待绝大部分细胞从培养板上脱落后，加入完全培养基终止消化，并用移液器将尚未脱落的细胞从培养板上吹下，并转移至15ml离心管中。1500rpm离心三分钟沉淀细胞后，弃上清。用完全培养基重悬细胞沉淀，并确保细胞分散均匀；
用血细胞计数板对细胞重悬液进行计数；
用RPMI 1640不完全培养基将所得到的细胞悬液的浓度调整到4x 105/mL；
6孔板的每个孔接种2mL细胞悬液，每孔的细胞总数为8x 105；
将6孔板置于37℃、5％CO2培养箱培养。
3.1.2给药(Day 2)
将每孔2ml完全培养基更换为2ml不完全培养基。以0.5％(v/v)的给药体积，根据标签和实验设计，每孔中加入10μL不同浓度的供试品溶液或溶媒。
各孔的处理如图1所示。
3.1.3大肠杆菌感染菌液制备(Day 2&3)
Day 2下午，解冻一支E.coli CFT073甘油管，吸取10μL甘油菌接种于10mL不含任何抗生素的LB液体培养基中，37℃震荡培养过夜；
Day 3早上，吸取50μL培养过夜的菌液(按1:100比例)转接至5mL不含任何抗生素的LB液体培养基，37℃震荡培养至OD600读数0.9-1.0，进入下一步的抗菌实验。
3.1.4抗菌实验(Day 3)
实验开始前，取出事先制备好的含0.8％琼脂平板(下层培养基)，平衡至室温(或现配琼脂平板)；
溶化上层琼脂培养基(含0.8％琼脂)，置于50℃水浴锅保温待用；
将适量OD600为0.9～1.0的E.coli菌液稀释至OD600为0.83(相当于5X108CFU/mL)；
药物作用24小时后，每孔加入10μL PBS稀释后的细菌悬液(约3.125X105个细菌)；
将加入了细菌的培养细胞置于37℃培养箱中孵育2小时。
孵育结束后，吸取上清于灭菌EP管中待用。用灭菌的PBS冲洗细胞三次，以除去未黏附于细胞表面的细菌。用500μL含有1％Triton X-100和蛋白酶抑制剂的PBS溶液裂解细胞。
铺板：用PBS逐级稀释细胞裂解液，稀释倍数为10000倍。取100μL已稀释的细胞裂解液，放入1.5mLEP管中，加入900μL0.8％LB培养基，吹打均匀，分别加入到刚铺好一层LB培养基的6孔板中。注意确保均匀的铺板。当培养基凝固后，在其上层再铺一层1mL/孔的LB培养基，注意确保均匀的铺板。
待上层琼脂培养基冷却凝固后，所有6孔板(琼脂平板)一起放入37℃培养箱培养，培养过夜。
3.1.5菌落计数(Day 4)
铺板后次日，从培养箱中取出6孔板进行菌落计数。
3.2 1,25(OH)2D3影响LL-37产生的ELISA实验
3.2.1接种(Day 1)、给药(Day 2)、以及大肠杆菌感染菌液制备(Day 2&3)同3.1.1，3.1.2，3.1.3。
3.2.2 LL-37的ELISA检测
用3.1.4中的方法获得细胞裂解液，13000rpm离心5min去除细菌和细胞残渣，取裂解液的上清，用Human LL-37 ELISA Kit(Hycult,Cat.No.HK321)定量其中的LL-37含量。
4实验结果
4.1抗菌实验
用记号笔标出各个剂量组5637细胞裂解液的菌落形成单位(CFU)，代表性结果如图2所示。
计数5637细胞各剂量组裂解液中CFU，并计算各组与乙醇溶剂对照组CFU的比值，得到相对CFU。重复多次实验的统计结果如图3所示。
统计检验方法为One-Way ANOVA LSD Test。*表示与乙醇对照组相比t<0.05，**表示与乙醇对照组相比t<0.01。
从现有数据看，不同浓度的VD3刺激5637细胞后，其显示出明显的抗菌作用。
4.2 1,25(OH)2D3影响LL-37产生的ELISA实验
对各剂量组裂解液中LL-37进行ELISA检测的结果如图4所示。
从现有数据看，不同浓度的VD3刺激5637细胞后，其细胞裂解液中LL-37水平有显著的剂量依赖的升高。
5.讨论
为了考察维生素D3在预防和治疗膀胱炎方面的作用，本实验采用维生素D3刺激来源于人膀胱细胞5637并与大肠杆菌共孵育，考察其细胞裂解液的抗菌效果。
该实验体系稳定，实验结果控制在合理的误差范围内，数据可信。实验结果显示，5637细胞经VD3刺激并与大肠杆菌共孵育后，其细胞裂解液显示出明显的抗菌作用。
实施例3：VD3刺激尿道上皮细胞的抗菌实验
1实验目的
本研究以维生素D的最终活化形式1，25-二羟维生素D3[1,25(OH)2D3]为供试品、以人尿道上皮永生化细胞SV-HUC1为实验系统，以大肠杆菌E.coli CFT073为抑菌对象，将经1,25(OH)2D3作用后的细胞与大肠杆菌E.coli CFT073共孵育，以E.coli CFT073的菌落形成单位(CFU)来考察1,25(OH)2D3在体外是否可以刺激人来源细胞株分泌抗菌肽，以达到抗菌效果。
2实验材料
2.1供试品1,25(OH)2D的信息
Figure PCTCN2014094893-appb-000004
使用无水乙醇溶解1,25(OH)2D3，配制成浓度为200x l0-6M的储备液，分装后，保存于-20℃冰箱中待用。
2.2实验仪器
MODEL 3111型CO2培养箱
XDS-1B型倒置显微镜
SDG-D2型超净工作台
DK-6000型水浴锅
Biofuge Stratos型离心机
SMARTSPEC 3000型紫外分光光度计
SCS-24恒温振荡摇床
2.3细菌和细胞培养基
F12基础培养基：购于Hyclone，货号SH30026.01B，每瓶500mL，保存于2-8℃冰箱待用。
含10％FBS的F12完全培养基：每450mL基础培养基加入50mL胎牛血清(FBS)。
无FBS的F12基础培养基即不完全培养基。
5XLBE液体培养基：即增加了Medium E的LB培养基。准确称取胰蛋白胨5g，酵母提取物2.5g，氯化钠5g，MgSO4·7H2O 0.0986g，Citric Acid·H2O 1.0087g，K2HPO4 6.55g，NaNH4HPO4·4H2O 1.6726g溶解于90mL去离子水中。用5N的NaOH调pH至7.0后，用去 离子水定容至100mL，121℃高压蒸汽灭菌15min。
0.8％琼脂平板：每1000mL LB液体培养基加入8g琼脂粉，121℃高压蒸汽灭菌15min。6孔板每孔加入1mL含0.8％琼脂的LB培养基，待培养基凝固后，将0.8％琼脂平板放入2-8℃冰箱保存(或可实验当天现配)。
0.8％上层琼脂培养基：每100mL LB液体培养基加入0.8g琼脂粉，制备成含0.8％琼脂的上层琼脂培养基，121℃高压蒸汽灭菌15min，待培养基冷却后，将上层培养基放入2-8℃冰箱保存。
3实验方法
3.1抗菌实验
3.1.1接种(Day 1)
将处于指数增长期的，汇合度90％左右的SV-HUC1细胞用胰酶(GIBCO，Cat.No.25200-056)消化5min，待绝大部分细胞从培养板上脱落后，加入完全培养基终止消化，并用移液器将尚未脱落的细胞从培养板上吹下，并转移至15ml离心管中。1500rpm离心三分钟沉淀细胞后，弃上清。用完全培养基重悬细胞沉淀，并确保细胞分散均匀；
用血细胞计数板对细胞重悬液进行计数；
用F12不完全培养基将所得到的细胞悬液的浓度调整到4x 105/mL；
6孔板的每个孔接种2mL细胞悬液，每孔的细胞总数为8x 105；
将6孔板置于37℃、5％CO2培养箱培养。
3.1.2给药(Day 2)
将每孔2ml完全培养基更换为2ml不完全培养基。以0.5％(v/v)的给药体积，根据标签和实验设计，每孔中加入10μL不同浓度的供试品溶液或溶媒。
各孔的处理如图1所示
3.1.3大肠杆菌感染菌液制备(Day 2&3)
Day 2下午，解冻一支E.coli CFT073甘油管，吸取10μL甘油菌接种于10mL不含任何抗生素的LB液体培养基中，37℃震荡培养过夜；
Day 3早上，吸取50μL培养过夜的菌液(按1:100比例)转接至5mL不含任何抗生素的LB液体培养基，37℃震荡培养至OD600读数0.9-1.0，进入下一步的抗菌实验。
3.1.4抗菌实验(Day 3)
实验开始前，取出事先制备好的含0.8％琼脂平板(下层培养基)，平衡至室温(或现配琼脂平板)；
溶化上层琼脂培养基(含0.8％琼脂)，置于50℃水浴锅保温待用；
将适量OD600为0.9～1.0的E.coli菌液稀释至OD600为0.83(相当于5X108CFU/mL)；
药物作用24小时后，每孔加入10μL PBS稀释后的细菌悬液(约3.125X105个细菌)；
将加入了细菌的培养细胞置于37℃培养箱中孵育2小时。
孵育结束后，吸取上清于灭菌EP管中待用。用灭菌的PBS冲洗细胞三次，以除去未黏附于细胞表面的细菌。用500μL含有1％Triton X-100和蛋白酶抑制剂的PBS溶液裂解细胞。
铺板：用PBS逐级稀释细胞裂解液和上清液，稀释倍数分别为10000倍和100000倍。取100μL已稀释的细胞裂解液或上清液，放入1.5mLEP管中，加入900μL0.8％LB培养基，吹打均匀，分别加入到刚铺好一层LB培养基的6孔板中。注意确保均匀的铺板。当培养基凝固后，在其上层再铺一层1mL/孔的LB培养基，注意确保均匀的铺板。
待上层琼脂培养基冷却凝固后，所有6孔板(琼脂平板)一起放入37℃培养箱培养，培养过夜。
3.1.5菌落计数(Day 4)
铺板后次日，从培养箱中取出6孔板进行菌落计数。
4实验结果
4.1抗菌实验
用记号笔标出各个剂量组SV-HUC1细胞裂解液的CFU，代表性结果如图5所示。
5.讨论
为了考察维生素D3在预防和治疗尿道炎方面的作用，本实验采用维生素D3刺激来源于人的SV-HUC1细胞并与大肠杆菌共孵育，考察其细胞裂解液的抗菌效果。
该实验体系稳定，实验结果控制在合理的误差范围内，数据可信。实验结果显示，SV-HUC1细胞经VD3刺激并与大肠杆菌共孵育后，其细胞裂解液显示出明显的抗菌作用。
为了更清楚地描述和理解本发明，我们通过举例的方式详细描述了本发明，但显而易见的是，本技术领域中普通技术人员可以在不背离权利要求的精神和范围的前提下，对本发明做出适当的改变或修改。

Claims (20)

  1. 维生素D化合物在制备用于预防或治疗膀胱炎的药物中的用途。
  2. 根据权利要求1所述的用途，其中所述维生素D化合物包括维生素D2、维生素D3、维生素D3的活化形式或类似物。
  3. 根据权利要求2所述的用途，其中所述维生素D3的活化形式是25-羟基维生素D3或1,25-二羟基维生素D3。
  4. 根据权利要求2所述的用途，其中所述维生素D化合物是1,25-二羟基维生素D3。
  5. 用于预防或治疗膀胱炎的药物组合物，其特征在于其含有维生素D化合物和其它可用于预防或治疗膀胱炎的药物。
  6. 根据权利要求5所述的药物组合物，其中所述维生素D化合物包括维生素D2、维生素D3、维生素D3的活化形式或类似物。
  7. 用于预防或治疗膀胱炎的药物制剂，其特征在于其含有维生素D化合物以及药学上可接受的载体。
  8. 根据权利要求7所述的药物制剂，其中所述维生素D化合物包括维生素D2、维生素D3、维生素D3的活化形式或类似物。
  9. 根据权利要求7所述的药物制剂，其剂型可以是片剂、胶囊、注射剂、输注剂、吸入剂软膏剂或栓剂。
  10. 用于预防或治疗膀胱炎的药盒，其特征在于其含有如权利要求7所述的药物制剂以及如何使用该药物制剂的说明书。
  11. 维生素D化合物在制备用于预防或治疗尿道炎的药物中的用途。
  12. 根据权利要求11所述的用途，其中所述维生素D化合物包括维生素D2、维生素D3、维生素D3的活化形式或类似物。
  13. 根据权利要求12所述的用途，其中所述维生素D3的活化形式是25-羟基维生素D3或1,25-二羟基维生素D3。
  14. 根据权利要求12所述的用途，其中所述维生素D化合物是1,25-二羟基维生素D3。
  15. 用于预防或治疗尿道炎的药物组合物，其特征在于其含有维生素D化合物和其它可用于预防或治疗尿道炎的药物。
  16. 根据权利要求15所述的药物组合物，其中所述维生素D化合物包括维生素D2、维生素D3、维生素D3的活化形式或类似物。
  17. 用于预防或治疗尿道炎的药物制剂，其特征在于其含有维生素D化合物以及药学上可接受的载体。
  18. 根据权利要求17所述的药物制剂，其中所述维生素D化合物包括维生素D2、维生素D3、维生素D3的活化形式或类似物。
  19. 根据权利要求17所述的药物制剂，其剂型可以是片剂、胶囊、注射剂、输注剂、吸入剂软膏剂或栓剂。
  20. 用于预防或治疗尿道炎的药盒，其特征在于其含有如权利要求17所述的药物制剂以及如何使用该药物制剂的说明书。
PCT/CN2014/094893 2014-01-02 2014-12-25 维生素d及其组合物的抗菌用途 WO2015101208A1 (zh)

Priority Applications (7)

Application Number Priority Date Filing Date Title
JP2016544430A JP2017501203A (ja) 2014-01-02 2014-12-25 抗生作用のためのビタミンｄ及びその組成物の使用
AU2014375577A AU2014375577A1 (en) 2014-01-02 2014-12-25 Vitamin D and antibacterial uses of composition thereof
EP14876219.8A EP3111939A4 (en) 2014-01-02 2014-12-25 Vitamin d and antibacterial uses of composition thereof
RU2016131454A RU2016131454A (ru) 2014-01-02 2014-12-25 Применения витамина d и его композиций для антибиоза
CN201480071325.7A CN105916510A (zh) 2014-01-02 2014-12-25 维生素d及其组合物的抗菌用途
US15/109,110 US20160324876A1 (en) 2014-01-02 2014-12-25 Vitamin d and antibacterial uses of compositions thereof
CA2935597A CA2935597A1 (en) 2014-01-02 2014-12-25 Uses of vitamin d and compositions thereof for antibiosis

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201410001937.0A CN104758300A (zh) 2014-01-02 2014-01-02 维生素d及其组合物的抗菌用途
CN201410001937.0 2014-01-02

Publications (1)

Publication Number Publication Date
WO2015101208A1 true WO2015101208A1 (zh) 2015-07-09

Family

ID=53493204

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2014/094893 WO2015101208A1 (zh) 2014-01-02 2014-12-25 维生素d及其组合物的抗菌用途

Country Status (8)

Country Link
US (1) US20160324876A1 (zh)
EP (1) EP3111939A4 (zh)
JP (1) JP2017501203A (zh)
CN (2) CN104758300A (zh)
AU (1) AU2014375577A1 (zh)
CA (1) CA2935597A1 (zh)
RU (1) RU2016131454A (zh)
WO (1) WO2015101208A1 (zh)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10441633B2 (en) 2014-09-24 2019-10-15 Zensun (Shanghai) Science & Technology, Co., Ltd. Uses of neuregulin in preventing, treating or delaying ventricular arrhythmia, and composition thereof
US10561709B2 (en) 2014-10-17 2020-02-18 Zensun (Shanghai) Science & Technology, Co., Ltd. Methods and compositions of neuregulins for preventing, treating or delaying preserved ejection fraction cardiac failure
US10702585B2 (en) 2014-01-03 2020-07-07 Zensun (Shanghai) Science & Technology, Co., Ltd. Formula of neuregulin preparation
CN111904966A (zh) * 2019-05-09 2020-11-10 上海泽生科技开发股份有限公司 维生素d及其组合物的抗菌用途
US10894815B2 (en) 2012-10-08 2021-01-19 Zensun (Shanghai) Science & Technology, Co., Ltd. Compositions and methods for treating heart failure in diabetic patients
US11253573B2 (en) 2011-10-10 2022-02-22 Zensun (Shanghai) Science & Technology, Co., Ltd. Compositions and methods for treating heart failure

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20230132635A (ko) 2013-05-22 2023-09-15 젠순 (상하이) 사이언스 앤드 테크놀로지 캄파니 리미티드 심부전을 치료하기 위한 뉴레귤린의 연장 방출

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1953752A (zh) * 2004-03-01 2007-04-25 拜奥克塞尔有限公司 用维生素d化合物治疗间质性膀胱炎
US20130143845A1 (en) * 2011-12-05 2013-06-06 William Francis Supple Vitamin d compounds and methods for enhancing muscle strength, and prevention and treatment of disease in human beings

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6548545B1 (en) * 2001-10-01 2003-04-15 40 J's Llc Treatment of interstitial cystitis using topical application of menthol and L-arginine
CA2524026A1 (en) * 2003-04-30 2004-11-18 Bioxell S.P.A. Gemini vitamin d3 compounds and methods of use thereof
WO2004098612A2 (en) * 2003-05-07 2004-11-18 Ab Science Calcitriol analogs of uses thereof
WO2005030222A1 (en) * 2003-09-24 2005-04-07 Bioxell S.P.A. 1,3-diaclyated,26,27-alkyl/haloalkyl vitamin d3 compounds and methods of use thereof
BRPI0508333A (pt) * 2004-03-01 2007-07-17 Bioxell Spa uso de um composto de vitamina d, método de prevenir e/ou tratar cistite intersticial, formulação farmacêutica, composto de vitamina d, e, kit
BRPI0616330A2 (pt) * 2005-09-26 2011-06-14 Novacea Inc prevenÇço e tratamento de distérbios gastrintestinais e da bexiga associados com quimioterapia ou radioterapia usando compostos ativos com vitamina d
WO2008115531A1 (en) * 2007-03-21 2008-09-25 Novacea, Inc. Prevention and treatment of infectious conditions with active vitamin d compounds or mimics thereof
FR2972328A1 (fr) * 2011-03-07 2012-09-14 Walmark A S Complement alimentaire augmentant la protection de l'epithelium des voies urinaires basses contre les infections a repetition
US20140155358A1 (en) * 2011-04-11 2014-06-05 Akthelia Pharmaceuticals Therapeutic Compounds

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1953752A (zh) * 2004-03-01 2007-04-25 拜奥克塞尔有限公司 用维生素d化合物治疗间质性膀胱炎
US20130143845A1 (en) * 2011-12-05 2013-06-06 William Francis Supple Vitamin d compounds and methods for enhancing muscle strength, and prevention and treatment of disease in human beings

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11253573B2 (en) 2011-10-10 2022-02-22 Zensun (Shanghai) Science & Technology, Co., Ltd. Compositions and methods for treating heart failure
US12076370B2 (en) 2011-10-10 2024-09-03 Zensun (Shanghai) Science & Technology, Co., Ltd. Compositions and methods for treating heart failure
US10894815B2 (en) 2012-10-08 2021-01-19 Zensun (Shanghai) Science & Technology, Co., Ltd. Compositions and methods for treating heart failure in diabetic patients
US10702585B2 (en) 2014-01-03 2020-07-07 Zensun (Shanghai) Science & Technology, Co., Ltd. Formula of neuregulin preparation
US11969458B2 (en) 2014-01-03 2024-04-30 Zensun (Shanghai) Science & Technology, Co., Ltd. Formula of neuregulin preparation
US10441633B2 (en) 2014-09-24 2019-10-15 Zensun (Shanghai) Science & Technology, Co., Ltd. Uses of neuregulin in preventing, treating or delaying ventricular arrhythmia, and composition thereof
US10561709B2 (en) 2014-10-17 2020-02-18 Zensun (Shanghai) Science & Technology, Co., Ltd. Methods and compositions of neuregulins for preventing, treating or delaying preserved ejection fraction cardiac failure
CN111904966A (zh) * 2019-05-09 2020-11-10 上海泽生科技开发股份有限公司 维生素d及其组合物的抗菌用途

Also Published As

Publication number Publication date
RU2016131454A (ru) 2018-02-07
EP3111939A1 (en) 2017-01-04
CN105916510A (zh) 2016-08-31
EP3111939A4 (en) 2017-11-08
JP2017501203A (ja) 2017-01-12
RU2016131454A3 (zh) 2018-08-31
US20160324876A1 (en) 2016-11-10
AU2014375577A1 (en) 2016-07-14
CN104758300A (zh) 2015-07-08
CA2935597A1 (en) 2015-07-09

Similar Documents

Publication Publication Date Title
WO2015101208A1 (zh) 维生素d及其组合物的抗菌用途
CN106539092B (zh) 一种绿藻多糖化纳米硒及其制备方法与应用
RU2479316C2 (ru) Пероральные композиции, продукты и способы применения
US20080051459A1 (en) Long term 24-hour intestinal administration of levodopa/carbidopa
WO2018064978A1 (zh) 一种抑菌剂配伍在制备阴道用组合物中的用途与阴道用组合物
US11202799B2 (en) Use of thiosulfate to potentiate the anti-pathogenic effect of lactobacilli
MX2010010866A (es) Composiciones y metodos para inmunoterapia.
AU2014413288B2 (en) Use of composition containing iron (II) amino acid chelate in preparing drug for regulating and controlling fat metabolism
CN114470008A (zh) 短乳杆菌或罗伊氏乳杆菌在制备治疗卵巢早衰的产品中的应用
US12005085B2 (en) Probiotic method and composition for maintaining a healthy vaginal microbiome
TWI482632B (zh) 醫藥用載體及使用該載體的藥物結構
US11337959B1 (en) Pharmaceutical composition Mix B to treat health conditions associated with elevated glucose levels
CN111904966A (zh) 维生素d及其组合物的抗菌用途
CN101780266A (zh) 防治畜禽呼吸道疾病的复方制剂
CN104352511A (zh) 一种粒细胞缺乏患者口腔溃疡糊剂及其制备方法
KR102439760B1 (ko) 상기도 감염균 질환 예방 및 치료를 위한 조성물
RU2760674C1 (ru) Применение препаратов растения polyscias filicifolia или его фрагментов для лечения элементозов и их последствий
CN103263425B (zh) 磷霉素氨丁三醇在制备治疗慢性细菌性前列腺炎药物中的应用
Febriza et al. Curcumin Effects in Inducing Vitamin D Receptor (VDR) and Inhibiting of Salmonella Typhi Growth in vivo
SK1602019A3 (sk) Suspenzia na prípravu probiotického nápoja a probiotický nápoj
Savitskaya et al. Prospective Use of Probiotics Immobilized on Sorbents with Nanostructured Surfaces
CN116531350A (zh) 一种口服递送罗伊式乳杆菌微胶囊及其制备和应用
CN103736075B (zh) 锌硒宝片及保妇康栓联合锌硒宝片在制备治疗老年性阴道炎药物中的应用
US20130150320A1 (en) Method of inhibiting increase of immunoglobulin a
CN116531394A (zh) 黄芩苷在制备对免疫检查点抑制剂治疗不适用的肿瘤的药物组合物中的应用

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: 14876219

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 15109110

Country of ref document: US

ENP Entry into the national phase

Ref document number: 2935597

Country of ref document: CA

ENP Entry into the national phase

Ref document number: 2016544430

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

REEP Request for entry into the european phase

Ref document number: 2014876219

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2014876219

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2014375577

Country of ref document: AU

Date of ref document: 20141225

Kind code of ref document: A

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112016015659

Country of ref document: BR

ENP Entry into the national phase

Ref document number: 2016131454

Country of ref document: RU

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 112016015659

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20160704