CN107417747A - It is a kind of to be used to treat bacterium infection or the compound of superbacteria infection and combinations thereof - Google Patents
It is a kind of to be used to treat bacterium infection or the compound of superbacteria infection and combinations thereof Download PDFInfo
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- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
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Abstract
The present invention disclose it is a kind of be used to treating compound that bacterium infection or superbacteria infect and combinations thereof, the compound name is the oxygen α L of Kaempferol 3 (2,3 two pairs of coumaric acyls) rhamnoside, referred to as KCR, molecular formula C38H31O14With four kinds of stereoisomers, respectively KCR EZ, KCR ZE, KCR ZZ and KCR EE, the composition are mutually mixed by any two kinds, three kinds or four kinds in KCR EZ, KCR ZE, KCR ZZ or KCR EE compounds and formed, and mutual mass ratio does not limit.The compound and combinations thereof can kill the bacterium that presently, there are and superbacteria, including MSRA, VRSA and other bacteriums or superbacteria, the compound and combinations thereof belongs to antibiotic, bacterium infection, especially superbacteria can be treated by the mode such as muscle or intravenous injection, oral, topical administration to infect.
Description
Technical field
The invention belongs to field of pharmacology, and in particular to a kind of chemical combination for being used to treat bacterium infection or superbacteria infection
Thing and combinations thereof.
Background technology
Antibiotic (antibiotics) is in life by microorganism (including bacterium, fungi, actinomyces) or high animals and plants
There is antipathogen or other active a kind of secondary metabolites caused by during work, other living cells can be disturbed to send out
Educate the chemical substance of function.Existing clinical conventional antibiosis is known as in transgenic engineered bacteria nutrient solution liquid extract and with chemistry side
Method synthesizes or semi-synthetic compound.Be currently known not lower ten thousand kinds of natural antibiotics, be mainly used in treating various bacterium infections or
Pathogenic microorganism infection class disease, serious side effect will not be generally produced to its host, but still suffered from present a lot
The bacterium of antibiotic ineffective, such as methicillin-resistant staphylococcus aureus (methicillin-resistant
Staphylococcus aureus, MRSA), vancomycin resistance staphylococcus aureus (vancomycin-resistant
Staphylococcus aureus, VRSA), quick staphylococcus aureus (vancomycin- in vancomycin
Intermediate Staphylococcus aureus, VISA), vancomycin resistant enterococci (vancomycin-
Resistant Enterococcus faecalis and Enterococcus faecium, VRE), multi-drug resistant pneumonia streptococcus
Bacterium (multi drug-resistant streptococcus pneumoniae, MDRSP), Carbapenem-resistant kerekou pneumonia primary
Bacterium (carbapenem-resistant Klebsiella pneumonia, CRKP) etc..These are produced to existing all antibiotic
The bacterium of drug resistance, because once infection just pasts medical help, lethal is high, and this kind of bacterium is collectively referred to as superbacteria again
(superbug).The antibiotic that these superbacterias can be killed is referred to as Zyvox (super- by us
antibiotics)。
Methicillin-resistant staphylococcus aureus (methicillin-resistant Staphylococcus aureus,
MRSA), the beta-lactam antibiotic of routine can be resisted, is to cause patient to infect one of dead main bacteria.MRSA is
Global quick sprawling, it has also become global public health problem.Global about 2,000,000,000 people (25-30% of population in the world) carry certain
The staphylococcus aureus of kind type, what about 200 ten thousand to 5,300 ten thousand people carried is superbacteria.50% golden yellow in global range
It is as caused by MRSA that staphy lococcus infection, which is caused a disease,.The MRSA infection for most starting to find belongs to Hospital-acquired MRSA infection
(health care-associated MRSA, HA-MRSA), have now been found that the Community-acquired in the places such as stadium, community
MRSA infects (community-associated pathogen, CA-MRSA).MRSA infection rate high mortalities are very high,
In the U.S., death toll is at 19000 or so in the institute because infecting MRSA every year, more than AIDS death toll.In China, according to
The Monitoring Data of Chinese Bacterial resistance surveillance net (CHINET) shows that recall rate of the MRSA infection in hospital just reaches
42.2%.
Infected for serious MRSA, vancomycin (vancomycin) is still current choice drug.But
There is quick staphylococcus aureus (VISA) in vancomycin resistance staphylococcus aureus (VRSA) and vancomycin.For this
The antibiotics research and development of a little drug-fast bacterias are slow, and newest antibiotic is the Daptomycin of U.S. FDA approval in 2003
(daptomycin), infected for serious golden yellow Portugal coccus.Daptomycin has serious side reaction, as striated muscle is molten
Solution, renal failure, the eosinophilic pneumonia of threat to life.In addition, Daptomycin cannot be used for pneumonia infection disease.
Multidrug resistance microorganism (multi drug-resistant microbial organisms, MDROs) it is quick
It is to threaten the serious problems of human health to occur with sprawling.Almost annual, all someone can not obtain because of antibiotic resistance
Effectively treatment, death.Since 2015, the annual whole world is about just 700,000 people because of the dead number of drug resistance.According to current antibiosis
The dead speedup of plain drug resistance, to the year two thousand fifty, this numeral will reach 10,000,000, more than the people for dying from cancer every year at present
Number.There is an urgent need to research and develop new antimicrobial agents effectively to contain these emerging superbacterias.
The content of the invention
For problems of the prior art, the present invention provides a kind of for treating bacterium infection or superbacteria infection
Compound and combinations thereof.
The present invention provide it is a kind of be used for treat bacterium infection or superbacteria infection compound, the compound it is entitled
Kaempferol 3- oxygen-α-L- (2,3- bis--to coumaric acyl) and rhamnoside, referred to as KCR, molecular formula C38H31O14, there are four kinds to be stood
Body isomers, respectively KCR-EZ, KCR-ZE, KCR-ZZ and KCR-EE, wherein R1 are that E-p- coumaric acyls group or Z-p- are fragrant
Beans acyl group group, R2 are E-p- coumaric acyls group or Z-p- coumaric acyl groups.
Preferably, it is golden yellow to include methicillin-resistant staphylococcus aureus (MRSA), vancomycin resistance for the superbacteria
Quick staphylococcus aureus (VISA) and vancomycin-resistant enterococcus and excrement intestines in color staphylococcus (VRSA), vancomycin
Coccus (VRE).
The present invention provides a kind of with the described antibacterial for being used to treat bacterium infection or the compound of superbacteria infection
Biocide preparation.
Preferably, the form of the antibiotic and sterilizing preparation is oral formulations, ejection preparation or preparation for external application to skin.
Preferably, the ejection preparation is contains 100-350mg/mL institutes in Emulsifier EL-60 polyethylene glycol mixed liquor
State compound；The external preparation meets to contain compound described in 4-100mg per in 100g paste；The oral formulations meet every
0.25g oral formulations contain compound described in 0.1-0.225g.
The present invention also provides a kind of to be formed by the described compound for being used to treat bacterium infection or superbacteria infection
Composition, the composition is by any two kinds, three kinds or four in KCR-EZ, KCR-ZE, KCR-ZZ and KCR-EE compound
Kind is mutually mixed to be formed, and mutual mass ratio does not limit.
Preferably, the form of the antibiotic and sterilizing preparation is oral formulations, ejection preparation or preparation for external application to skin, the note
Preparation is penetrated to contain composition described in 100-350mg/mL in Emulsifier EL-60 polyethylene glycol mixed liquor；The external preparation
Meet to contain composition described in 4-100mg per in 100g paste；The oral formulations meet to contain 0.1- per 0.25g oral formulations
Compound described in 0.225g.
The present invention also provides a kind of by the described compound or described for being used to treat bacterium infection or superbacteria infection
The compound formulation that is formed of composition for being used to treat bacterium infection or superbacteria infection, the compound formulation is except including use
The compound infected in treatment bacterium infection or superbacteria or the composition for treating bacterium infection or superbacteria infection
In addition, in addition to other antibiotic, bacteriostatic agent or reinforcing agent.
Preferably, the antibiotic includes but is not limited to Novel Quinolone class and cephalo-type antimicrobial, and the bacteriostatic agent includes
But beta-lactamase inhibitor is not limited to, the reinforcing agent includes but is not limited to carboxylesterase inhibitor.
Present invention has the advantage that：The present invention provides a kind of change for being used to treat bacterium infection or superbacteria infection
Compound and combinations thereof, the bacterium that presently, there are and superbacteria, including MSRA, VRSA and other bacteriums or super can be killed
Level bacterium, the compound and combinations thereof belongs to antibiotic, can pass through the mode such as muscle or intravenous injection, oral or topical administration
Bacterium infection, especially superbacteria is treated to infect.
Brief description of the drawings
Fig. 1：The structural representation of Kapp mycin (KCR) in the present invention；
Fig. 2：The influence comparison diagram that Kapp mycin (KCR) is expressed s. aureus protein with OXA；
Fig. 3：Kapp mycin (KCR) is with OXA to bacterioprotein differentiation comparison diagram；
Fig. 4：The HPLC appearance comparison diagrams of four kinds of stereoisomers of Kapp mycin；
Fig. 5：The HPLC examination criteria curve maps of four kinds of stereoisomers of Kapp mycin；
Fig. 6：The comparison diagram of the bioavilability of different preparations；
Fig. 7：Kapp mycin (KCR) pharmacokinetic curve during intravenous injection；
Fig. 8：The toxicity detection figure of Kapp mycin (KCR)；
Fig. 9：BNPP suppresses Kapp mycin (KCR) metabolic map；
Figure 10：The species difference comparison diagram of Kapp mycin (KCR) metabolism；
Figure 11：The stability comparison diagram of Kapp mycin (KCR) under different condition.
Embodiment
To make the purpose, technical scheme and advantage of the embodiment of the present invention clearer, below in conjunction with the embodiment of the present invention
In accompanying drawing, the technical scheme in the embodiment of the present invention is clearly and completely described, it is clear that described embodiment is
The part of the embodiment of the present invention, rather than whole embodiments.Based on the embodiment in the present invention, ordinary skill people
The every other embodiment that member is obtained on the premise of creative work is not made, belongs to the scope of protection of the invention.
Kapp mycin (Kapomycin, abbreviation KCR), i.e. Kaempferol-3-O-Alpha-L- (2 ", 3 "-di-p-
Coumaroyl) rhamnoside, the entitled Kaempferol 3- oxygen-α-L- of Chinese (2,3- bis--to coumaric acyl) rhamnoside is (referred to as
KCR)。KCR(C38H31O14) it is powdery non-water soluble substance, molecular weight 711.The present invention under study for action find Kapp mycin from
There are four kinds of stereoisomers when being extracted in plant, as shown in figure 1, being respectively：KCR-EE (R1=R2=E-p- coumaric acyls),
KCR-ZZ (R1=R2=Z-p- coumaric acyls), KCR-ZE (R1=Z-p- coumaric acyls, R2=E-p- coumaric acyls) and KCR-EZ (R1=
E-p- coumaric acyls, R2=Z-p- coumaric acyls).Therefore, KCR is typically expressed as the mixing that four kinds of stereoisomers of the above are formed
Thing.
Kapp mycin is not soluble in water, physiological saline and glucose solution, and being highly soluble in ethanol, (maximum concentration is up to 350mg/
mL).In ethanol/glycerine, (volume ratio is:10:100,5:100 or 1:100) favorable solubility in mixed solution.Ethanol/glycerine mixes
Close in solution, the volume ratio of ethanol and glycerine does not influence the antibacterial activity of Kapp mycin.Kapp mycin (KCR) can be from Chinese parasol tree
Extraction obtains in tree, and specific extracting method is to be extracted at normal temperatures with ethanol after crushing Chinese parasol leaf skin or branch, obtains second
Alcohol primary extract, then ethanol primary extract is separated with silica gel and vacuum liquid phase chromatography method, then gradient elution, gradient elution
When respectively with following solution：N-hexane, ethyl acetate hexane mixed liquor, ethyl acetate, ethyl acetate methanol mixed liquor, first
Alcohol, methanol aqueous solution.Kapp mycin (KCR) purifies (C8 posts and ammonia mainly in ethyl acetate methanol mixed liquor component with HPLC
Pilum) i.e. obtain high-purity Kapp mycin (KCR).
Pass through in vitro test tested K CR minimum inhibitory concentrations (minimum inhibitory concentration, MIC)
Patient's collection of microorganism (bacterium) from bacteremia, wound infection either urethral infection obtains or medical phase
The related bacterium in close and community.These bacteriums include methicillin-resistant staphylococcus aureus (methicillin-
Resistant Staphylococcus aureus, MRSA), vancomycin resistance staphylococcus aureus (vancomycin-
Resistant Staphylococcus aureus, VRSA), quick staphylococcus aureus (vancomycin- in vancomycin
Intermediate Staphylococcus aureus, VISA), vancomycin-resistant enterococcus and enterococcus faecalis
(vancomycin-resistant Enterococcus faecalis and Enterococcus faecium, VRE).KCR
Four kinds of stereoisomerses be dissolved in ethanol, adjust caseinhydrolysate peptone meat soup with aseptic deionized water and cation
(Mueller Hinton II meat soups) (CAMHB) is diluted to concentration.Operating procedure is as follows：
Step a：Prepare MIC culture dishes：The sterile round bottom culture dish (Microdilution of micro liquid-based dilution pallet
trays)。
Step b：Add 50 microlitres of CAMHB meat soups and (often show 8 holes) into each hole of No. 1-12 row of 96 orifice plates, its
In No. 1 row be set to negative control.
Step c：50 microlitres of KCR (mixture of tetra- kinds of stereoisomers of KCR), KCR stereoisomers and tradition is added to resist
Raw element is into the hole of No. 2 row.The stock concentration of antibiotic is 4 times of concentration in No. 2 row holes.For example, the concentration in No. 2 row holes is
32 μ g/mL, then the stock concentration of the antibiotic is 128 μ g/mL.
Antibiotic concentration 2 times of dilution proportions successively, it is added to and last 50 microlitres of antibiosis is abandoned in the hole in No. 3-11 row
Element.
Order according to No. 12 row holes to No. 1 row hole, adds 50 microlitres of CAMHB meat soups into all holes.
Step d：Prepare inoculum (bacterium)：Prepare 0.5 Ma Kefalanshi McFarland of 2mL aseptic deionized water
Suspension (1.5x108cfu/mL).66 microlitres of aseptic deionized waters for being added to 2mL are taken, it is 5x10 to obtain concentration6Cfu/mL's
Suspension (suspension take 10 microlitres be added to micro titre trays (microtiter tray) after obtain whole concentration
5x105cfu/mL)。
Step e：Add 10 microlitres of inoculum (5x106Cfu/mL suspension) into the hole of 12 row, the hole is as growth
Control hole.
Step f：Add 10 microlitres of inoculum (5x106Cfu/mL suspension) into each hole of 11 to No. 2 row.
Step g：It is incubated at 37 DEG C, in 16-20 hours, records clump count, obtain minimum inhibitory concentration MIC.
Test result：KCR and KCR four kinds of stereoisomers KCR-EE, KCR-EZ, KCR-ZE and KCR-ZZ have bright
Aobvious anti-MRSA activity.In four kinds of stereoisomers, KCR-EE activity is most strong, remaining about 2-4 times weak compared with its of 3 kinds activity.
The minimum inhibitory concentration MIC of MRSA and VRSA, KCR and its four kinds of compounds to various sources are in 2-8 μ g/mL, such as table 1 below institute
Show：
Table 1：KCR and its four kinds of stereoisomers antibacterial activity in vitro
Remarks：Wherein S.aureus is staphylococcus aureus.MRSA is methicillin-resistant staphylococcus aureus；VRSA
For vancomycin resistance staphylococcus aureus；VISA is quick staphylococcus aureus in vancomycin；MIC, minimum inhibitory concentration,
μg/mL；IP represents inpatient；OP represents out-patient；HA represents Nosocomial infection；CA represents Community-acquired sense
Dye.
In addition to MRSA and VRSA, KCR and its each stereoisomers also have obvious anti-VRE activity, especially
To the VREF (Enterococcus faecium) in VRE.VREF is to vancomycin and ampicillin all resistances
(MIC is both greater than 32 μ g/mL).Most active to VREF is KCR-EE and KCR-ZE, and MIC is in 0.5-1.0 μ g/mL, such as
Shown in table 2：
Table 2：The antibacterial activity in vitro of KCR and its four kinds of stereoisomers to VRE
Remarks：E.faecalis is enterococcus faecalis；E.faecium is VREF；VRE be vancomycin-resistant enterococcus and
Enterococcus faecalis；MIC is minimum inhibitory concentration, unit μ g/mL；IP represents inpatient；OP represents out-patient, and HA represents hospital
Acquired Infection；CA represents Community Acquired Infections.
KCR study on mechanism
The therapeutic response of bacterial antibiotic forms change by the protein of bacterium to be caused, and shows as the protein system of bacterium
System is disorderly.The influence changed is formed to bacterioprotein by monitoring antibiotic, can reveal that the mechanism of action of antibiotic.We
Influences of the KCR to the aureus protein composition of methicillin-sensitivity is detected with proteomics.OXA
(oxacillin) bactericidal action is played by suppressing bacteria cell wall synthesis.We using OXA as with reference to antibiotic,
See whether KCR is same mechanism of action, and operating procedure is as follows：
Step 1：The staphylococcus aureus of methicillin-sensitivity is respectively at the KCR and OXA with 5 times of MIC dosage
Reason；
Step 2：Set up without antibiotic treatment group as control；
Step 3：After 4 hours, every group of bacterium, cracking, tandem mass spectrum mark (Tandem Mass Tag, TMT) etc. are collected
LC-MS-MS (Liquid Chromatography-Mass Spectrometry, LC-MS) is analyzed after reason
The change of albumen composition.
In the quantitative analytical data of 1144 albumen obtained from all 9 samples, KCR have impact on 626 albumen, benzene azoles
XiLin have impact on 207 albumen (5% false discovery rate FDR, relaxing test moderated t-test).Using principal component analysis
(Principal Component Analysis, PCA) method determines the similitude of 3 groups of total protein groups.Protein science shows KCR
Treatment group differs markedly from OXA (as shown in Figure 2), and this shows that KCR mechanism of action is different from OXA, be not with
OXA equally plays bactericidal action by suppressing bacteria cell wall synthesis.
We use ontological analysis (rank-based ontology analysis) method based on order, analyze KCR
Group and the expression of control group protein diversityization, it is determined that the protein classes most changed by force as caused by KCR.Ribosomal protein is most obvious
The albumen of change, in 54 ribosomal subunit's compositions, the expression for having 46 subunit's compositions is remarkably reinforced.On the contrary, benzene azoles is western
Woods causes 13 ribosomal subunit's composition expression to decline, only 1 composition expression enhancing, as shown in Figure 3.In addition, aminoacyl-
The expression of tRNA ligases is also remarkably reinforced in KCR groups.The evident characteristic of protein synthesis inhibitor is exactly that they cause bacterium excellent
Ribosomal protein is first expressed, even if overall protein expression is also such when declining.Accordingly, it is concluded that KCR is by suppressing bacterium
The synthesis of protein and play bactericidal action.
Utilize high performance liquid chromatography (HPLC) detection KCR blood concentration
Gather mouse blood and (add 50 microlitres of 36mg/mL's in every milliliter of blood in the test tube of the anti-coagulants containing EDTA
EDTA).14000rpm is centrifuged 15 minutes (collect blood plasma can be stored in -20 DEG C standby) at 4 DEG C.
25 microlitres of KCR storing solutions are added in 225 microlitres of blood plasma, are mixed, and add 800 microlitres of absolute ethyl alcohols afterwards.Set up
Control tube without KCR, i.e. 200 microlitres of blood plasma are added in 800 microlitres of absolute ethyl alcohols.It is vortexed and mixes 10 seconds, 14000rpm is in room
The lower centrifugation of temperature 15 minutes, collects supernatant in 1.5mL Ai Bende Eppendorf centrifuge tubes.After 45 DEG C of evaporation dryings, add
250 microlitres of absolute ethyl alcohol dissolvings, centrifugation, take 10 microlitres of sample introduction HPLC systems of supernatant to be detected.
Ethyl acetate can be as absolute ethyl alcohol of the replacement in above-mentioned as recycling design, and the rate of recovery is respectively 81% He
75%.
Liquid chromatogram Waters Alliance HPLC system testing conditions：The photodiode array of Waters 2998 is examined
Survey device, the C18 reverse-phase chromatographic columns (3.9 × 300mm) of 4 μm of particle diameter.5 microlitres or 10 microlitres of sampling volume.Normal temperature detects.Flowing
Phase：Formic acid (the volume ratio 60 of methanol/acetonitrile/0.3%:3:37), flow velocity 0.75mL/min.Detection wavelength 313nm.
The HPLC peak sequences of KCR 4 compounds are followed successively by KCR-EZ, KCR-ZZ, KCR-EE, KCR-ZE (such as Fig. 4 institutes
Show).As shown in figure 5, the concentration of KCR examination criteria curves is 18.75,6.25,2.5,1.25,0.25 μ g/ml.The detection
Under the conditions of, standard curve is linearly good, coefficient R 2=0.9978.Test limit (LOD) is 0.21 μ g/mL, quantitative limit (LOQ)
For 0.272 μ g/mL.
The present invention also provides a kind of composition for being used to treat bacterium infection or superbacteria infection, specially four kinds of solids
Any two kinds, three kinds or four kinds mixing in isomers KCR-EE, KCR-ZZ, KCR-ZE and KCR-EZ, any mass ratio mix
Close can, without ratio limit.In four kinds of stereoisomers, KCR-ZZ antibacterial activities are most strong, and when selecting, four kinds of solids are different
When structure body combines to form the composition, optimal mass percent KCR-EZ is 26%, KCR-ZZ 22%, KCR-EE are
36% and KCR-ZE is 16%.When selecting any two kinds or three kinds of stereoisomers combine to form the composition, each group
Divide mass percent proportional allocations.In the composition of specific matched combined thing, in the acceptable preferably table 3 below of the present invention
Mass percent collocation form the composition.
Table 3：Four kinds of stereoisomers mass percent when forming composition contrasts
The form of said composition can be oral formulations, ejection preparation or preparation for external application to skin (such as paste or paint)
Deng KCR can also be prepared into Liposomal formulation (injection).
The present invention, which also provides a kind of composition, has compound or other antibiotic, the suppression of synergy with other
Microbial inoculum or reinforcing agent, joint prepare the compound formulation formed, and the compound formulation can be compound oral formulations or composite injection
Preparation etc., these antibiotic, bacteriostatic agent or reinforcing agent include but is not limited to Novel Quinolone class and cephalo-type antimicrobial etc., Shu Ba
The beta-lactam enzyme level such as smooth (Sulbactam), clavulanic acid (Clavulanic acid), Tazobactam Sodium (Tazobactam)
Agent, or carboxylic acid enzyme inhibitor etc..With carboxylic acid enzyme inhibitor, with compound formulation is combined into, KCR can be suppressed and digested in vivo, from
And maintain the time of effective blood drug concentration to be obviously prolonged, KCR sterilization antibacterial efficacy can be strengthened.
Provided by the present invention for treatment bacterium infection or the compound of superbacteria infection and combinations thereof, the chemical combination
Thing or composition prepare to be formed one of preparation method of ejection preparation be will contain KCR (four kinds stereoisomer any one) or
The ethanol solution (compound or the concentration of composition be 100-350mg/mL) of composition is dissolved in 0.5-1 times of volume polyoxy second
Alkene castor oil polyethylene glycol mixed liquor (Cremophor/polyethylene glycol, Crem/PG, volume ratio 20:80-
80:20, preferably 60:40) concentrated type ejection preparation, is formed.After the concentrated type ejection preparation is formed, 10-30 times can be used
Volume is carried out in normal saline solution, G/NS parenteral solution or glucose injection of concentrated type ejection preparation etc.
Dilution uses, and is used with adapting to intravenous injection etc..Add for example with the ethanol solution 1.7mL (i.e. 0.5g KCR) containing KCR
Enter 1.7mL Emulsifier EL-60 polyethylene glycol (Crem/PG) mixed liquor, 50mL or 100mL physiology is added after mixing
Salt solution medium sized vein is administered.
Provided by the present invention for treatment bacterium infection or the compound of superbacteria infection and combinations thereof, the chemical combination
When preparation forms the paste of external preparation, paste can be used for skin infection, such as skin injury and burn for thing or composition.Should
Formulation effective ingredient be KCR-EE, KCR-ZZ, KCR-ZE and KCR-EZ in any one compound, or wherein any two kinds or
Three kinds or four kinds compounds of person form constituents, can add certain proportion auxiliary material, including ethanol, glycerine and other are necessary
Auxiliary material such as cupu oil, vaseline, cotton seed oil, coconut oil or liquid paraffin etc., the auxiliary material specifically added do not limit, as long as full
Foot can prepare to form paste, the one kind that can be added thereto, two kinds or more, and mutual quality does not limit.Institute
The content of KCR compounds or its composition in paste is stated to meet to contain 2-200mg active ingredients (KCR chemical combination per in 100g paste
Thing or its composition).Above-mentioned substance is well mixed at 70 DEG C, and paste is made.According to clinical needs, this can be adjusted and matched somebody with somebody
Side so that the paste can liquefy at 37 DEG C, be easy to be applied to wound (37 DEG C of wound temperature).KCR dissolve in ethanol or
Paste is prepared in ethanol/glycerite and with 5-125 times of MIC concentration, by taking anti-MRSA (MIC is 8 μ g/mL) as an example, 100g paste
The mass range of middle KCR contents is 4-100mg.The composition of typical 100g paste is as shown in table 4 below：
Table 4：100g paste compositions
| a | KCR | 0.1g |
| Ethanol 96% | 1.0g | |
| Glycerine | 34.0g | |
| b | Cupu oil | 54.9g |
| c | Coconut oil | 10.0g |
| It is total: | 100.0g |
Provided by the present invention for treatment bacterium infection or the compound of superbacteria infection and combinations thereof, the chemical combination
Thing and combinations thereof selects conventional formulation, such as tablet, capsule, dispersible tablet, sustained release tablets when preparation forms oral formulations,
Binder, filler, disintegrant, lubricant etc. are added in the preparation, the type and quality specifically added is not limited, according to
Reasonable selection is actually needed, but is needed to meet per 0.25g oral formulations containing 0.1-0.225g (being preferably 0.1-0.2g) institute
Compound or composition are stated, remaining is auxiliary material.Oral formulations regular size is 0.25g or 0.5g, for the people of 70kg body weight, often
Secondary oral 1-2 pieces.
KCR pharmacokinetics
Animal used is CD-1 mouse.The final concentration of 1.0mg/mL of KCR preparations.2 kinds of KCR preparations are as intravenous injection
(intravenous injection, IV) is used：(1) Emulsifier EL-60/polyethylene glycol (Cremophor/
polyethylene glycol,Crem/PG,v/v:40/60) preparation；(2) 25% human serum albumin (hAlb) preparations.Note in abdominal cavity
When penetrating (intraperitoneal injection, IP), KCR is dissolved in physiological saline (saline).(oral is administered orally
Administration, PO) when, OraPlus is prepared into KCR oral formulations.All formulations dosage is 0.25mL.5,
30 and 120 minutes collection blood samples, separated plasma and red blood cell, KCR (mobile phases are detected with HPLC：Volume ratio is 58:04:38
The formic acid of methanol/acetonitrile/0.2%, flow velocity 0.6mL/min, Detection wavelength 313nm, the μ L of sample introduction 10).
Testing result：KCR is mainly distributed in blood plasma, can detect that in red blood cell a small amount of KCR (<0.1%).Difference system
The bioavilability descending order of agent is：Crem/PG IV>hAlb IV>saline IP>OraPlus PO (see Fig. 6).
In intravenously administrable, the KCR in blood declines rapidly, and half-life period is 2.26 minutes (see Fig. 7).
KCR toxicity test
By KCR ethanol solutions be dissolved in isometric Emulsifier EL-60 polyethylene glycol mixed liquor (Crem/PG, 60:
40v/v), afterwards with 20 times of normal saline dilution.It is respectively using 6-8 weeks female CD-1 mouse, intravenous injection administration, dosage
62.5mg/kg body weight and 6.25mg/kg body weight, administered volume 0.25mL.Control group parenteral solution is free of KCR.Mouse is the 1st, 4
Put to death with carbon dioxide suction in 7 days, gather marrow and blood, leucocyte (White Blood Cell) and blood platelet
(Platelet) count.Detect the aspartic acid Cyklokapren (AST) in blood plasma and glutamic-pyruvic transaminase (ALT) assess hepatotoxicity,
Blood urea nitrogen (BUN) (BUN) assesses Toxicity of Kidney.Marrow grain/macrophage colony forms unit (colony forming unit-
Granulocyte/macrophage, CFU-GM) analysis and evaluation blood (marrow) toxicity.
Single injection KCR does not have found blood (marrow), liver and Toxicity of Kidney, sees Fig. 8.
The species difference of KCR metabolism
The KCR not only energy rapid metabolizations (half-life period only has 2.26 minutes) in Mice Body, and KCR is in mice plasma
Can be degraded rapidly disappearance.But esterase inhibitor BNPP [double (4- nitrobenzophenones) phosphates, bis (4- are added in mice plasma
Nitrophenyl) phosphate] after, retention times of the KCR in mice plasma is obviously prolonged.KCR can be by carboxy-lesterase
(carboxylesterase) digest, it is particularly sensitive to the carboxy-lesterase of mouse.Carboxy-lesterase cracks the C-O keys enzymolysis of No. 3 positions
KCR.Carboxylesterase inhibitor can suppress KCR metabolism, extend the KCR effective blood drug concentration times, strengthen KCR pharmacological action,
Therefore carboxylesterase inhibitor can be used in combination or form combination formulations with KCR.
Test procedure：The carboxylesterase inhibitor BNPP of 5 microlitres of various concentrations is added in 180 microlitres of mice plasmas:0、
0.005th, 0.05,0.5 and 5mM.20 microlitres of KCR (100 μ g/mL) are added to above-mentioned each pipe, 37 DEG C are incubated 24 hours.Add
800 microlitres of ethyl acetate extract KCR, HPCL methods detection KCR.Compare (KCR contents at i.e. 0 hour) with KCR initial value,
After 24 hours, not plus in BNPP blood plasma, KCR only remains 11.03%, adds the BNPP that 5 μ L concentration are 5mM, and KCR surplus ratios are
94.20%, such as shown in Figure 9.KCR is respectively placed in the blood plasma of different genera (mouse, people, pig and dog), 6 hours and 24 small
The KCR amounts in blood plasma are constantly detected, and KCR initial value compares, and obtains KCR retention rates.After 6 hours, KCR in mice plasma
Surplus about 25%, about 44% in people, about 82% in pig, about 74% in dog.KCR metabolism has obvious species difference, such as sees figure
Shown in 10.
KCR stability tests
KCR is dissolved separately in absolute ethyl alcohol or ethanol glycerine mixed liquor, and (ethanol is 1 with glycerine volume ratio:99), final concentration
For 1mg/mL.KCR solution is placed in screw cap bottle and sealed.Every kind of KCR solution is individually positioned in 37 DEG C or room temperature.0th day (i.e.
After KCR solution prepares, sampling detection at once), when the 1st, 2,6 and 12 week (Week) and 9th month (Month), taking-up 30 is micro-
KCR solution is risen, is added in the HPLC bottles for filling 270 microlitres of ethanol, the measurement of HPLC methods KCR (5 microlitres of sampling volume, stream
Fast 0.6mL/min).
KCR ethanol solution, when room temperature and 37 DEG C preserve 9 months, KCR four kinds of stereoisomer things are still
It is stable.KCR ethanol/glycerite (1/99, v/v) can be stablized at room temperature to be saved to 9 months.At 37 DEG C, Ke Yiwen
Surely preserve 2-6 weeks, but after 6 weeks, KCR degradeds are obvious, as shown in figure 11.
The preferred embodiments of the present invention are these are only, are not intended to limit the invention, for those skilled in the art
For member, the present invention can have various modifications and variations.Any modification within the spirit and principles of the invention, being made,
Equivalent substitution, improvement etc., should be included in the scope of the protection.
Claims (10)
1. it is a kind of be used for treat bacterium infection or superbacteria infection compound, it is characterised in that the compound it is entitled
Kaempferol 3- oxygen-α-L- (2,3- bis--to coumaric acyl) and rhamnoside, referred to as KCR, molecular formula C38H31O14, there are four kinds to be stood
Body isomers, respectively KCR-EZ, KCR-ZE, KCR-ZZ and KCR-EE, each counter structure formula are as follows：
R1 in above structural formula is E-p- coumaric acyls group or Z-p- coumaric acyl groups, R2 be E-p- coumaric acyls group or
Z-p- coumaric acyl groups.
2. the compound according to claim 1 for being used to treat bacterium infection or superbacteria infection, it is characterised in that institute
State superbacteria include methicillin-resistant staphylococcus aureus (MRSA), vancomycin resistance staphylococcus aureus (VRSA),
Quick staphylococcus aureus (VISA) and vancomycin-resistant enterococcus and enterococcus faecalis (VRE) in vancomycin.
A kind of 3. antibiotic and sterilizing for being used to treat bacterium infection or the compound of superbacteria infection with described in claim 1
Preparation.
4. according to claim 3 have the antibiotic and sterilizing for being used to treat bacterium infection or the compound of superbacteria infection
Preparation, it is characterised in that the form of the antibiotic and sterilizing preparation is oral formulations, ejection preparation or preparation for external application to skin.
5. according to claim 4 have the antibiotic and sterilizing for being used to treat bacterium infection or the compound of superbacteria infection
Preparation, it is characterised in that the ejection preparation is to contain 100-350mg/mL in Emulsifier EL-60 polyethylene glycol mixed liquor
The compound；The preparation for external application to skin meets to contain compound described in 4-100mg per in 100g paste；The oral formulations
Meet to contain compound described in 0.1-0.225g per 0.25g oral formulations.
A kind of 6. combination that compound for treating bacterium infection or superbacteria infection as described in claim 1 is formed
Thing, it is characterised in that the composition is by any two kinds, three kinds in KCR-EZ, KCR-ZE, KCR-ZZ and KCR-EE compound
Or four kinds be mutually mixed to be formed, mutual mass ratio does not limit.
7. the antibiotic and sterilizing system according to claim 6 for being used to treat the composition of bacterium infection or superbacteria infection
Agent, it is characterised in that the form of the antibiotic and sterilizing preparation is oral formulations, ejection preparation or preparation for external application to skin.
8. the antibiotic and sterilizing system according to claim 7 for being used to treat the composition of bacterium infection or superbacteria infection
Agent, it is characterised in that the ejection preparation is contains 100-350mg/mL institutes in Emulsifier EL-60 polyethylene glycol mixed liquor
State composition；The external preparation meets to contain composition described in 4-100mg per in 100g paste；The oral formulations meet every
0.25g oral formulations contain composition described in 0.1-0.225g.
A kind of 9. compound or claim 6 for being used to treat bacterium infection or superbacteria infects as described in claim 1
The compound formulation that the described composition for treating bacterium infection or superbacteria infection is formed, it is characterised in that described
Compound formulation is removed including being used to treat the compound of bacterium infection or superbacteria infection or for treating bacterium infection or super
Beyond the composition of bacterium infection, in addition to other antibiotic, bacteriostatic agent or reinforcing agent.
10. the composition according to claim 9 for being used to treat bacterium infection or superbacteria infection or compound are formed
Compound formulation, it is characterised in that the antibiotic includes but is not limited to Novel Quinolone class and cephalo-type antimicrobial, described antibacterial
Agent includes but is not limited to beta-lactamase inhibitor, and the reinforcing agent includes but is not limited to carboxylesterase inhibitor.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100234311A1 (en) * | 2009-03-16 | 2010-09-16 | The University Of Mississippi | Methicillin-Resistant Staphylococcus Aureus Active Metabolites |
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2017
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100234311A1 (en) * | 2009-03-16 | 2010-09-16 | The University Of Mississippi | Methicillin-Resistant Staphylococcus Aureus Active Metabolites |
Non-Patent Citations (3)
| Title |
|---|
| MOHAMED A. IBRAHIM,ET AL.: "Methicillin-Resistant Staphylococcus aureus (MRSA)-Active Metabolites from Platanus occidentalis (American Sycamore)", 《J. NAT. PROD.》 * |
| SHENGXIN CAI,ET AL.: "Identification of Compounds with Efficacy against Malaria Parasites from Common North American Plants", 《J. NAT. PROD.》 * |
| 孟胜男，等: "《药剂学》", 31 January 2016 * |
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