WO2020143535A1 - 利福霉素-喹嗪酮偶联分子及其盐的应用和制剂 - Google Patents

利福霉素-喹嗪酮偶联分子及其盐的应用和制剂 Download PDF

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WO2020143535A1
WO2020143535A1 PCT/CN2020/070162 CN2020070162W WO2020143535A1 WO 2020143535 A1 WO2020143535 A1 WO 2020143535A1 CN 2020070162 W CN2020070162 W CN 2020070162W WO 2020143535 A1 WO2020143535 A1 WO 2020143535A1
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rifamycin
quinazinone
coupling molecule
infection
biofilm
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PCT/CN2020/070162
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French (fr)
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马振坤
威廉∙J∙韦斯
安东尼∙西蒙∙林奇
袁鹰
刘宇
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丹诺医药(苏州）有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • 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
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents

Definitions

  • the invention relates to an application and preparation of a rifamycin-quinazinone coupling molecule and its salt, and belongs to the technical field of medicine.
  • the prosthesis needs to be removed by surgery and accompanied by long-term antibiotic treatment, resulting in huge pain and huge medical expenses for the patient.
  • the average cost of treatment for each joint infection is as high as more than $100,000.
  • IDSA American Infectious Disease Association
  • DAIR Debridement, Antibiotics and Implant Retention
  • the object of the present invention is to provide an application and preparation of rifamycin-quinazinone coupling molecule and its salt, rifamycin
  • the preparation of quinoxazone coupling molecule and its pharmaceutically acceptable salts can effectively treat biofilm infections.
  • the rifamycin-quinazinone coupling molecule has the formula I structure:
  • the pharmaceutically acceptable salt of the rifamycin-quinazinone coupling molecule may be its alkali metal salt or alkaline earth metal salt, such as potassium salt, sodium salt, double potassium salt, double sodium salt , Sodium potassium salt, etc.; preferably, the pharmaceutically acceptable salt of the rifamycin-quinazinone coupling molecule is the disodium salt of the rifamycin-quinazinone coupling molecule.
  • the disodium salt of the rifamycin-quinazinone coupling molecule is the reaction of the rifamycin-quinazinone coupling molecule represented by Formula I with sodium carbonate or sodium hydroxide After adding sodium formaldehyde sulfoxylate reaction prepared.
  • the pH value of the reaction solution is controlled to 7-11.
  • this conversion of the in-situ formation of the disodium salt with NaOH is a slow dissolution process and may lead to an increase in impurities.
  • the degradation products are induced by alkali and are pH-dependent. The higher the pH value, the greater the amount of degradation. Therefore, the setting of the PH value here can maximize the synthesis of disodium salt and minimize the degradation.
  • the biofilm infection includes a biofilm infection caused by bacteria using a medical device.
  • the biofilm infection includes one or a combination of venous catheter infection, artificial heart valve infection, artificial blood vessel infection, orthopedic implant infection, fracture fixation infection and artificial joint infection .
  • the bacteria include coagulase-negative staphylococci, enterococci, corynebacterium parvum, streptococcus megaterium, clostridium, clostridium, bacillus, methicillin-sensitive and/or alpha One or a combination of oxicillin-resistant Staphylococcus aureus.
  • the present invention also provides a specific application form, that is, a pharmaceutical preparation for the treatment of biofilm infections, the pharmaceutical component of which includes the rifamycin-quinazinone coupling represented by Formula I Molecules and their pharmaceutically acceptable salts.
  • the biofilm infection here includes one or a combination of venous catheter infection, artificial heart valve infection, artificial blood vessel infection, orthopedic implant infection, fracture fixation infection and artificial joint infection.
  • Bacteria that cause biofilm infections here include methicillin-sensitive and/or methicillin-resistant Staphylococcus aureus or coagulase-negative staphylococci, streptococcus, enterococcus, anaerobic bacteria such as Corynebacterium parvum, large digestion Streptococcus, Clostridium, Clostridium and Bacillus.
  • the type of the pharmaceutical preparation includes spray, aerosol, injection, tincture, ointment, powder injection or patch.
  • each unit of the pharmaceutical preparation contains the following raw material components:
  • the rifamycin-quinazinone coupling molecule shown in formula I and its pharmaceutically acceptable salts 100-105mg, mannitol 60-80mg, sodium formaldehyde sulfoxylate 2-6mg, polysorbate 80 0.1-1mg, Anhydrous ethanol 4-10 ⁇ L, sodium hydroxide to adjust PH9.5-10, water 3-10.5mL.
  • the present invention also provides a pharmaceutical combination for the treatment of human biofilm infections, the effective components of which include the rifamycin-quinazinone coupling molecule represented by Formula I and a pharmaceutically acceptable salt thereof.
  • the effective component may also be an antibacterial drug, such as rifampicin, levofloxacin, fluoroquinolone, gatifloxacin, etc.
  • the rifamycin-quinazinone coupling molecule of the present invention and its pharmaceutically acceptable salt, especially the disodium salt, and the prepared pharmaceutical preparation can effectively treat the human body including venous catheters, artificial heart valves, artificial blood vessels, Biofilm infections associated with orthopedic implants, fracture fixation, and artificial joint infections.
  • FIG. 1 is a comparison diagram of the dose-effect relationship of rifamycin-quinazinone-coupling molecule I against the detection of wild bacteria, rifampin-resistant bacteria and fluoroquinolone-resistant Staphylococcus aureus biofilm infection in Example 5;
  • FIG. 2 is a graph comparing the reduction in average log10CFU caused by the treatment of four staphylococcus aureus with rifamycin-quinolinone coupling molecule I and reference reagent in Example 5 for 24 hours;
  • Fig. 3a is a comparison chart of the average log 10 CFU reduction of the biofilm of S. aureus CB969 strain caused by the mixture of rifamycin-quinazinone coupling molecule I and rifampicin + tigecycline in Example 5;
  • FIG. 3b is a comparison chart of the average log 10 drug-resistant CFU of S. aureus CB969 strain biofilm caused by the mixture of rifamycin-quinazinone coupling molecule I and rifampicin + tigecycline in Example 5;
  • Example 4 is a graph of the average Log 10 CFU change generated by the rifamycin-quinazinon coupling molecule I and the control reagent on S. epidermidis CB191 colony biofilm treatment for 24 hours in Example 6;
  • FIG. 5 is a graph comparing the reduction in average Log 10 CFU generated by 4 ⁇ g/mL rifamycin-quinazinon coupling molecule I or control reagent on S. epidermidis CB191 colony biofilm treatment for 24 hours in Example 6;
  • FIG. 6 is a comparison graph of the average Log 10 resistance CFU generated by 4 ⁇ g/mL rifamycin-quinazinone coupling molecule I or a control reagent on S. epidermidis CB191 colony biofilm treatment for 24 hours in Example 6.
  • FIG. 6 is a comparison graph of the average Log 10 resistance CFU generated by 4 ⁇ g/mL rifamycin-quinazinone coupling molecule I or a control reagent on S. epidermidis CB191 colony biofilm treatment for 24 hours in Example 6.
  • Example 7 is a comparison diagram of log 10 CFU changes of a rifamycin-quinolinone coupling molecule I and a control antibiotic on a rat model of C. aureus CVC infection after 4 days of treatment in Example 7;
  • Example 8 is a graph comparing the results of 24 hours of rifamycin-quinazinone conjugated molecule I and control drug treatment of wild-type Staphylococcus epidermidis CB191 (ATCC#35984) in a constant flow reaction system in Example 9;
  • Example 9 is the development of 24-hour drug resistance of rifamycin-quinazinone conjugated molecule I and a control drug in wild type Staphylococcus epidermidis CB191 (ATCC#35984) in Example 9 in a constant flow reaction system Comparison chart
  • FIG. 10a is a comparison graph of rebound growth in a 72-hour test cycle of rifamycin-quinazinone conjugated molecule I and a control drug in a constant flow reaction system in Example 9 for the treatment of wild-type Staphylococcus epidermidis CB191 (ATCC#35984) ;
  • Figure 10b is the comparison of drug resistance development in the 72-hour trial period of rifamycin-quinazinon coupling molecule I and the control drug in the treatment of wild-type Staphylococcus epidermidis CB191 (ATCC#35984) in the constant flow reaction system in Example 9.
  • FIG. 11a is a comparison graph of rebound growth in a 72-hour test period of rifamycin-quinazinone coupling molecule I and a control drug in the treatment of Staphylococcus epidermidis CB1103 (ParC S80F ) in a constant flow reaction system in Example 9;
  • FIG. 11b is a comparison chart of drug resistance development in a 72-hour test cycle of rifamycin-quinazinone -coupling molecule I and a control drug in a constant flow reaction system in the treatment of Staphylococcus epidermidis CB1103 (ParC S80F ) in Example 9;
  • Example 12 is a comparison diagram of the results of 14 days of the treatment of wild-type Staphylococcus aureus CB192 of the rifamycin-quinazinon coupling molecule I and the control drug of Example 10;
  • FIG. 13 is a comparison graph of the time curve of the treatment of wild-type Staphylococcus aureus CB192 with rifamycin-quinazinone coupling molecule I and the control drug in Example 10;
  • This embodiment provides a disodium salt of a rifamycin-quinazinone coupling molecule and a preparation method thereof.
  • the preparation method of the disodium salt of the rifamycin-quinazinone coupling molecule is as follows:
  • the filtrate was placed in a dry ice/acetone bath and placed under nitrogen for 30 minutes.
  • the frozen solid was lyophilized with a vacuum freeze dryer at 33 ⁇ 10e -3 mBar and a temperature of -52°C overnight to obtain a fluffy orange solid (about 137 mg, yield >98%, content 82.7%).
  • It is the disodium salt of the rifamycin-quinazinon coupling molecule.
  • the solubility of the disodium salt in sterile water is greater than 20 mg/mL.
  • the pH of the aqueous solution is 10.3. (Note: 2.2 equivalents of sodium carbonate is necessary, the lower amount is not sufficient to be completely converted into the disodium salt of the rifamycin-quinazinone coupling molecule.
  • Sodium bicarbonate is a by-product and can maintain a high pH value .)
  • This embodiment provides a disodium salt of a rifamycin-quinazinone coupling molecule and a preparation method thereof.
  • the preparation method of the disodium salt of the rifamycin-quinazinone coupling molecule is as follows:
  • the filtrate was placed in a dry dry ice/acetone bath and placed under nitrogen for 30 minutes.
  • the frozen solid was lyophilized with a vacuum freeze dryer at 33 ⁇ 10e -3 mBar and a temperature of -52°C overnight to obtain a fluffy orange solid (about 165 mg, a recovery rate of 90%, and a content of 96%).
  • It is the disodium salt of the rifamycin-quinazinon coupling molecule.
  • the solubility of the sodium salt thus prepared in sterile water is greater than 20 mg/mL.
  • the rifamycin-quinazinon coupling molecule is an acid that is almost insoluble in water.
  • 1N NaOH was used to form the disodium salt in situ.
  • Crystalline APIs are difficult to convert to the disodium salt because this conversion is a slow dissolution process and may lead to an increase in impurities.
  • the degradation products are induced by alkali and are pH-dependent. The higher the pH value, the greater the amount of degradation. According to the rate and amount of NaOH added in the formulation, the study was conducted to enhance the solubility of the API and limit the amount of degradation. At the same time, pH and degradation studies are needed.
  • the rifamycin-quinazinone coupling molecule is light-stable and thermally unstable at temperatures greater than 60°C; when exposed to air, the rifamycin-quinazinone coupling molecule is slowly oxidized, ascorbic acid and formaldehyde Sodium sulfate can inhibit its oxidation in solution.
  • the disodium salt of the rifamycin-quinazinon coupling molecule When heated at a high humidity of 45°C, the disodium salt of the rifamycin-quinazinon coupling molecule has 20% decomposition within 6 days. However, when stabilized with 1% sodium formaldehyde sulfoxylate, its decomposition is greatly inhibited. Degradation is the result of air oxidation, and the main degradation product is quinone type.
  • the disodium salt of the rifamycin-quinazinone coupling molecule was stored at room temperature for 6 days with a decomposition rate of 2%; when stored at 4
  • This embodiment provides the application of the rifamycin-quinazinone coupling molecule and its pharmaceutically acceptable salt in the preparation of a medicament for the treatment of biofilm infections as an active ingredient of a preparation for injection or infusion Powder injection.
  • the powder injection is dissolved by adding 5 ml of water for injection to the content of the powder injection, which can be used for injection; or diluted with 5% glucose injection or 0.9% sodium chloride injection for infusion.
  • the powder injection in this embodiment is a 10 mL lyophilized vial (rifamycin-quinazinone coupling molecule 100 mg/vial).
  • the vial unit contains the following raw material components:
  • Rifamycin-quinazinone coupling molecule disodium salt 100mg (Rifamycin-quinazinone coupling molecule disodium salt is prepared in Example 1 or 2; the filling weight is 95%-105% Theoretical filling weight), mannitol 65-70mg, sodium formaldehyde sulfoxylate 2.1-2.3mg (sodium formaldehyde sulfoxylate exists in the form of dihydrate, the formulation contains 0.5mg/mL sodium formaldehyde sulfoxylate), polysorbate 80 0.30 -0.35mg, absolute ethanol 0-7 ⁇ L (absolute ethanol is used to dissolve polysorbate 80, it can be adjusted according to the production equipment without using absolute ethanol), sodium hydroxide is adjusted to pH9.5-10.5, water for injection 3.5mL (The theoretical filling volume of 3.5mL includes an excess of 5%, and the actual filling volume can be adjusted according to the content of the rifamycin-quinazinone
  • freeze-dried powder injection of the rifamycin-quinazinone coupling molecule of this embodiment, used for anti-infection is prepared by the following process steps:
  • This embodiment provides the application of the preparation of rifamycin-quinazinone coupling molecule in the preparation of antibacterial drugs for treating colony biofilm infection formed in vitro.
  • the study drug is the rifamycin-quinazinone coupling molecule represented by Formula I, which is obtained by the method of Example 3 (abbreviated as rifamycin-quinazinone coupling molecule I). Supplied by Danuo Pharmaceutical and stored at -20°C.
  • the control compound was purchased commercially. After pre-cultivating the S. aureus CB192 (ATCC#6538) colony biofilm for 24 hours, it was treated with the test drug for 24 hours, and the colony biofilm analysis was performed by a conventional method. And through the growth of the treated biofilm on the agar containing the same concentration of test drug, to track the development of biofilm resistance to drugs.
  • the rifampicin, linezolid, oxacillin, vancomycin, azithromycin and ciprofloxacin in the examples of the present invention
  • the data is basically consistent with other in vitro anti-staphylococcal biofilm activity test results.
  • the unanimous conclusion in most studies is that rifamycin antibiotics have better anti-biofilm activity than other antibiotics. These activities may reflect the key role of DNA continuous transcription in the development, maintenance, and/or maturation of in vitro biofilms, as well as the important role of RNA polymerase.
  • the efficient penetration of rifamycin antibiotics into pre-formed biofilms also contributes to its activity.
  • Co-administration of rifampicin and secondary antibiotics provides an effective treatment for certain types of biofilm-associated infections in indwelling medical devices, where the rifampicin component may be the main efficacy component, while secondary antibiotics can be used to eliminate Fuping resistant strains.
  • the rifamycin-quinazinone coupling molecule I is superior to the effect of separately acting on individual targets (RNA polymerase or type II DNA topoisomerase) Fumycin or fluoroquinolone antibiotics provide a better comprehensive effect in killing drug efficacy and preventing bacterial resistance.
  • This example provides the application of rifamycin-quinazinone coupling molecules in the treatment of human biofilm infections.
  • the rifamycin-quinazinone coupling molecule represented by formula I was used to form a specific rifamycin and/or fluoroquinolone resistant mutant strain derived from Staphylococcus aureus CB192 (ATCC#6538) in vitro The antibacterial activity of the colony biofilm.
  • the study drug was rifamycin-quinazinon coupling molecule I or the preparation prepared in Examples 1-3.
  • the preparations prepared from the pharmaceutically acceptable salts of the rifamycin-quinazinone coupling molecules in Examples 1-3, especially the preparations of the disodium salt, will first decay into the benefits shown in molecular form I in the body Formycin-quinazinone coupling molecule.
  • the control compound was purchased commercially.
  • the four isogenic S. aureus strains include wild-type strain CB0192 (ATCC#6538), rifamycin-resistant strain CB0785 (rpoB His481Tyr variant), fluoroquinolone-resistant strain CB0969 (parC Ser80Phe and gyrA Ser84Leu variant) and Fumycin and fluoroquinolone dual-resistant strain CB0974 (rpoB His481Tyr , parC Ser80Phe and gyrA Ser84Leu variants).
  • S. aureus CB0785, CB0969, or CB0974 colonies were formed into biofilms, and treated with test reagents for 24 hours to perform colony biofilm analysis.
  • the treated biofilm sample is placed on agar containing the same reagent and concentration to develop drug resistance.
  • the data for the wild-type strain CB0192 (ATCC#6538) is from Example 4. Reagents tested on all four strains included rifamycin-quinazinon coupling molecule I, ciprofloxacin, gatifloxacin, and rifampicin.
  • the rifampin+gatifloxacin and rifampin+ciprofloxacin mixtures were all mixed in a 1:1 weight ratio (eg, 4g/mL rifampicin+4g/mL gatifloxacin).
  • RESULTS Four isobaric-resistant Staphylococcus aureus strains were tested for rifamycin-quinazinon coupling molecule I and rifamycin (rifampin) and fluoroquinolone (ciprofloxacin and (Tifloxacin) anti-biofilm efficacy determination. The results showed that the rifamycin-quinazinon coupling molecule I had a good dose-effect relationship between the detected wild bacteria, rifampicin-resistant bacteria and fluoroquinolone-resistant Staphylococcus aureus biofilms (see Figure 1, Table 3) .
  • the rifamycin-quinazinone coupling molecule I caused more log 10 CFU reduction for each isogenic strain than the control drug ( Figure 2). Even at 4 and 16g/mL, rifampicin will lead to a rapid growth of drug-resistant mutants, which is not the case with the rifamycin-quinazinone coupling molecule I. Unlike rifampicin and other control reagents tested, the rifamycin-quinazinone coupling molecule I can prevent bacterial resistance at effective concentrations (shown in Figures 3 and 4).
  • Table 3 The average log 10 CFU reduction of the four reagents for S. aureus
  • This example provides the application of rifamycin-quinazinone coupling molecules in the treatment of bacterial biofilm infections.
  • This example reveals the antibacterial activity of the rifamycin-quinazinon coupling molecule and related reagents against the in vitro colony biofilm formed by Staphylococcus epidermidis ATCC#35984.
  • the study drug was rifamycin-quinazinon coupling molecule I or the preparation prepared in Examples 1-3. Supplied by Danuo Pharmaceutical and stored at -20°C. The control compound was purchased commercially. Experiments have shown that the preparations prepared from the pharmaceutically acceptable salts of the rifamycin-quinazinone coupling molecules in Examples 1-3, especially the preparations of the disodium salt, will first decay into the benefits shown in molecular form I in the body Formycin-quinazinone coupling molecule.
  • CB191 was pre-cultured for 24 hours to form a colony biofilm, and then treated with test reagents for 24 hours.
  • test reagents for 24 hours.
  • the development of drug resistance after treatment can be obtained by tracking the processed biofilm samples.
  • the rifamycin-quinazinone coupling molecule I can inhibit the development of surface Staphylococcus aureus resistance within its effective concentration range for killing bacteria.
  • Table 4 Reduction of the average Log10CFU caused by rifamycin-quinazinone conjugated molecule I and control reagents on S. epidermidis CB291 colony biofilm treatment for 24 hours
  • Table 5 Average Log 10 resistant CFU generated by rifamycin-quinazinone conjugated molecule I and control reagent on S. epidermidis CB191 colony biofilm treatment for 24 hours
  • This example provides the application of rifamycin-quinazinone coupling molecules in the treatment of biofilm infections.
  • a rat venous catheter model was used to verify the efficacy of rifamycin-quinoxazone coupling molecule I on a rat model of Central Venous Catheter (CVC) infection.
  • CVC Central Venous Catheter
  • a stable rat model of chronic central venous catheter infection and drug efficacy is established, which is used to evaluate the in vivo efficacy of antibacterial drugs on bacterial infections in blood vessels related to medical devices.
  • the bacteria used are isogenic S. aureus groups that can form biofilms.
  • the wild type strain is CB192 (ATCC#6538), and its two isogenic derivatives are the rifampin-resistant CB785 strain (rpoB H481Y ) and the fluoroquinolone-resistant CB969 strain (gyrA S84L , parC S80F ).
  • Table 6 Mean log 10 CFU reduction of rifamycin-quinazinone conjugated molecule I and control antibiotics for 7 days on rat central venous catheter S. aureus infection model
  • a is administered twice daily by tail vein injection
  • * is oral administration.
  • Table 7 Changes in mean log10CFU of 10 mg/mL rifamycin-quinazinone conjugated molecule I and control antibiotics in the treatment of wild and drug-resistant Staphylococcus aureus infections in rats
  • This example provides a comparison of the efficacy of rifamycin-quinazinone coupling molecule I and clinically used fluoroquinolone + rifampicin in the treatment of methicillin-resistant and fluoroquinolone-resistant Staphylococcus aureus experimental endocarditis.
  • Staphylococcus aureus MRSA 67/0 was used to establish the endocarditis model in this example.
  • Animal model The experimental aortic valve bacterial endocarditis model described in this example was established in New Zealand white rabbits
  • results Compared with untreated controls, after the end of treatment and after 3 days of relapse, treatment with rifamycin-quinazinone conjugated molecule I (40 mg/kg, intravenous injection, twice daily for 3 days) All reduced the density of Staphylococcus aureus in the three tissues.
  • Table 8 Effect of rifamycin-quinazinone conjugated molecule I and fluoroquinolone + rifampicin treatment on the mean CFU of methicillin-resistant and fluoroquinolone-resistant Staphylococcus aureus experimental endocarditis
  • rifamycin-quinazinone conjugate molecule I has the best reduction of S. aureus in all three tissues compared to levofloxacin + rifampin or ciprofloxacin + rifampin The effect of density.
  • the rifamycin-quinazinone-coupling molecule I produced a >5 log reduction in the titers of methicillin-resistant and fluoroquinolone-resistant Staphylococcus aureus in cardiac tissue.
  • the minimum inhibitory concentrations of rifamycin-quinazinone coupling molecule I, rifampicin, ciprofloxacin and levofloxacin against Staphylococcus aureus were 0.03, 0.015, 8 and 2 ⁇ g/mL (data not available) display).
  • CB1834 The minimum inhibitory concentrations of rifamycin-quinazinone coupling molecule I, rifampicin, ciprofloxacin and levofloxacin against Staphylococcus aureus
  • the rifamycin-quinazinone coupling molecule I has been shown to be effective for endocarditis caused by methicillin-resistant Staphylococcus aureus that is sensitive or resistant to fluoroquinolones.
  • This example provides the anti-biofilm efficacy study of the rifamycin-quinazinon coupling molecule in the trickle reactor model.
  • strains used included S. epidermidis CB191 (ATCC#35984), S. epidermidis CB1038 (rifampin resistance, rpoB H481Y variation), S. epidermidis CB1106 (fluoroquinolone resistance, grlA S80F and gyrA S84Y variation).
  • the minimum inhibitory concentration (MIC) test is performed according to the recommended guidelines of CLSI (Clinical and Laboratory Standards Institute).
  • Static or constant trickle device a 20-cell trickle reactor commonly used in the art is used.
  • results The rifamycin-quinazinone conjugated molecule I, rifampicin, two fluoroquinolones, and the rifampicin + ciprofloxacin cocktail protocol were used to study the biological properties of Staphylococcus epidermidis using a constant-trickle biofilm system.
  • the assessment included 48-hour growth of the biofilm, followed by 24 hours of treatment with the study drug, or 72 hours of treatment with the 48-hour drug-free rebound phase.
  • the wild-type Staphylococcus epidermidis CB191 and the isogenic drug-resistant derivatives containing nucleotide substitutions in ParC S80F with reduced susceptibility to some quinolones were tested for 72 hours.
  • rifamycin-quinazinone conjugated molecule I is superior to all control drugs, including rifampicin + ciprofloxacin cocktail therapy. Especially when the rifamycin-quinazinone coupling molecule I was administered at doses of 1 ⁇ g/mL and 4 ⁇ g/mL, respectively, the total Log10CFU/cm 2 reduction was 3.33 and 4.84, respectively.
  • biofilms treated with rifamycin-quinazinone conjugated molecule I for 72 hours or rifampicin plus ciprofloxacin cocktail were recovered (rebound) for 48 hours in a drug-free trial, and then sampled again To observe its drug resistance and survivability.
  • biofilms treated with rifampicin plus ciprofloxacin cocktail showed a rapid and significant increase in activity count after antibiotic removal, while samples treated with rifamycin-quinazinone coupling molecule I did not Any significant increase in activity count is shown. No resistance to rifamycin-quinazinone coupled molecule I, rifampicin, or ciprofloxacin was observed in any rebound samples (as shown in Figure 11).
  • Table 9 Treatment with wild-type Staphylococcus epidermidis CB191 (ATCC#35984) in a 72-hour experimental period (including a 48-hour drug-free period) in a constant flow reaction system with rifamycin-quinazinon coupling molecule I and a control drug Average total log10, log10 reduction and drug resistance CFU/cm 2
  • Table 10 In the constant flow response system caused by the rifamycin-quinazinone coupling molecule I and the control drug for the treatment of quinolone-resistant Staphylococcus epidermidis CB1103 (ParC S80F ) during the 72-hour experimental period (including the 48-hour drug-free period) Average total log10, log10 reduction and drug resistance CFU/cm 2
  • This example provides the application of rifamycin-quinazinone coupling molecules in the treatment of biofilm infections.
  • Study drug and material the preparation prepared in the rifamycin-quinazinone coupling molecule I or Example 1-3. Supplied by Danuo Pharmaceutical and stored at -20°C. Control compounds or formulations are purchased commercially.
  • the bacteria used are S. aureus strains. Wild type CB192 (ATCC#6538), its two isogenic resistance derivatives CB785 (rifampin-resistant rpoB H481Y ) and CB969 (fluoroquinolone-resistant gyrA S84L /parC S80F ).
  • Test animal In this example, 5-6 week old BALB/c mice were used. During the experiment, the mice were free to eat/drink water, and all operations were in accordance with the guidelines of the Research Animal Use and Care Committee (IACUC).
  • IACUC Research Animal Use and Care Committee
  • the bacteria used were the isogenic S. aureus forming biofilms.
  • the wild-type strain is CB192 (ATCC#6538), and its two isogenic resistance derivatives are rifampin-resistant strain CB785 (rpoB H481Y ) and fluoroquinolone-resistant strain CB969 (gyrA S84L , parC S80F ). All three strains showed very similar growth kinetics in vivo, and the average values of Teflon implants were 6.6-6.7 and 7.1-7.2 log10 CFU/implant at 7 and 21 days after infection, respectively.
  • rifamycin-quinazinone conjugated molecule I and 4 control antibiotics were evaluated by intraperitoneal injection twice daily 7 days after infection.
  • rifampicin was intraperitoneally injected at 25 mg/kg twice daily for at least 7 days, and a significant reduction in bacterial cells at the site of subcutaneous infection was observed.
  • the log reductions were vancomycin-0.1, daptomycin 0.7, gatifloxacin 1.7, rifampicin 2.1, and rifamycin-quinazinone coupling molecule I 5.0.
  • Combination therapy with fuping and gatifloxacin requires 10+10mg/kg.
  • rifamycin-quinazinone coupling molecule I requires 10 mg/kg to significantly inhibit the emergence of rifampicin resistance in the body, while rifampicin + gatifloxacin combination therapy requires 10 + 10 mg/kg to inhibit.
  • rifamycin-quinazinone conjugated molecule I monotherapy or rifampin + gatifloxacin combination therapy is the only way to remove bacteria from most implants (16 rifamycin-quinazine 15 of the ketone-coupled molecule I-treated implants detected ⁇ 2.09log10CFU, and 30 of the 30 rifampicin+gatifloxacin-treated implants detected ⁇ 2.09log10CFU).
  • rifampicin can effectively reduce the CFU of sensitive bacteria in this model, its efficacy is limited by the continued emergence and expansion of spontaneous rifampicin-resistant variants during long-term treatment.
  • the efficacy of rifamycin-quinazinone conjugated molecule I against wild-type, rifampin-resistant, and fluoroquinolone-resistant Staphylococcus aureus implanted infections is generally comparable to twice the dose of rifampicin + Combination therapy with gatifloxacin is comparable.
  • Table 11 14-day treatment of wild-type Staphylococcus aureus CB192
  • This example provides the application of rifamycin-quinazinone coupling molecules in the treatment of biofilm infections.
  • Study drug and material the preparation prepared in the rifamycin-quinazinone coupling molecule I or Example 1-3. Supplied by Danuo Pharmaceutical and stored at -20°C. Control compounds or formulations are purchased commercially.
  • Staphylococcus aureus NT111-3 (NRS249 or HT20020341), congenital valvular endocarditis isolate, p-ciprofloxacin, clindamycin, erythromycin, gentamicin, oxacillin, Penicillin resistant.
  • Test animal In this example, C57BL/6 female mice aged 8-10 weeks were used. During the experiment, the mice were free to eat/drink water, and all operations were in accordance with the guidelines of the Research Animal Use and Care Committee (IACUC).
  • IACUC Research Animal Use and Care Committee
  • Table 12 The efficacy of rifamycin-quinazinone coupling molecule I, rifampicin and vancomycin in mouse PJI infection model
  • the rifamycin-quinazinon coupling molecule and the pharmaceutical preparation or composition thereof according to the embodiments of the present invention can effectively treat or prevent medical devices including artificial joints, venous catheters, fracture fixation, heart valves or artificial blood vessels. Biofilm infection.

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Abstract

一种利福霉素-喹嗪酮偶联分子及其药学上可接受的盐在制备治疗生物膜感染的药物中的应用，该利福霉素-喹嗪酮偶联分子具有式I所示结构。还提供了一种用于治疗生物膜感染的药物制剂，其药用组分包括式I所示的利福霉素-喹嗪酮偶联分子及其药学上可接受的盐。利福霉素-喹嗪酮偶联分子及其药学上可接受的盐，尤其是双钠盐，以及制备得到的药物制剂能够有效治疗人体包括静脉导管、人工心脏瓣膜、人造血管、整形外科植入物、骨折固定和人工关节感染相关的生物膜感染。

Description

利福霉素-喹嗪酮偶联分子及其盐的应用和制剂 技术领域
本发明涉及一种利福霉素-喹嗪酮偶联分子及其盐的应用和制剂，属于医药技术领域。
背景技术
医疗器械相关生物膜感染是当前抗感染领域最迫切的未满足的需求之一。随着人口的老龄化和假体植入手术的增加，与之相关的生物膜感染的发病率正处于快速增长阶段。据统计，美国在2010年进行了约100万例髋关节或膝关节植入手术，预计2030年这类手术将增加到400万例，加强感染预防措施并未明显降低发病率，相反从2001年到2009年髋关节植入术感染率反而从1.99％升高到2.18％，膝关节植入术感染率从2.05％增加到2.18％。在中国，人工关节植入手术也从2013年的28万例增加至2014年的40万例，处于快速增长阶段。生物膜感染的治疗非常困难，一般需要通过手术取出假体并伴随长期抗菌素治疗，导致患者的巨大痛苦和庞大的医疗费用。在美国，每例人工关节感染的平均治疗费用高达10万美元以上。对于早期感染(关节植入后30天内或感染症状出现3周内)，如果是无窦道并且人工关节稳固的患者，美国感染病协会(Infectious Diseases Society of America，IDSA)发表的诊治指南建议对患者实施清创、抗菌素治疗和人工关节保留(Debridement,Antibiotics and Implant Retention，DAIR)的策略，然而这一策略的有效率目前只有30-50％；而其他的患者则必需进行一步或两步人工关节的置换(修复)手术。对于某些病人，置换手术已经无法实施，截肢则成为了最后的治疗手段。除了人工关节外，还有许多其它类型医疗器械相关的生物膜感染如中心静脉导管、人工心脏瓣膜、骨折修复固定、人造血管植入和心脏起搏器感染等，目前均缺乏有效的治疗手段。
发明内容
鉴于上述现有技术存在的缺陷和临床治疗上存在的尚未满足的重大需求，本发明的目的是提供一种利福霉素-喹嗪酮偶联分子及其盐的应用和制剂，利福霉素-喹嗪酮偶联分子及其药学上可接受的盐的制剂能够有效治疗生物膜感染。
本发明的目的是通过如下方案实现的：
一种利福霉素-喹嗪酮偶联分子及其药学上可接受的盐在制备治疗生物膜感染的药物中的应用，该利福霉素-喹嗪酮偶联分子具有式Ⅰ所示结构：
Figure PCTCN2020070162-appb-000001
Figure PCTCN2020070162-appb-000002
上述的应用中，所述利福霉素-喹嗪酮偶联分子的药学上可接受的盐可以是其碱金属盐或碱土金属盐，如钾盐、钠盐、双钾盐、双钠盐、钠钾盐等；优选的，所述利福霉素-喹嗪酮偶联分子的药学上可接受的盐为利福霉素-喹嗪酮偶联分子的双钠盐。
上述的应用中，优选的，所述利福霉素-喹嗪酮偶联分子的双钠盐为式Ⅰ所示的利福霉素-喹嗪酮偶联分子与碳酸钠或氢氧化钠反应后、加入甲醛次硫酸钠反应制备得到的。
上述的应用中，将碳酸钠或氢氧化钠替换成其他金属的对应的盐或碱，甲醛次硫酸钠替换成其他金属的有机盐，可以得到其他利福霉素-喹嗪酮偶联分子的药学上可接受的盐。
上述的应用中，优选的，在制备得到利福霉素-喹嗪酮偶联分子的盐时，控制反应液的pH值为7-11。在制备过程中，用NaOH原位形成双钠盐的这种转化是缓慢的溶出过程，可能导致杂质的增加。降解产物是由碱诱导并且是pH依赖性的，pH值越高，降解的量越大。所以这里的PH值的设定能够最大限度地合成双钠盐、又能够最大限度地降低降解。
上述的应用中，优选的，所述生物膜感染包括使用医疗器械、由细菌引起的生物膜感染。
上述的应用中，优选的，所述生物膜感染包括静脉导管感染、人工心脏瓣膜感染、人造血管感染、整形外科植入物感染、骨折固定感染和人工关节感染中的一种或几种的组合。
上述的应用中，优选的，所述细菌包括凝固酶阴性葡萄球菌、肠球菌、短小棒状杆菌、大消化链球菌、梭形杆菌、梭状芽孢杆菌、类杆菌、甲氧西林敏感和/或甲氧西林耐药的金黄色葡萄球菌中的一种或几种的组合。
根据上述的应用，本发明还提供一种具体的应用形式，即一种用于治疗生物膜感染的药物制剂，其药用组分包括式Ⅰ所示的利福霉素-喹嗪酮偶联分子及其药学上可接受的盐。
这里的生物膜感染包括静脉导管感染、人工心脏瓣膜感染、人造血管感染、整形外科植入物感染、骨折固定感染和人工关节感染中的一种或几种的组合。引起这里的生物膜感染的细菌包括甲氧西林敏感和/或甲氧西林耐药金黄色葡萄球菌或凝固酶阴性葡萄球菌，链球菌属，肠球菌属，厌氧菌例如短小棒状杆菌、大消化链球菌、梭形杆菌属、梭状芽孢杆菌属和类杆菌属。
上述的药物制剂中，优选的，该药物制剂制剂类型包括喷剂、气雾剂、注射剂、酊剂、膏剂、粉针剂或贴剂。
上述的药物制剂中，优选的，每单位的所述药物制剂中包含如下原料组分：
式Ⅰ所示的利福霉素-喹嗪酮偶联分子及其药学上可接受的盐100-105mg，甘露醇60-80mg，甲醛次硫酸钠2-6mg，聚山梨酯80 0.1-1mg，无水乙醇4-10μL，氢氧化钠调节PH9.5-10，水3-10.5mL。
本发明还提供一种用于治疗人体生物膜感染的药物组合，其有效组分包括式Ⅰ所示的利福霉素-喹嗪酮偶联分子及其药学上可接受的盐。
上述的药物组合中，有效组分还可以是抗菌药物，例如利福平、左氧氟沙星、氟喹诺酮和加替沙星等。
本发明的突出效果为：
本发明的利福霉素-喹嗪酮偶联分子及其药学上可接受的盐，尤其是双钠盐，以及制备得到的药物制剂能够有效治疗人体包括静脉导管、人工心脏瓣膜、人造血管、 整形外科植入物、骨折固定和人工关节感染相关的生物膜感染。
附图说明
图1是实施例5中，利福霉素-喹嗪酮偶联分子I对检测的野生菌，利福平耐药菌和氟喹诺酮耐药金葡菌生物膜感染的量效关系对比图；
图2是实施例5中，利福霉素-喹嗪酮偶联分子I和参照试剂处理四种金黄色葡萄球菌24小时造成的平均log10CFU的减少对比图；
图3a是实施例5中，利福霉素-喹嗪酮偶联分子I和利福平+替加环素混合物造成的金葡菌CB969株生物膜平均log 10CFU的减少对比图；
图3b是实施例5中，利福霉素-喹嗪酮偶联分子I和利福平+替加环素混合物造成的金葡菌CB969株生物膜平均log 10耐药CFU的对比图；
图4是实施例6中，利福霉素-喹嗪酮偶联分子I和对照试剂对表皮葡萄球菌CB191菌落生物膜处理24小时生成的平均Log 10CFU变化图；
图5是实施例6中，4μg/mL利福霉素-喹嗪酮偶联分子I或对照试剂对表皮葡萄球菌CB191菌落生物膜处理24小时生成的平均Log 10CFU的减少对比图；
图6是实施例6中，4μg/mL利福霉素-喹嗪酮偶联分子I或对照试剂对表皮葡萄球菌CB191菌落生物膜处理24小时生成的平均Log 10耐药CFU对比图。
图7是实施例7中，利福霉素-喹嗪酮偶联分子I和对照抗生素对大鼠野生金葡菌CVC感染模型治疗4天后的log 10CFU变化对比图；
图8是实施例9中，在恒流量反应系统中利福霉素-喹嗪酮偶联分子I和对照药物治疗野生型表皮葡萄球菌CB191(ATCC#35984)，24小时的结果比较图；
图9是实施例9中，在恒流量反应系统中利福霉素-喹嗪酮偶联分子I和对照药物治疗野生型表皮葡萄球菌CB191(ATCC#35984)，24小时的耐药性的发展对比图；
图10a是实施例9中，在恒流量反应系统中利福霉素-喹嗪酮偶联分子I和对照药物治疗野生型表皮葡萄球菌CB191(ATCC#35984)72小时试验周期中反弹生长对比图；
图10b是实施例9中，在恒流量反应系统中利福霉素-喹嗪酮偶联分子I和对照药物治疗野生型表皮葡萄球菌CB191(ATCC#35984)72小时试验周期中耐药发展对比图；
图11a是实施例9中，在恒流量反应系统中利福霉素-喹嗪酮偶联分子I和对照药物治疗表皮葡萄球菌CB1103(ParC S80F)72小时试验周期中反弹生长对比图；
图11b是实施例9中，在恒流量反应系统中利福霉素-喹嗪酮偶联分子I和对照药物治疗表皮葡萄球菌CB1103(ParC S80F)72小时试验周期中耐药发展对比图；
图12是实施例10的利福霉素-喹嗪酮偶联分子I和对照药物处理野生型金黄色葡萄球菌CB192 14天结果对比图；
图13是实施例10利福霉素-喹嗪酮偶联分子I和对照药物处理野生型金黄色葡萄球菌CB192的时间曲线对比图；
具体实施方式
为了对本发明的技术特征、目的和有益效果有更加清楚的理解，现对本发明的技术方案进行以下详细说明，但不能理解为对本发明的可实施范围的限定。下述实施例中所述实验方法，如无特殊说明，均为常规方法；所述试剂和材料，如无特殊说明，均可从商业途径获得。
实施例1
本实施例提供一种利福霉素-喹嗪酮偶联分子的双钠盐及其制备方法。该利福霉素-喹嗪酮偶联分子的双钠盐的制备方法如下：
将式Ⅰ所示的利福霉素-喹嗪酮偶联分子(游离酸，丙酮结晶)(115mg，0.095mmol)置于圆底烧瓶中，在室温下加入乙醇(0.2mL，无水或USP级)混合均匀，加入0.22mL 1M碳酸钠溶液，混合物搅拌5分钟，用4.5mL HPLC水(或USP级无菌水)稀释，搅拌30分钟或直至搅拌均匀，加入甲醛次硫酸钠(HOCH 2SO 2Na·2H 2O)(1.2mg)，搅拌5分钟，过滤均相溶液。滤液放置在干冰/丙酮浴中，于氮气下放置30分钟。冷冻固体用真空冷冻干燥器在33×10e -3mBar，温度-52℃下过夜冻干，得到蓬松的橙色固体(约137mg，收率>98％，含量为82.7％)。即为利福霉素-喹嗪酮偶联分子的双钠盐。该双钠盐在无菌水中的溶解度大于20mg/mL。水溶液的pH值为10.3。(注意：2.2当量的碳酸钠是必需的，较低的量不足以完全转化为利福霉素-喹嗪酮偶联分子的双钠盐。碳酸氢钠是副产物，可以保持高的pH值。)
Figure PCTCN2020070162-appb-000003
实施例2
本实施例提供一种利福霉素-喹嗪酮偶联分子的双钠盐及其制备方法。该利福霉素-喹嗪酮偶联分子的双钠盐的制备方法如下：
将式Ⅰ所示的利福霉素-喹嗪酮偶联分子(游离酸，丙酮结晶)(172mg，0.14mmol)置于圆底烧瓶中，在室温下加入乙醇(0.4mL，无水或USP级)，搅拌混合均匀，搅拌下缓慢加入0.1N氢氧化钠溶液2.8mL，将溶液pH值控制在9.5-10.5之间，混合物搅拌15分钟，并用2.3mL HPLC水(或USP级无菌水)稀释，搅拌15分钟或直至搅拌均匀，加入甲醛次硫酸钠(HOCH 2SO 2Na·2H 2O)(1.7mg)，搅拌5分钟，过滤均相溶液。滤液放置在干燥的干冰/丙酮浴中，在氮气下放置30分钟。冷冻固体用真空冷冻干燥器在33×10e -3mBar，温度-52℃下过夜冻干，得到蓬松的橙色固体(约165mg，回收率90％，含量为96％)。即为利福霉素-喹嗪酮偶联分子的双钠盐。由此制备的钠盐在无菌水中的溶解度大于20mg/mL。
对实施例1、2得到的利福霉素-喹嗪酮偶联分子的双钠盐进行稳定性分析。
利福霉素-喹嗪酮偶联分子是一种几乎不溶于水的酸。在制剂制备过程中，用1N NaOH原位形成双钠盐。结晶原料药很难转化为双钠盐，因为这种转化是缓慢的溶出过程，可能导致杂质的增加。降解产物是由碱诱导并且是pH依赖性的，pH值越高，降解的量越大。根据配方中添加的NaOH的速率和用量，对增强原料药的溶解性并限制降解量进行研究。同时需要进行pH值与降解的研究。利福霉素-喹嗪酮偶联分子光稳定，在温度大于60℃时，热不稳定；当暴露于空气中，利福霉素-喹嗪酮偶联分子 被缓慢氧化，抗坏血酸和甲醛次硫酸钠可以抑制其在溶液中的氧化。在高湿度45℃下加热时，利福霉素-喹嗪酮偶联分子的双钠盐在6天内有20％的分解。但用1％甲醛次硫酸钠稳定时，其分解受到很大抑制。降解是空气氧化的结果，主要降解物为醌型。利福霉素-喹嗪酮偶联分子的双钠盐在室温下贮存6天，分解率为2％；在4℃储存6天，分解率为1％。
实施例3
本实施例提供利福霉素-喹嗪酮偶联分子及其药学上可接受的盐在制备治疗生物膜感染的药物中的应用，作为一种制剂的有效成分，该制剂为注射或输液用粉针剂。
该粉针剂在静脉输液给药前，向该粉针剂中的内容物加入5毫升注射用水溶解，可用于注射；或者再用5％葡萄糖注射液或0.9％氯化钠注射液稀释，进行输液。
本实施例的粉针剂为10mL冻干小瓶(利福霉素-喹嗪酮偶联分子100mg/瓶)，小瓶单元包含如下原料组分：
利福霉素-喹嗪酮偶联分子的双钠盐100mg(利福霉素-喹嗪酮偶联分子的双钠盐为实施例1或2所制得；填充重量为95％-105％的理论填充重量)，甘露醇65-70mg，甲醛次硫酸钠2.1-2.3mg(甲醛次硫酸钠以二水合形式存在，该配方含有0.5mg/mL的甲醛次硫酸钠)，聚山梨酯80 0.30-0.35mg，无水乙醇0-7μL(无水乙醇用以溶解聚山梨酯80，可以不使用无水乙醇，根据生产设备进行调整)，氢氧化钠调节pH9.5-10.5，注射用水3.5mL(3.5mL的理论填充体积包括5％的过量，实际填充体积可以根据生产过程中利福霉素-喹嗪酮偶联分子的含量进行调整)。
本实施例的利福霉素-喹嗪酮偶联分子的冻干粉针剂，用于抗感染，是通过如下工艺步骤制备得到的：
在符合规范的洁净区内将甘露醇溶解于注射用水中，加入甲醛次硫酸钠，再加入用无水乙醇溶解的聚山梨酯80，加入利福霉素-喹嗪酮偶联分子I，混合搅拌10分钟，得到第一溶液；
将第一反应液用氢氧化钠调节pH 9.5-10.5，然后搅拌直到完全溶解，定容到10mL，得到第二溶液；
将第二溶液过滤，测定利福霉素-喹嗪酮偶联分子的浓度，调整其含量为100mg/10mL，低压冻干，即得到利福霉素-喹嗪酮偶联分子的冻干粉针剂。
实施例4
本实施例提供利福霉素-喹嗪酮偶联分子的制剂在制备治疗体外形成的菌落生物膜感染的抗菌药物中的应用。
材料和方法：研究药物为式I所示的利福霉素-喹嗪酮偶联分子按实施例3的方法得到的制剂(简称利福霉素-喹嗪酮偶联分子I)。由丹诺医药提供，并在-20℃下储存。对照化合物由商业途径采购得到。将金黄色葡萄球菌CB192(ATCC#6538)菌落生物膜预培养24小时之后，用试验药物处理24小时，用常规方法进行菌落生物膜分析。并通过处理后的生物膜在含相同浓度试验药物的琼脂上的生长情况，来跟踪生物膜对药物耐药性的发展。
结果：在对已经形成的金黄色葡萄球菌CB192菌落生物膜的24小时处理中，利福霉素-喹嗪酮偶联分子I在4和16μg/mL浓度时分别造成1.6和1.9log 10CFU的减少(表1)，与参比试剂比具有更好的抗生物膜药效。在处理期间，即使是在4和16μg/mL时，利福平仍产生大量利福平耐药突变菌(表2)。相反，在相同条件下没有观察到对利福霉素-喹嗪酮偶联分子I和其他对照试剂耐药性的发展。因此，与利福平不同，利福霉素-喹嗪酮偶联分子I在其杀伤细菌的有效浓度范围内可抑制细菌耐药性的发展。
根据所检测的抗生素杀死在生物膜状态下金黄色葡萄球菌的相对药效，本发明实施例中的利福平、利奈唑胺、苯唑西林、万古霉素、阿奇霉素和环丙沙星的数据与其它体外抗葡萄球菌生物膜活性的试验结果基本一致。大多数研究中得出的一致结论是，利福霉素类抗生素对比其他抗生素类具有更优异的抗生物膜活性。这些活性可能反映了在体外生物膜的发育、维持和/或成熟中DNA持续转录的关键作用，以及其中RNA聚合酶的重要作用。此外，利福霉素类抗生素对预先形成的生物膜中的高效渗透也有助于其活性的提高。将利福平与二级抗生素联合给药提供了对某些类型的留置医疗设备生物膜相关感染的有效治疗方案，其中利福平组分可能是主要功效成分，而二级抗生素可用于消除利福平耐药菌株。
综上所述，在本实施例所用的实验条件下，利福霉素-喹嗪酮偶联分子I优于分别作用于单独靶向(RNA聚合酶或II型DNA拓扑异构酶)的利福霉素或氟喹诺酮类抗生素，其在杀灭药效和预防细菌耐药性方面提供了更好的综合效果。这些数据为利福霉素-喹嗪酮偶联分子I在治疗金黄色葡萄球菌引起的生物膜相关感染中的潜在用途提供了证据。
表1使用利福霉素-喹嗪酮偶联分子I和参比试剂处理24小时的金黄色葡萄球菌CB192菌落生物膜(预培养24小时)的平均Log10减少值
Figure PCTCN2020070162-appb-000004
表2使用利福霉素-喹嗪酮偶联分子I和参比试剂处理24小时的金黄色葡萄球菌CB192菌落生物膜(预培养24小时)的平均Log10耐药CFU
Figure PCTCN2020070162-appb-000005
实施例5
本实施例提供利福霉素-喹嗪酮偶联分子在治疗人体生物膜感染中的应用。
本实施例使用式Ⅰ所示利福霉素-喹嗪酮偶联分子对金黄色葡萄球菌CB192(ATCC#6538)衍生的具有特异性利福霉素和/或氟喹诺酮耐药突变菌株体外形成的菌落生物膜的抗菌活性。
材料和方法：研究药物为利福霉素-喹嗪酮偶联分子I或实施例1-3中制备的制剂。实验显示实施例1-3中利福霉素-喹嗪酮偶联分子的药学上可接受的盐制备的制剂，尤其是双钠盐的制剂在体内会先蜕变成分子形式Ⅰ所示的利福霉素-喹嗪酮偶联分子。
对照化合物由商业途径采购得到。四个等基因金黄色葡萄球菌菌株包括野生型菌株CB0192(ATCC#6538)、利福霉素耐药株CB0785(rpoB His481Tyr变异)，氟喹诺酮耐药株CB0969(parC Ser80Phe和gyrA Ser84Leu变异)和利福霉素和氟喹诺酮双耐药株CB0974(rpoB His481Tyr,parC Ser80Phe和gyrA Ser84Leu变异)。
培养24小时使金黄色葡萄球菌CB0785、CB0969或CB0974菌落形成生物膜，并用试验试剂处理24小时，进行菌落生物膜分析。将处理过的生物膜样品置于含有相同试剂和浓度的琼脂上，得到耐药性的发展。野生型菌株CB0192(ATCC#6538)的数据来自实施例4。对所有四株菌株进行检测的试剂包括利福霉素-喹嗪酮偶联分子I、环丙沙星、加替沙星和利福平。为研究CB0969菌株，利福平+加替沙星和利福平+环丙沙星混合物均以1:1重量比混合(例如，4g/mL利福平+4g/mL加替沙星)。
结果：分别对四个等基因耐药金黄色葡萄球菌菌株进行了对利福霉素-喹嗪酮偶联分子I和利福霉素(利福平)和氟喹诺酮(环丙沙星和加替沙星)的抗生物膜药效测定。结果显示利福霉素-喹嗪酮偶联分子I对检测的野生菌，利福平耐药菌和氟喹诺酮耐药金葡菌生物膜有良好的量效关系(见图1,表3)。在4g/mL治疗浓度下，利福霉素-喹嗪酮偶联分子I对每个等基因菌株都造成了比对照药物更多的log 10CFU减少(图2)。即使在4和16g/mL下，利福平也会导致耐药突变体的快速增长，而利福霉素-喹嗪酮偶联分子I没有发生这种情况。与利福平不同而与其他测试的对照试剂一样，利福霉素-喹嗪酮偶联分子I在有效的浓度下可以防止细菌耐药性产生(图3、图4所示)。
表3：检测试剂对四种金葡菌的平均log 10CFU减少量
Figure PCTCN2020070162-appb-000006
Figure PCTCN2020070162-appb-000007
实施例6
本实施例提供利福霉素-喹嗪酮偶联分子在治疗细菌生物膜感染中的应用。
本实施例揭示了利福霉素-喹嗪酮偶联分子和相关试剂对表皮葡萄球菌ATCC#35984形成的体外菌落生物膜的抗菌活性。
材料和方法：研究药物为利福霉素-喹嗪酮偶联分子I或实施例1-3中制备的制剂。由丹诺医药提供，并在-20℃下储存。对照化合物由商业途径采购得到。实验显示实施例1-3中利福霉素-喹嗪酮偶联分子的药学上可接受的盐制备的制剂，尤其是双钠盐的制剂在体内会先蜕变成分子形式Ⅰ所示的利福霉素-喹嗪酮偶联分子。
表皮葡萄球菌ATCC#35984菌株，即本实施例中的CB191，用于本实施例中的生物膜研究。将CB191预培养24小时形成菌落生物膜，然后用试验试剂处理24小时。在含有相同浓度的试验试剂琼脂上，通过跟踪处理过的生物膜样品得到处理后耐药性的发展。
结果：在对已经形成的表皮葡萄球菌CB191菌落生物膜的24小时处理中，利福霉素-喹嗪酮偶联分子I显示出比对照试剂更强的量效关系(表4，图4)。在4μg/mL浓度时造成3.2log10CFU的减少，抗生物膜药效上明显强于于与参比试剂(表4，图5)。在处理期间，即使是在4和16μg/mL时，利福平仍产生大量(>4.6log 10CFU)的利福平耐药突变体(表5，图6)。相反，在相同条件下没有观察到对利福霉素-喹嗪酮偶联分子I和其他对照试剂耐药性的发展。因此，与利福平不同，利福霉素-喹嗪酮偶联分子I在其杀伤细菌的有效浓度范围内可抑制表面金葡菌耐药性的发展。
表4：利福霉素-喹嗪酮偶联分子I和对照试剂对表皮葡萄球菌CB291菌落生物膜处理24小时造成的平均Log10CFU的减少
Figure PCTCN2020070162-appb-000008
Figure PCTCN2020070162-appb-000009
表5：利福霉素-喹嗪酮偶联分子I和对照试剂对表皮葡萄球菌CB191菌落生物膜处理24小时生成的平均Log 10耐药CFU
Figure PCTCN2020070162-appb-000010
实施例7
本实施例提供了利福霉素-喹嗪酮偶联分子在治疗生物膜感染中的应用。
本实施例使用大鼠静脉导管模型，验证利福霉素-喹嗪酮偶联分子I对大鼠中心静脉导管(Central Venous Catheter，CVC)感染模型的疗效。
本实施例建立了一种稳定的慢性中心静脉导管感染和药效大鼠模型，用于评价抗菌药物对医疗器械相关血管内细菌感染的体内疗效。所使用的细菌是可形成生物膜上的等基因金黄色葡萄球菌组。野生型菌株为CB192(ATCC#6538)，其两个等基因衍生物为耐利福平菌株的CB785株(rpoB H481Y)和耐氟喹诺酮菌株的CB969株(gyrA S84L,parC S80F)。
结果：在感染后3天开始每日两次静脉注射。对野生型菌株CB192，每日两次10mg/kg静脉注射7天后的平均log减少量分别为利福平3.3和利福霉素-喹嗪酮偶联分子I3.5，而同时进行的其他6种对照抗生素(加替沙星、万古霉素、达托霉素、环丙沙星、阿奇霉素和利奈唑胺)的治疗效果均很低(log减少量<0.8)(如表6所示)。值得注意的是，在4天、每天两次的静脉注射处理后,只有利福平或利福霉素-喹嗪酮偶联分子I治疗可以减少导管相关的细菌量达到或接近2.09log10CFU/导管的检出低限(图7)。虽然在10mg/kg静脉注射利福平处理后2天和4天内检测到自发的体内利福平耐药变异体(如表7所示)，但在利福霉素-喹嗪酮偶联分子I处理利福平敏感菌感染后没有检测到利福平耐药菌。与利福平耐药菌株CB785相应升高的MIC一致，10mg/kg的利福平和利福霉素-喹嗪酮偶联分子I对利福平耐药菌株CB785形成的CVC感染无效(log减少<0.3)。对高度氟喹诺酮类耐药菌株CB969形成的CVC感染，10mg/kg的利福平和利福霉素-喹嗪酮偶联分子I处理仍然有效(log减少2.8-3.1)，而 10mg/kg加替沙星是无效的。
总之，利福霉素-喹嗪酮偶联分子I对野生型和氟喹诺酮耐药的金黄色葡萄球菌导管相关感染的体内药效在该模型中与利福平相似，但在利福霉素-喹嗪酮偶联分子I处理后未检测到利福平耐药变异体。这些结果表明，利福霉素-喹嗪酮偶联分子I处理血管内装置相关葡萄球菌感染具有的应用潜力。
表6：利福霉素-喹嗪酮偶联分子I和对照抗生素7天治疗对大鼠中心静脉导管金黄色葡萄球菌感染模型的平均log 10CFU减少
Figure PCTCN2020070162-appb-000011
a通过尾静脉每天两次注射给药，*为口服给药。
表7：10mg/mL利福霉素-喹嗪酮偶联分子I和对照抗生素治疗大鼠中心静脉导管野生和耐药金葡菌感染的平均log10CFU的变化
Figure PCTCN2020070162-appb-000012
*：通过尾静脉每天两次注射给药。
实施例8
本实施例提供利福霉素-喹嗪酮偶联分子I与临床使用的氟喹诺酮+利福平治疗耐甲氧西林和氟喹诺酮耐药金黄色葡萄球菌实验性心内膜炎的疗效比较。
菌株：金黄色葡萄球菌MRSA 67/0用于本实施例心内膜炎模型的建立。
动物模型：本实施例所述实验性主动脉瓣细菌性心内膜炎模型是在新西兰白兔中建立
研究药物：为利福霉素-喹嗪酮偶联分子I或实施例1-3中制备的制剂。由丹诺医 药提供，并在-20℃下储存。对照化合物或制剂由商业途径采购得到。
结果：与未经处理的对照相比，在治疗结束后和3天复发后，用利福霉素-喹嗪酮偶联分子I(40mg/kg，静脉注射，每天两次共3天)治疗均降低了三种组织中金黄色葡萄球菌密度。利福霉素-喹嗪酮偶联分子I在心脏组织产生>5log减少，肾脏3-4log减少和脾脏2.4-2.9log减少(如表8所示)。结果与对照动物的细菌密度相比有统计学意义(p＝0.002-0.04)，特别是对复发组。
表8：利福霉素-喹嗪酮偶联分子I与氟喹诺酮+利福平治疗对耐甲氧西林和氟喹诺酮耐药金黄色葡萄球菌实验性心内膜炎各个组织平均CFU的影响
Figure PCTCN2020070162-appb-000013
这些结果表明，与左氧氟沙星+利福平或环丙沙星+利福平相比，利福霉素-喹嗪酮偶联分子I在所有三种组织中均有最佳的降低金黄色葡萄球菌密度的效果。在本模型中使用的当前剂量方案中，利福霉素-喹嗪酮偶联分子I在心脏组织中对甲氧西林耐药和氟喹诺酮耐药金黄色葡萄球菌的滴度产生>5log减少。利福霉素-喹嗪酮偶联分子I、利福平、环丙沙星和左氧氟沙星对金黄色葡萄球菌(CB1834)的最低抑菌浓度分别为0.03、0.015、8和2μg/mL(数据未显示)。除了观察到利福霉素-喹嗪酮偶联分子I对心脏组织的疗效外，治疗的动物肾脏和脾脏中的细菌计数减少了3-4log。
MRSA的患病率和这些对氟喹诺酮类耐药的菌株的比例对单剂使用或与另一种药物联合治疗葡萄球菌心内膜炎有直接的影响。利福霉素-喹嗪酮偶联分子I已被证明对氟喹诺酮类药物敏感或耐药的耐甲氧西林金黄色葡萄球菌引起的心内膜炎有效。
实施例9
本实施例提供利福霉素-喹嗪酮偶联分子在滴流反应器模型中的抗生物膜药效研究。
研究药物：为利福霉素-喹嗪酮偶联分子I或实施例1-3中制备的制剂。由丹诺医药提供，并在-20℃下储存。对照化合物或制剂由商业途径采购得到。
菌株：所用菌株包括表皮葡萄球菌CB191(ATCC#35984)，表皮葡萄球菌CB1038(利福平耐药，rpoB H481Y变异)，表皮葡萄球菌CB1106(氟喹诺酮耐药，grlA S80F和gyrA S84Y变异)。最低抑菌浓度(MIC)测试依据CLSI(Clinical and Laboratory Standards Institute)推荐指南进行操作。
静态或恒定滴流装置：采用本领域常用的20格滴流反应器。
结果：用恒定滴流生物膜系统研究了利福霉素-喹嗪酮偶联分子I、利福平、两种氟喹诺酮类药物和利福平+环丙沙星鸡尾酒方案对表皮葡萄球菌生物膜的药效。评估包括生物膜的48小时生长，然后用研究药物进行24小时治疗，或者用48小时无药物反弹阶段进行72小时疗程治疗。用野生型表皮葡萄球菌CB191和含有对一些喹诺酮类药物敏感性降低的在ParC S80F核苷酸取代的等基因耐药衍生物进行了72小时的 时间杀菌实验。
对于野生型表皮葡萄球菌CB191的24小时治疗，利福霉素-喹嗪酮偶联分子I优于所有对照药物，包括利福平+环丙沙星鸡尾酒疗法。特别在利福霉素-喹嗪酮偶联分子I分别以1μg/mL和4μg/mL剂量给药时，总的Log10CFU/cm 2减少分别为3.33和4.84。相比之下，其他单一治疗包括利福平(剂量为4μg/mL)、环丙沙星(剂量为4μg/mL)或加替沙星(剂量为2μg/mL)导致log10CFU/cm 2降低1.9至2.7范围。此外，利福平处理导致利福平耐药变异体的快速选择和生长，使得在处理结束时，基本上100％的生物膜群体对测试药物具有耐药性。相反，其它测试药物没有观察到明显的耐药发展。当以鸡尾酒形式进行试验时，利福平(4μg/mL)加环丙沙星(4μg/mL)比单独使用利福平或环丙沙星更能减少生物膜的活菌数，可能反映了该混合物抗耐药性的改善。在剂量为4μg/mL时，利福平+环丙沙星鸡尾酒疗法的总体抗生物膜药效(平均CFU/cm 2减少3.18)仍低于利福霉素-喹嗪酮偶联分子I(平均CFU/cm 2减少4.84)(如表9，图8和图9所示)。
在72h对野生型表皮葡萄球菌CB191时间-杀菌研究中，利福霉素-喹嗪酮偶联分子I优于利福平，并且显示出与环丙沙星、加替沙星或由利福平加环丙沙星组成的鸡尾酒等同或更好的整体抗生物膜药效。利福霉素-喹嗪酮偶联分子I、环丙沙星和加替沙星治疗第3天(24h治疗)的CFU/cm 2减少分别为6.08、5.16和6.21。当在利福霉素-喹嗪酮偶联分子I、环丙沙星或加替沙星处理的样品各自的CLSI耐药折点进行筛选时，未检测到显著的治疗耐药性(如表10，图10所示)。
在治疗结束时，用利福霉素-喹嗪酮偶联分子I处理72小时或利福平加环丙沙星鸡尾酒处理的生物膜在无药物试验中恢复(反弹)48小时，然后再次取样，以观察其耐药性和存活性。在这些研究中，用利福平加环丙沙星鸡尾酒处理的生物膜在除去抗生素后显示出活性计数的快速和显著增加，而利福霉素-喹嗪酮偶联分子I处理的样品没有显示出活性计数的任何显著增加。在任何反弹样品中未观察到对利福霉素-喹嗪酮偶联分子I、利福平或环丙沙星的耐药性(如图11所示)。
总的来说，这些组合的滴流生物膜数据表明，利福霉素-喹嗪酮偶联分子I处理的生物膜显示出延长的后抗生素效应，这于单剂量利福霉素和喹诺酮类药物所观察到的不同，这种影响不能简单地用良好的耐药特性来解释。
这些研究证实了多功能药物利福霉素-喹嗪酮偶联分子I在治疗临床葡萄球菌生物膜感染中具有的潜在应用。
表9：在恒流量反应系统中利福霉素-喹嗪酮偶联分子I和对照药物治疗野生型表皮葡萄球菌CB191(ATCC#35984)72小时实验期(包括48小时无药期)造成的平均log10总数，log10减少数和耐药CFU/cm 2
Figure PCTCN2020070162-appb-000014
Figure PCTCN2020070162-appb-000015
表10：在恒流量反应系统中利福霉素-喹嗪酮偶联分子I和对照药物治疗喹诺酮耐药表皮葡萄球菌CB1103(ParC S80F)72小时实验期(包括48小时无药期)造成的平均log10总数，log10减少数和耐药CFU/cm 2
Figure PCTCN2020070162-appb-000016
实施例10
本实施例提供利福霉素-喹嗪酮偶联分子在治疗生物膜感染中的应用。
本实施例检测利福霉素-喹嗪酮偶联分子I对皮下生物膜植入感染小鼠模型的药效。
研究药物和材料：为利福霉素-喹嗪酮偶联分子I或实施例1-3中制备的制剂。由丹诺医药提供，并在-20℃下储存。对照化合物或制剂由商业途径采购得到。
菌株：所用细菌为金黄色葡萄球菌菌株。野生型CB192(ATCC#6538)，其两个等基因耐药衍生物CB785(利福平耐药rpoB H481Y)和CB969(氟喹诺酮耐药gyrA S84L/parC S80F)。
试验动物：本实施例使用5-6周龄BALB/c小鼠。试验期间小鼠可以自由进食/饮水，所有操作符合研究动物使用和护理委员会(IACUC)指南。
结果：所使用的细菌是形成生物膜的等基因金黄色葡萄球菌。野生型菌株为CB192(ATCC#6538)，其两个等基因耐药衍生物为利福平耐药菌株CB785(rpoB H481Y)和氟喹诺酮耐药菌株CB969(gyrA S84L，parC S80F)。三株菌株在体内均表现出非常相似的生长动力学，特氟隆植入物相关平均值在感染后7天和21天分别为6.6-6.7和 7.1-7.2log10CFU/植入物。
通过在感染后7天每日两次腹腔注射给药来评估利福霉素-喹嗪酮偶联分子I和4种对照抗生素的药效。针对野生型菌株CB192，以25mg/kg每天两次腹腔注射利福平给药至少7天，观察到皮下感染部位的细菌细胞的显著减少。25mg/kg单药治疗14天后，Log减少分别为万古霉素-0.1、达托霉素0.7、加替沙星1.7、利福平2.1、利福霉素-喹嗪酮偶联分子I 5.0。2 5mg/kg利福平+25mg/kg加替沙星的联合治疗也显示Log下降5.0。对于野生型菌株，只有利福平+加替沙星的联合治疗或利福霉素-喹嗪酮偶联分子I的单一治疗将细菌量降低到或接近检测低限(表11，图12)。在时间-杀菌研究中，加替沙星治疗7天后的Log减少为0.8，利福平为2.2，利福平+加替沙星联合治疗为2.6，利福霉素-喹嗪酮偶联分子I为4.0(图13所示)。然而，在长期的利福平单药治疗后，大量自发耐药利福平细菌持续出现，从而限制了其有效性。相比之下，在25mg/kg利福霉素-喹嗪酮偶联分子I单药治疗以及25+25mg/kg利福平+加替沙星联合治疗后，未检测到利福平耐药变异体。对CB192感染的14天治疗的量效关系研究进一步表明，加替沙星需要25mg/kg，利福平需要10mg/kg，利福霉素-喹嗪酮偶联分子I需要10mg/kg，利福平+加替沙星联合治疗需要10+10mg/kg。同时，利福霉素-喹嗪酮偶联分子I需要10mg/kg来显著抑制体内利福平耐药性出现，而利福平+加替沙星联合治疗需要10+10mg/kg来抑制。此外，利福霉素-喹嗪酮偶联分子I单药治疗或利福平+加替沙星联合治疗是清除大多数植入物中细菌的唯一方法(16个利福霉素-喹嗪酮偶联分子I治疗的植入物中15个检测到<2.09log10CFU，30个利福平+加替沙星治疗植入物中18个检测到<2.09log10CFU)。
对耐药细菌感染的治疗研究中，对利福平耐药株CB785，利福平单药治疗无效，而单用利福霉素-喹嗪酮偶联分子I和利福平+加替沙星的联合治疗效果比野生型菌株活性低(CB785LogCFU分别降低2.5和3.6)。对氟喹诺酮耐药菌株CB969单用加替沙星治疗无效，而单用利福霉素-喹嗪酮偶联分子I治疗和利福平+加替沙星联合治疗有效(CB969Log分别减少4.0和2.0)。虽然利福平明显能有效地降低该模型中的敏感细菌的CFU，但其疗效受到长期治疗期间自发性利福平耐药变异体的持续出现和扩增的限制。总体而言利福霉素-喹嗪酮偶联分子I对野生型、利福平耐药和氟喹诺酮类耐药金黄色葡萄球菌植入感染的体内药效一般与两倍剂量的利福平+加替沙星的联合治疗相媲美。这些结果表明利福霉素-喹嗪酮偶联分子I治疗周围组织器械相关葡萄球菌感染的具有应用潜力。
表11：14天处理野生型金黄色葡萄球菌CB192的药效和耐药性的出现
Figure PCTCN2020070162-appb-000017
实施例11
本实施例提供利福霉素-喹嗪酮偶联分子在治疗生物膜感染中的应用。
本实施例实验利福霉素-喹嗪酮偶联分子I对小鼠假体关节感染(Prosthetic Joint Infection，PJI)模型的药效。
研究药物和材料：为利福霉素-喹嗪酮偶联分子I或实施例1-3中制备的制剂。由丹诺医药提供，并在-20℃下储存。对照化合物或制剂由商业途径采购得到。
菌株：金黄色葡萄球菌NT111-3(NRS249或HT 20020341)，先天性瓣膜心内膜炎分离株，对环丙沙星，克林霉素，红霉素，庆大霉素，苯唑青霉素，青霉素耐药。
试验动物：本实施例使用8-10周龄C57BL/6雌性小鼠。试验期间小鼠可以自由进食/饮水，所有操作符合研究动物使用和护理委员会(IACUC)指南。
结果：利福平或万古霉素处理对感染动物的钢丝或股骨的细菌总滴度几乎没有影响。利福霉素-喹嗪酮偶联分子I(25mg/kg BID×7或14天)对钢丝和股骨均有疗效。7天方案中，在所有的样本内，钢丝细菌滴度降低到两种处理方案的检测低限。在第14天，股骨的细菌滴度降低到2.99log 10CFU，但在第21天恢复到4.73log10CFU。利福霉素-喹嗪酮偶联分子I给药14天的方案中，第21天时钢丝和股骨的金黄色葡萄球菌滴度分别减少到2.05和2.72log10CFU(如表12所示)。
表12：利福霉素-喹嗪酮偶联分子I、利福平和万古霉素在小鼠PJI感染模型中的药效
Figure PCTCN2020070162-appb-000018
由上可见，本发明实施例的利福霉素-喹嗪酮偶联分子及其药物制剂或组合物能够有效治疗或预防医疗器械包括人工关节、静脉导管、骨折固定、心脏瓣膜或人造血管相关生物膜感染。

Claims (10)

  1. 一种利福霉素-喹嗪酮偶联分子及其药学上可接受的盐在制备治疗生物膜感染的药物中的应用，该利福霉素-喹嗪酮偶联分子具有式Ⅰ所示结构：
    Figure PCTCN2020070162-appb-100001
  2. 根据权利要求1所述的应用，其特征在于：所述利福霉素-喹嗪酮偶联分子的药学上可接受的盐为利福霉素-喹嗪酮偶联分子的双钠盐。
  3. 根据权利要求2所述的应用，其特征在于：所述利福霉素-喹嗪酮偶联分子的双钠盐为式Ⅰ所示的利福霉素-喹嗪酮偶联分子与碳酸钠或氢氧化钠反应后、加入甲醛次硫酸钠反应制备得到的。
  4. 根据权利要求1-3任一项所述的应用，其特征在于：所述生物膜感染包括使用医疗器械、由细菌引起的生物膜感染。
  5. 根据权利要求4所述的应用，其特征在于：所述生物膜感染包括静脉导管感染、人工心脏瓣膜感染、人造血管感染、整形外科植入物感染、骨折固定感染和人工关节感染中的一种或几种的组合。
  6. 根据权利要求4所述的应用，其特征在于：所述细菌包括凝固酶阴性葡萄球菌、肠球菌、短小棒状杆菌、大消化链球菌、梭形杆菌、梭状芽孢杆菌、类杆菌、甲氧西林敏感和/或甲氧西林耐药的金黄色葡萄球菌中的一种或几种的组合。
  7. 一种用于治疗生物膜感染的药物制剂，其药用组分包括式Ⅰ所示的利福霉素-喹嗪酮偶联分子及其药学上可接受的盐。
  8. 根据权利要求7所述的药物制剂，其特征在于，该药物制剂制剂类型包括喷剂、气雾剂、注射剂、酊剂、膏剂、粉针剂或贴剂。
  9. 根据权利要求7或8所述的药物制剂，其特征在于，每单位的所述药物制剂中包含如下原料组分：
    式Ⅰ所示的利福霉素-喹嗪酮偶联分子及其药学上可接受的盐100-105mg，甘露醇60-80mg，甲醛次硫酸钠2-6mg，聚山梨酯80 0.1-1mg，无水乙醇4-10μL，氢氧化钠调节PH9.5-10，水3-10.5mL。
  10. 一种用于治疗人体生物膜感染的药物组合，其有效组分包括式Ⅰ所示的利福霉素-喹嗪酮偶联分子及其药学上可接受的盐。
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