CN102503994A - Method of treating clostridium difficile-associated diarrhea - Google Patents

Abstract

A method of treating a disease or disorder caused by the presence of a bacterium selected from the group consisting Clostridium species, Staphylococcus species, Enterococcus species and combinations thereof comprising administering to a patient in need an effective amount of a mixture, which comprises tiacumicin B, lipiarmycin A4, and at least one of other macrocyclic compounds .

Description

The method of the diarrhoea that the treatment difficile toxins is relevant

The application be International Application PCT/US2006/041436 of proposing on October 23rd, 2006 (national applications number: 200680047986.1, denomination of invention: the dividing an application method of the diarrhoea that the treatment difficile toxins is relevant).

Related application

The rights and interests of U.S. Provisional Patent Application series number 60/729,135 that the application requires to submit on October 21st, 2005 and the U.S. Provisional Application series number 60/749,641 submitted on December 12nd, 2005.

The disclosure of above-mentioned related application is incorporated herein fully by reference.

Background of invention

1. invention field

The present invention relates to treatment by being selected from by fusobacterium (Clostridium) species; The disease that the existence of the bacterium of the group that Staphylococcus (Staphylococcus) species and enterococcus spp (Enterococcus) species and combination thereof are formed causes; Particularly by being selected from by difficile toxins (Clostridium difficile, " C.difficile "), clostridium perfringens (Clostridium perfringens; " C.perfringens "); The disease that the existence of the bacterium of the group that streptococcus aureus (Staphylococcus aureus, " S.aureus ") and their combination are formed causes is more specifically treated the disease that the existence by difficile toxins causes.Said disease can be a colitis, pseudomembranous colitis, or diarrhoea.

2. Description of Related Art

The diarrhoea (AAD) that microbiotic is correlated with comprise the streptococcus aureus of the streptococcus aureus (MRSA) of X-1497 resistance, and toxigenic bacterial strain of clostridium perfringens (C.perfringens) causes by difficile toxins.AAD is the main economical load of health care system, according to the annual extra hospital expenses that only just produce hundred million dollars of 30-60 in the U.S. of conservative estimation.

AAD is the serious problem in hospital and long term care facilities and the group.Difficile toxins is the common cause of AAD in the hospital facility, causes about 20% AAD case colitis (AAC) case relevant with most microbiotic.The incidence of the increase of the diarrhoea (CDAD) that difficile toxins is relevant is owing to the prescription [Wilcox etc., Lancet (lancet) 1996,348:767-8] of opening Broad spectrum antibiotics continually to inpatient.

The severe form of said disease is the colitis (PMC) of pseudomembrane property, and its main performance on histology is the colitis with mucous patch, and main performance clinically is serious diarrhoea, abdominal colic, and general toxicity.Total lethality rate from CDAD is low, but much higher in the patient who suffers from serious colitis or system toxicity.Nearest research shows, in addition when death be not during directly owing to difficile toxins, compare with the contrast that case matches, the mortality ratio in CDAD patient is also much higher.

Diarrhoea and colitis are caused by the generation of one or more clostridium difficile toxins.Organism breeds in the patient's of administration Broad spectrum antibiotics colon, or breeds in more uncommon patient's at cancer chemotherapy the colon.After with said pharmaceutical treatment, in about 20% the inpatient of suffering from diarrhoea, diagnose out CDAD.

Present treatment for AAD or CDAD comprises the related antimicrobial medicament or the termination of chemotherapeutics, non-specific supportive measurement and with treating to the microbiotic of difficile toxins.Modal antimicrobial therapy selects to comprise vancomyein and metronidazole.Relevant with antibiotic therapy CDAD with the clinical recurrence of disease.It is reported that the frequency of recurrence is 5-50%, wherein the recurrence rate of 20-30% is modal reference numerals.No matter medicine, dosage, or with perdurability of any above-mentioned antibiotic initial treatment how, recurrence is with almost equal frequency generation.Main challenge in the treatment is in the treatment that has repeatedly the patient of recurrence, and wherein antibiotic control is debatable.

Two kinds of concrete treatments of the most often using are vancomyein and metronidazole, although vancomyein is by FDA unique medicine of indication approval for this reason.Yet vancomyein is not to recommend for first line treatment of CDAD, mainly is because it only has antibiotic activity to some serious life-threatening multidrug resistance bacteriums.Therefore, in the minimized effort of appearance that makes vancomyein resistance faecalis (VRE) or vancomyein resistance streptococcus aureus (VRSA), only if when the sin qua non, medical group does not encourage to use this medicine.

Start from about vancomyein resistance archenteric flora, especially enterococcal promotion and selection purpose are recommended metronidazole as initial treatment.Although the frequency of report difficile toxins resistance in some countries can be＞6%, it is almost effective as vancomyein that metronidazole remains, considerably more cheap and can be used for oral or the intravenously use.Metronidazole and significant side effects comprise nauseating, neuropathy, and leukopenia, epileptic seizures is with relevant to the alcoholic acid toxic reaction.In addition, it is used for children or the pregnant woman is unsafe.

Although two kinds of medicaments all are effectively in treatment is infected, the increment rate of treatment failure and diarrhoea recurrence is the defective of standard treatment in about 20% the patient who begins to react.Reported that the treatment of carrying out with metronidazole is the important risk factor of transplanting (colonization) and infecting for VRE.In addition, present regimen quite bothered, and required many to 500mg four times on the one (qid) at 10-14 days.Therefore, existence is for the demand of the better treat-ment of CDAD case and other AAD and AAC case.

Therefore, need the exploitation antibacterials, the tendency that it has low generation resistance, having does not have crossed resistance and/or has the post antibiotic effect of prolongation what existing biocide reduced.

Summary of the invention

The present invention provides treatment by being selected from by the fusobacterium species, the staphylococcus species, and enterococcus spp species, and the disease that causes of the existence of the bacterium of the group formed of combination or the method for illness, said method comprises mixture from significant quantity to its patient of needs that use.Said mixture comprises that the platform of significant quantity colludes mycin B and is selected from the other macrocylc compound by the following group of forming:

III (OP-1416, RT ratio 0.71),

IV (OP-1415, RT ratio 0.81),

V (OP-1417, the RT ratio: 0.84),

IX (OP-1435, the RT ratio: 1.13),

X (OP-1437, the RT ratio: 1.19),

XI (OP-1402, the RT ratio: 1.24),

XII (OP-1433, the RT ratio: 1.39),

XIII (OP-1438, the RT ratio: 1.48),

XIV (lipiarmycin A4, OP-1405, the RT ratio: 0.89), and combination.When having the compound of formula XIV, said mixture comprises the compound of about 0.1 to about 5% formula XIV.

Preferably, said mixture comprises that the platform of at least 90 weight % colludes mycin B.More preferably, said mixture comprises that the platform of at least 95 weight % colludes mycin B.

Preferably, said mixture comprises at least 1 weight %, and more preferably about 2 weight % are to the other macrocylc compound of about 5 weight %.

Preferably, said mixture comprises about 0.1 weight % to about 5 weight %, and more preferably 0.3 weight % is to 3 weight %, and 0.3 weight % is to 1.5 weight % especially, the lipiarmycin A4 of especially about 1 weight %.

Preferably, when having lipiarmycin A4, said mixture also comprises at least a of following compounds:

VI (OP-1431, Tiacumicins F, the RT ratio: 0.92),

VII (Op-1432, platform collude mycin C, the RT ratio: 0.95) and

VIII (OP-1434, Tiacumicins A, RT ratio: 1.10).

Preferably, said mixture shows the HPLC collection of illustrative plates of in Fig. 5, describing basically.

Preferably, disease or the illness and the difficile toxins of treatment according to the present invention, clostridium perfringens, streptococcus aureus and combination thereof are relevant.More preferably, the disease or the illness of treatment are relevant with difficile toxins according to the present invention.

Preferably, the disease of treatment is diarrhoea or colitis according to the present invention, and particularly diarrhoea more specifically is CDAD.

Preferably, the method preparation of mixture according to the present invention through comprising the following steps:

With microorganism culturing in nutritional medium in nutritional medium, to accumulate mixture; With

From nutritional medium, separate said mixture;

Said nutritional medium comprises the sorbent material that adsorbs said mixture.

Said nutritional medium preferably includes the sorbent material of 0.5-15 weight %.Said sorbent material is polymeric adsorbent preferably.More preferably; Said polymeric adsorbent is selected from the group of being made up of following: ion exchange resin

XAD16; XAD16HP; XAD2; XAD7HP, XAD1180, XAD1600; IRC50 and

XAD761.Said mikrobe is dactylosporangium aurantiacum (Dactylosporangium aurantiacum) hamdenensis subspecies preferably.Based on weight, nutritional medium comprises 0.2% to 10% glucose, 0.02% to 0.5% K ₂HPO ₄, 0.02% to 0.5% MgSO ₄7H ₂O, 0.01% to 0.3% KCl, 0.1% to 2% CaCO ₃, 0.05% to 2% casamino acids, 0.05% to 2% yeast extract and 0.5% to 15% XAD-16 resin.Culturing step preferably carries out at about 25 pH to about 35 ℃ temperature and from about 6.0 to about 8.0.

Preferably, the disease of treatment is relevant with the use of microbiotic or cancer chemotherapy or antiviral therapy according to the present invention.

According to an embodiment preferred, said mixture is with the amount of about 50mg to 1000mg, and more preferably 100mg is to the amount of 400mg; Amount every day 1-3 time of 200mg especially, more preferably once a day or twice, twice of every day especially; At 3-15 days, use in about especially 10 days.Preferred oral is used.

Treatment of the present invention can be allowed effective treatment and difficile toxins; Streptococcus aureus; With the relevant diarrhea disease of product enterotoxin bacterial strain of clostridium perfringens, and can be beneficial to system's microbiotic and can not increase the vancomyein resistance faecalis (VRE) in the digestive tube.The present invention also reduces the existence of VRE in the digestive tube.

From the following detailed that combines the accompanying drawing consideration, other purpose of the present invention and characteristic will become obvious.Yet, it being understood that it is not in order to define scope of the present invention that said accompanying drawing just designs from illustrational purpose, should be for scope of the present invention with reference to accompanying Claim.It should also be understood that accompanying drawing need not be confined to certain limit, only and if point out that in addition they only tend to conceptually illustrate structure as herein described and method.

The accompanying drawing summary

In the accompanying drawings:

Fig. 1 shows 1B-MD stage administration time table.

Fig. 2 is presented at the bacteroid quantity after the treatment.Matching symbol rank check (Pairs signed-ranks test), two tails.For counting＜3log 10, use value 2.9.

Fig. 3 shows the effect of vancomyein to bacteroide fragilis (B.fragilis) crowd's treat-ment.

Fig. 4 is presented at after the MCC treatment, the quantitative minimizing of difficile toxins nourishing body counting.

Fig. 5 is the typical HPLC collection of illustrative plates of mixture, and said mixture can be used in the method for the invention.

The detailed description of the preferred embodiments of the invention

To provide as follows with in this application some abbreviation or the definition of term:

The diarrhoea that the AAD=microbiotic is relevant

ATCC=American type culture collection (American Type Culture Collection)

¹³C=carbon 13

CO ₂=carbonic acid gas

N ₂=nitrogen

H ₂=hydrogen

TAPS=N-three (methylol) methyl-3-aminopropanesulfonicacid acid

MOPS=3-(N-morpholino) propanesulfonic acid

The diarrhoea that the CDAD=difficile toxins is relevant

Clinical and the laboratory standard institute (Clinical and Laboratory Standards Institute) of CLSI=, preceding NCCLS

ED ₅₀The effective dose of=generation 50% reaction

The HPLC=performance liquid chromatography

The IR=infrared spectroscopy

The quantitative lower limit of LLOQ=

MCC=comprises the compsn of macrocylc compound

The MIC=minimum inhibition concentration

MIC ₅₀The minimum inhibition concentration of the bacterial isolates of the test of=inhibition 50%

MIC ₉₀The largest inhibition concentration of the bacterial isolates of the test of=inhibition 90%

MRSA=X-1497-resistance streptococcus aureus

Standard committee of clinical labororatory of NCCLS=country, present CLSI

The PMC=pseudomembranous colitis

VRE=vancomyein-resistance faecalis

VRSA=vancomyein-resistance streptococcus aureus

Term " microbiotic relevant illness " refers to disturb when antibiotic therapy the balance of gastral microorganism species, makes causal organism as producing the difficile toxins of enterotoxin, and streptococcus aureus and clostridium perfringens bacterial strain are bred when vigorous and the illness that causes.Except other symptom, these biologies can cause diarrhoea, pseudomembranous colitis and colitis and show as diarrhoea, urgent urination, abdominal colic, tenesmus and fever.Diarrhoea when serious, causes dewatering and the medical science complication relevant with dehydration.

Term " MCC " refers to for whole antibiotic substance, mainly comprises the preparation that platform colludes mycin B (for example, analyze through HPLC, at least 90%, 95%-98% preferably).MCC comprises also that in a small amount the platform of (for example, at least 1%, 2%-5% preferably) colludes mycin B related compound, i.e. the compound of lipiarmycin A4 and at least a formula III-XIV that shows in the above.PCT applies for PCT/US03/21977, and its international publication number is WO 2004/014295A2, provides preparation to comprise the method that platform colludes the mixture of mycin B.The full content of this PCT application is incorporated into this paper as a reference.Yet, be specifically designed to inhuman MCC and can comprise and be less than 80% platform and collude mycin B (, measuring) through HPLC to whole antibiotic substance.

Term " vehicle " refers to be added into pharmaceutical composition further to help the nonreactant of administered compound.The instance of vehicle includes but not limited to, lime carbonate, calcium phosphate, various sugar and various types of starch, derivatived cellulose, gelatin, vegetables oil and polyoxyethylene glycol.

Term " halogen " comprises F, Cl, Br and I.

Term " macrocylc compound " refers to have the organic molecule that comprises the macrocyclic structure that surpasses 10 atoms usually.

Term " 18-unit macrocylc compound " refers to have the organic molecule of the ring structure that comprises 18 atoms.

Term " unit's ring " can comprise any ring structure, comprises aforesaid carbocyclic ring and heterocycle.Term " unit " means the quantity of the skeletal atom of makeup ring.Therefore, pyridine for example, pyrans and sulfo-pyrans are 6 yuan of rings, and the pyrroles, furans and thiophene are 5 yuan of rings.

Term " MIC " or " minimum inhibition concentration " refer at the needed antibiotic minimum concentration of the growth of vitro inhibition bacterial isolates.The domestic method of confirming antibiotic MIC is to prepare some to comprise the pipe of microbiotic serial dilution thing, then it is inoculated with the purpose bacterial isolates.After inoculation under suitable normal atmosphere and the temperature, antibiotic MIC can not have the pipe of the minimum concentration of turbidity (not having growth) to confirm from demonstration.

Term " MIC ₅₀" refer in given bacterial species to suppress the needed antibiotic minimum concentration of growth of 50% bacteria tested bacterial strain.

Term " MIC ₉₀" refer in given bacterial species to suppress the needed antibiotic minimum concentration of growth of 90% bacteria tested bacterial strain.

Term " patient " need to refer to the human or animal of therapeutic treatment.For purposes of the present invention, people patient is typically taken care of in one-level health care institution such as hospital or sanatorium specially.Yet; After from one-level health institution, leaving hospital; Treatment can be carried out on outpatient's basis with using microbiotic or cancer chemotherapeutic or the relevant disease of antiviral therapy, or can be prescribed by the doctor of the home care that is independent of one-level health care institution.The animal that needs therapeutic treatment is typically under animal doctor's nursing.

Term " pharmaceutical carrier " refers to medicinal carrier or thinner.

Term " pharmaceutical salts " refers to from those of medicinal mineral alkali and organic bases.Salt from the alkali that is fit to comprises an alkali metal salt (for example, sodium salt or sylvite), alkaline earth salt (for example, magnesium salts), ammonium salt and N (C ₁-C ₄Alkyl) 4+ salt etc.The illustrational instance of in these some comprises sodium hydroxide, Pottasium Hydroxide, choline hydroxide, yellow soda ash etc.

Term " pharmaceutical composition " refers to that as herein described one or more collude mycin, or its physiologically acceptable salt, with other the chemical ingredients such as the mixture of physiology acceptable carrier and/or vehicle.The purpose of pharmaceutical composition is to promote compound administration in organism.

Term " physiology acceptable carrier " refers to not cause to the obvious stimulation of biology and does not eliminate the biological activity of the compound of being used and the carrier or the thinner of character.

Term " pseudomembranous colitis " or " enteritis " refer to because the inflammation of the mucous membrane of small intestine and large intestine causes forming pseudomembrane property material (that is, by fibrin, mucus, the material of downright bad epithelial cell and white corpuscle composition).

When being used for this paper, term " platform colludes mycin " refers to all be included in the compound family of 18 yuan of macrocylc compound that show among the following formula I:

Formula I

When being used for this paper, term " platform colludes mycin B " refers to the first macrocylc compound of the 18-that shows among the formula II below:

Formula II (RT ratio 1.0)

When being used for this paper, term lipiarmycin A4 refers to 18 yuan of macrocylc compound showing among the formula XIV below:

According to one embodiment of the invention, behind the oral administration multiple doses, in blood plasma, detect low MCC level, its great majority are under quantitative boundary.As contrast, the ight soil level in two researchs is very high, surpasses the MIC to difficile toxins ₉₀(0.125 μ g/mL) 10,000 times.

According to one embodiment of the invention, in the patient, the recurrence of the diarrhoea relevant with difficile toxins can still not have the amount of influence and use MCC perdurability to be inhibited to patient's normal stool road flora through the recurrence with effective inhibition difficile toxins.

According to an embodiment, for the MCC of CDAD every day oral dosage will be in promoting agent/sky scope of about 1.0g at about 50mg, preferably from about 100mg in about 600mg/ days scope.Generally speaking, treatment will continue from the time phase about 3 to about 15 days.As required, can use the medicine and treatment interval of more or less amount.For example, according to the clinical study result who reports hereinafter, from about 10 days process, the dosage of the MCC of the about 100-400 milligram of administration every day confirms in treatment CDAD it is effectively, has minimum clinical recurrence.

According to one embodiment of the invention, said mixture can be through following universal method preparation.

From shaking bottle in the scope of big " in batches " fermentation container, in container, cultivate the bacterium that produces MCC.In order to produce a large amount of basically MCC, in jar, use the deep layer aerobic fermentation.Yet, can obtain in a small amount through shake-flask culture.For the jar fermentation, preferably use the nourishing body inoculum.Through with biological spore form, the mycelium fragment, or the substratum of freeze dried throw out inoculation small volume prepares the nourishing body inoculum, and fresh to obtain, the organism culture of active growth.Then, the nourishing body inoculum is transferred in the bigger jar, wherein behind the incubation time that process is fit to, with the output generation MCC microbiotic of a large amount of raisings.If foaming becomes problem, so skimmer in a small amount being joined possibly be essential in the large-scale fermention medium.

In control medium, produce to improve output with other additive/composition.Use liquid-submergence, stir culture method to carry out the production of MCC.Fermentation is carried out in 25 ℃ to 37 ℃ TR.Carefully monitor the consumption of carbon source, and add the carbon source of additional quantity if desired.The pH of fermentation preferably maintains about 6.0 to about 8.0.After the fermented liquid inoculation, between 3-15 days, produce and accumulation MCC.

Discovery is purchased output and the organic efficiency that polymeric adsorbent increases MCC during the fermentation.Sorbent material preferably exists with 0.5-15 weight % scope.MCC absorbs through resin and reclaims from fermentation culture with extraordinary output (＞100mg/L nutrient solution), and through wash wash-out on resin and the mycelium with various polar solvents.

Use polymeric adsorbent such as ion exchange resin (Amberlite) (XAD-16) MCC to be caught out from nutrient solution at first, during the fermentation.After accomplishing fermentation, solid piece (comprising polymeric adsorbent) is separated from nutrient solution through screening.Solid piece is used eluent ethyl acetate, then under reduced pressure concentrate.

After fermentation ends, solid piece (comprising polymeric adsorbent) is separated from nutrient solution through screening.Mixture with organic solvent such as ETHYLE ACETATE, methyl alcohol, acetonitrile or two or more organic solvents elutes MCC from resin.Then, under reduced pressure extract is concentrated.This resistates is further purified through following manner, that is, through with low polar solvent such as hexane, heptane, methylcyclohexane grinds, or through in the two-phase solvent system like ETHYLE ACETATE/water; ETHYLE ACETATE/sodium chloride aqueous solution; Methyl alcohol/hexane; Distribute in the mixture of two or more solvents that acetonitrile/hexane or other exist with various ratios and combination, or through carrying out the column chromatography wash-out with the organic solvent system that is fit to.The purification process of MCC is based on the CH that uses 50: 50: 1 at present ₃CN/H ₂The MeOH/H of O/AcOH or 70: 30: 1 ₂O/AcOH carries out as reverse (C-18) post of the middle pressure of eluent.The level that comprises the MCC of needs with brine wash is divided and it is concentrated.Resistates is dissolved in the ETHYLE ACETATE, is evaporated to drying so that light yellow foam to be provided with water washing and with organic layer, it uses washed with isopropyl alcohol again, and drying under reduced pressure is to produce white powder.The level that merges purity＞88% is divided.The level branch is concentrated to the half the of initial volume.Filtering precipitate and water washing leaching cake.Under high vacuum, solid drying is spent the night to obtain white powder and to analyze with HPLC.Typically, the platform that mixture comprises the 90%-99% scope colludes mycin B as staple, the macrocylc compound of lipiarmycin A4 (0.1% to 5%) and at least a or multiple above-mentioned formula III-XIV.

Embodiment

Be not inclined to and limit scope of the present invention by any way the following example is provided through describing specific embodiments of the present invention.

Be prepared in the mixture that uses among the following embodiment according to above-mentioned preparation method.Following table shows the compsn of certain exemplary mixture prepared in accordance with the present invention.

Carrying out HPLC according to following method measures.

Mobile phase A: the trifluoroacetic acid of 2.0mL is joined in the 2L HPLC water, filter and the degassing.

Mobile phase B: the trifluoroacetic acid of 1.0mL is joined in the 2L acetonitrile, filter and the degassing.

Post: the 4.6X150mm post comprises Chemical bond in the cellular silica of diameter 3-10 μ m or the octyl group silane of ceramic particle (for example, Zorbax Eclipse XDB-C8,3.5 μ m).

Detector: 230nm.

Flow velocity: about 1.0mL/ minute.

Volume injected: about 10 μ L.

Working time: about 10 μ L.

Thinner: 100% acetonitrile.

Attention: the RT of mixture must be in 8-12 minute.

The standard fabrication thing: the mixture that accurately takes by weighing about 20mg in the 100mL volumetric flask, with it with thinner dissolving and be diluted to certain volume.

The specimen preparation thing: the mixture that accurately takes by weighing about 20mg is in the volumetric flask of 100mL.Add about 60mL thinner and vortex with dissolving.Also mix to certain volume with diluent.

The suitability of system: such as following method guidance, to the standard fabrication thing carry out chromatography and the record maximum response.For five reinjected RSDs of colluding the peak area of mycin B is NMT 2.0%, and the tailing factor that platform colludes mycin B area is NMT 2.0.

Method: the thinner of injecting about 10 μ L.Inject standard and the specimen preparation thing of equal-volume (about 10 μ L) respectively, record chromatogram and react for the main peak detector for measuring.

The relative residence time:

Calculate: use formula to calculate measured value:

Wherein: R _u=from measuring the peak area that platform that prepared product obtains colludes mycin B

R _s=the platform that obtains from the standard fabrication thing colludes the peak area of mycin B

The purity of P=reference standard comprises water factor.

W _Std=SW (mg)

StdDil=standard dilution (mL).

W _Smp=example weight (mg).

WF=sample water factor.

Discard the peak that derives from thinner, and pass through the w/w per-cent of the individual and total related substances of computes:

Wherein: R _iThe peak area of=the related substances that obtains from the specimen preparation thing.

R _u=the platform that obtains from the specimen preparation thing colludes the peak area of mycin B.

RF _i=related substances response factor is (for all related substances RF _i=1.0)

In addition, will be presented among Fig. 5 according to the typical HPLC collection of illustrative plates of mixture of the present invention.The compound that in mixture, comprises, for example the compound of formula II-XIV can find in the HPLC collection of illustrative plates according to their RT ratio.Par-101 in Fig. 5 representes that the RT ratio is that 1.0 platform colludes mycin B.

Then, with said mixture (50mg) and 100mg Avicel PH 102, FMC (Microcrystalline Cellulose) mixes in the capsule shell (size l capsule shell) of No. 1 size.

Embodiment 1. inoculums, pH and positively charged ion are to the influence of MCC to the external activity of difficile toxins

Known MIC value for many antibiotic measurements receives environmental variance such as pH, the concentration of divalent cation such as calcium and magnesium and the influence of bacterial density.Anti-microbial activity is important consideration to the dependency of these factors, and particularly for the microbiotic of target bacterium in digestive tube, wherein these parameters can be along with diet and morbid states and changed bigger.

When being designed for the method for vitro test in future, MIC also can be the important factor of considering to the susceptibility of these environmental variances.Clinical and laboratory standard institute, CLSI (preceding NCCLS) recommend to use and have replenished vitamin K ₁The minimum inhibition concentration (MIC) that is used for anaerobion with the Brucella agar of teichmann's crystals is measured.Yet the level of the divalent cation in this substratum is not by stdn.In addition, the pH at the substratum that uses under the anaerobism glove box also can change under different gaseous mixture.Anaerobion is incubation in the mixture of nitrogen, hydrogen and carbonic acid gas typically, and CO ₂Existence with the acidifying substratum and can be the important source of variability.Consider the variety (H of the air pressure conditions that can be used for the anaerobion sensitivity tests ₂/ CO ₂Generator vacuumizes/replacement method or anaerobic room), inoculation what also possibly be difficult to stdn.The obtainable anaerobic condition in each laboratory will confirm that organism is exposed to the time length that oxygen atmosphere is arranged in work top (bench top) operating process and anaerobism balance, and therefore influence the viability and the experimental result of culture.

In this research, we have checked the level of divalent cation calcium and magnesium, pH (from 5-8), and inoculum density (above 3 one magnitude) is to the variability of the different batches of the influence of MIC and Brucella nutrient solution.

Material and method

Bacterial isolates:

Laboratory strains difficile toxins 9689,700057,43255,17857 and eubacterium lentum (Eubacterium lentum) 43055 is available from American type culture collection (American Type Culture Collection (ATCC)).On the brucella agar plate, said flat board has replenished teichmann's crystals and vitamin K to all bacterial strains by streak inoculation, and it is from the freezing storage liquid that in 10% glycerine, remains on-78 ℃ before use.

The MIC test:

The CLSI method (4) that is used for anaerobism nutrient solution and agar dilution at present is used to carry out the MIC assessment.Test is not a kind of effective means for clostridial MIC in the nutrient solution dilution; Yet, since after the anaerobic room inner equilibrium potential inaccuracy of the pH of measure solid agar, use and compared two kinds of methods for assessment pH effect.

Inoculum density is to the influence of MIC value:

Use agar dilution method (4), measured inoculum density the influence of difficile toxins to the susceptibility of MCC and vancomyein.Through at first preparation～10 ⁸The suspension of cfu/mL prepares inoculum, and then with said suspension with 10 times of factor serial dilutions, thereby obtain 10 ⁵-10 ⁸Culture density range between the cfu/mL is to obtain 10 ²-10 ⁵The spot density of cfu/ spot.

PH is to the influence of MIC value:

Use agar dilution and micro-nutrient solution dilution process, in the susceptibility of the pH of 6-8 scope inner evaluation difficile toxins to MCC.

Use the agar dilution method, in two independent experiments, in the pH of 6.2-8.0 scope, confirm the MIC of MCC to the difficile toxins bacterial strain.In order to obtain to the needed oxyphobe pH of sensitivity tests, with the damping fluid (NaH of 100mM ₂PO ₄Or TAPS [N-three (methylol) methyl-3-aminopropanesulfonicacid acid]) adds in the substratum at pH 7 and 8 respectively.Even using strong shock absorption, pH changes a little after the balance in anaerobic gas, and therefore in some situations, with the substratum oxyphobe pH that titration extremely needs in ambient air.Actual pH is confirmed after the anaerobic room inner equilibrium always.Only detect with the vancomyein that compares at pH 7.

Use nutrient solution Microdilution method, in 3 independent series, confirm that in the pH of 6-8 scope MCC and vancomyein are directed against the MIC value of difficile toxins bacterial strain.In first series, in ambient air titration not buffered Brucella nutrient solution to obtain the pH scope of 5-9.Yet, at glove box environment (10%H ₂/ 5%CO ₂/ 85%N ₂) in; The anaerobism balance of substratum has reduced the pH of substratum; Cause the anaerobism pH scope (have the portable pH meter test of flat pH probe, said probe then is transferred to inside in glove box external application damping fluid standard calibration) in the 5-7.5 scope like use.For experiment subsequently, damping fluid is added the pH change that causes owing to the anaerobism balance with antagonism in the substratum.In second series, with 10mM damping fluid [NaH ₂PO ₄H ₂O pH7.0, MOPS pH 8.0, or TAPS pH 9.0, the pH value in ambient air) add pH value greater than in 6 the substratum with the pH scope of acquisition 6-7.6 after the anaerobism balance.In the 3rd series, handle for the pH that surpasses 6, buffer concentration is increased to 100mM, to obtain the anaerobism pH scope of 6-8.1.

Divalent cation concentration is to the influence of MIC value:

Use the agar dilution method to confirm that calcium and magnesium ion concentration are to the influence of difficile toxins bacterial strain to the susceptibility of MCC.(Laboratory Specialists Inc) confirms like the desired divalent cation level in the Brucella nutrient solution of manufacturer through laboratory Expert Co., Ltd.Add the divalent cation (with the form of calcium chloride or magnesium chloride) of additional quantity, thus obtain 2.1,3.0 with the calcium ion concn of 5.7mg/dL and 3.3,4.5 and the magnesium ion concentration of 7.5mg/dL.

MCC MIC value is about the reproducibility of different commercial batch substratum

Use CLSI agar dilution method; Also use Brucella agar (lot number #30768960 from three of BBL different commerce batch; 211086; With 3167036) check the susceptibility of difficile toxins to MCC, said Brucella agar has replenished the vitamin K (SIGMA (Sigma) lot number V-3501 and 0214010) and the teichmann's crystals (SIGMA lot number 072K1221 and 034K7656) of different batches.

The result

Inoculum density is to the influence of MIC value

Table 1 and 2 shows that the inoculum density are to MCC and the influence of vancomyein to the MIC of two bacterial strains (ATCC 9689 and ATCC 700057) of difficile toxins.Two difficile toxins bacterial strains do not receive 10 to the susceptibility of MCC ⁵-10 ⁸Cfu/ml (10 ²-10 ⁵The influence of the inoculum concentration CFU/ spot), such as the identical MIC value that obtained through inoculum concentration for all tests demonstration.Yet the MIC of vancomyein increases with inoculum concentration and increases progressively, and wherein compares with minimum inoculum density, and the highest inoculum density meter reveals on MIC four times increase.These results show that inoculum density is not result's the important factor that influences the MCC sensitivity tests of difficile toxins.

Table 1.MCC (μ g/mL) is to the difficile toxins ATCC 9689 (10 of different vaccination thing density ²-10 ⁵The CFU/ spot) external activity

Table 2.MCC (μ g/mL) is to the difficile toxins ATCC 700057 (10 of different vaccination thing density ²-10 ⁵The CFU/ spot) external activity

PH is to the influence of MIC value

Table 3 has been described different pH values to the effect of difficile toxins to the susceptibility of MCC, and it is two independent dates, and is measured through the agar dilution method.In experiment for the first time, for two difficile toxins bacterial strains, handle (pH 6.2 and pH 7.2) with lower pH and compare, the highest pH handles 8 times the increase that (pH 7.9) show the MIC value.When at the highest pH (pH 8.0) when repeating the experiment of confirmation property, the MIC value keeps higher for two bacterial strains.For arbitrary bacterial strain, between pH 6.2 and pH 7, in MCC MIC, do not observe increase.

The MIC value with the increase of pH not with the consistent dependency that grows into that increases, as if so pH is not only owing to the viability of organism in the increase of higher pH to the influence of MIC.Handle with pH 6.2 and pH 7.9 and to compare, pH 7 handles has more not intensive organism spot growth.

Table 3.pH is to the influence (buffered substratum) of agar dilution MIC value

Table 4,5 and 6 are presented at three independent dates, the MIC data of the nutrient solution Microdilution susceptibility method of carrying out with the pH scope of 5-8.1.In first series, wherein substratum is not cushioned, for two difficile toxins bacterial strains, at the MIC of the MCC of pH 7.5 than at big 8 times (table 4) of the MIC of pH 5.9.MIC at pH 5 can not be determined, because biology can not be grown on this pH.For difficile toxins ATCC 9689 and ATCC 700057, on MCC MIC, buffered (10mM) pH 7.6 handles respectively the increase (table 5) of revealing 8 times and 16 times than pH 6 processing lists.In this three strongest ones' buffering (100mM) series, observe similar result for two kinds of organisms, wherein the highest pH handles (pH 8.1) and on MIC, shows 16 times of increases (table 6) than minimum pH processing (pH 6).In three all experiments, vancomyein demonstrates similar trend, wherein handles with minimum pH and compares, and the highest pH handles and produces doubly bigger MIC of 4-8.

Table 4. uses not the buffered medium pH to the influence of MIC

Table 5. uses the influence of the pH of weak buffered substratum (10mM) to MIC

Table 6. uses the influence of the pH of strong buffered substratum (100mM) to MIC

Also visual inspection is carried out in the total growth to assay plate in all pH processing.In first series, it uses not buffered nutrient solution, total the cultivation turbidity increase with pH.Except identical with pH 7.6 culture turbidity for pH 7.2, in second series, observe identical trend, said second series is used the buffering nutrient solution of 10mM.In the 3rd series, in whole pH handled, the culture turbidity was more equal, exception be pH 7.5, it is the most muddy.

Generally speaking, for the two kinds of methods of sensitivity tests and the buffering salt of various concentration, for two bacterial strains of difficile toxins, the MIC value of MCC and vancomyein all increases with pH.

Divalent cation concentration is to the influence of MIC value:

The calcium in commercial Brucella nutrient solution and the measurement of magnesium level show respectively 21 with calcium and the magnesium ion concentration of 33mg/L.The divalent cation that adds various additional quantity, and in the MCC MIC value of three different calcium ion concns (21,30 with 57mg/L) and three different magnesium ion concentrations (33,45 and 75mg/L) test for the difficile toxins bacterial strain.In all types of substratum, it is identical that the MIC value keeps.In having the substratum of different cation concns, difficile toxins 9689 has the MIC value of 0.063 μ g/ml, and difficile toxins 700057 has the MIC value of 0.125 μ g/ml.Test is as the vancomyein of contrast, and said test is carried out with the Brucella agar that replenishes, and in experimentation, it does not add any extra calcium or magnesium as contrast, is 1 μ g/ml (table 7 and 8) for the MIC value of all experiment confirm expections.

The external activity of the MCC of table 7. in the Brucella agar that replenishes different divalent cation concentration

The external activity of the MCC of table 8. in the Brucella agar that replenishes different divalent cation concentration

MCC MIC value about different commercial batch substratum:

Use the Brucella nutrient agar that replenishes of three kinds of different batches to come the activity of comparison MCC three different dates to the difficile toxins bacterial strain.Through test QC organism, eubacterium lentum is at CLSI (NCCLS) tolerance interval, and promptly the activity of the clindamycin in the 0.06-0.25 μ g/mL is controlled MIC and measured.Another contrast step of carrying out MIC mensuration is to comprise metronidazole and monitor its activity to the difficile toxins bacterial strain, and it demonstrates in our laboratory has the MIC value of scope in 0.25-0.5 μ g/mL.Such as in table 9 demonstration, MCC does not receive the influence of the Brucella agar that replenishes of different batches to the activity of difficile toxins.All contrasts confirm the activity in confirming scope.

The external activity of the MCC of the substratum test of three kinds of different batches of table 9. use

Conclusion

Opposite with vancomyein, MCC does not receive 10 to the activity of difficile toxins ²-10 ⁵The influence of the inoculum concentration in the cfu/ spot extension.

Difficile toxins does not receive the influence of cation concn (calcium ion in the 2.1-5.7mg/dL scope, the mg ion of concentration in the 3.3-7.5mg/dL scope) and different commercial batch substratum to the susceptibility of MCC.

In the pH of 6-8 scope, increase with pH for the two MIC value of MCC and vancomyein.In the high MIC value on the alkaline pH possibly be owing to when being higher than their pKa, the deprotonation form of the phenolic hydroxyl group of these two kinds of compounds, and in this situation, they form charged kind, expect that it has permeability still less to bacterial cell.On the contrary, when being lower than pKa (for MCC, 7.22), the microbiotic major part is by protonated, and therefore permeation cell film more effectively.

Biological density increases with the pH that increases usually; When having buffer reagent, stand density, but be not the dependency minimizing of MIC to pH.Although when lacking damping fluid, organism density becomes positive correlation with alkalescence, and MIC trend can not be the result of pH to the influence of biological density.This is that wherein in whole pH treating processes, organism density is more equal because in the buffering experiment, observe the identical relation between MIC and the pH, and this possibly be owing to the different effect of buffer type to biological growth causes.

Security, pharmacokinetics and the consequence of the MCC of embodiment 2. in the patient of health volunteer's diarrhoea relevant (CDAD) with suffering from difficile toxins

The 1B-MD stage.

Summary. this be oral, the multiple doses that carries out of the Miami University clinical pharmacology department of the Chinese Academy of Sciences of Miami, double blinding in the Florida State, at random, placebo is as research contrast, that dosage progressively rises.Richard Preston, M.D. is as the main investigator of this test.The tolerance of the many oral dosages of MCC and pharmacokinetics are assessed in the trial volunteer of 24 health altogether.(in 3 groups, each organizes 8 experimenters to the oral dosage of the MCC of assessment, wherein handles with promoting agent for 6; 2 placebo treatment) be 150,300, and 450mg (in the capsule that the powder of the research medicine that comprises 50mg is filled); It carried out 10 days early using every day after the meal continuously.The administration experimenter and the combination inpatient/out-patient monitor on the basis.In 10 days administration stage, let the experimenter get into research unit at the 0th day and the 9th day, and stopped nearly 48 hours in the back of being admitted to hospital at every turn.After hit the target table arrangement and program, let the experimenter leave hospital at the 2nd day and the 11st day.In out-patient's stage, the experimenter reports the administration situation and stays to research unit and observed 3 hours every day.

In the multiple dosing phase process, collect serial blood at different time points/interval, urine and faecal samples.Confirm the blood plasma of MCC, urine and ight soil concentration are carried out the pharmacokinetics analysis.Before the experimenter is excluded from research, at the planning chart of arranging follow-up examination on the 17th day of each conceptual phase.At whole treatment stage and in follow-up investigation, closely the generation of any spinoff of study on monitoring experimenter or unusual lab investigation are found.See Fig. 1,1B-MD stage administration time table.

The 2A stage

Summary. this is to select the safety of MCC and the dosage of effective dose to find research.To experimenter's administration 100 at random (50mg/12 hour), 200 (100mg/12 hours), or 400 (200mg/12 hours) mg/ days continue 10 days, carry out clinical assessment subsequently.The experimenter has write down all symptoms on daily record every day card.To stool frequency and denseness, existence and the abdominal discomfort of blood in ight soil given and special attention.Get into and (10-12 days) or (the more early whichsoever) chamber of experimentizing assessment when withdrawing from when treatment finishes in screening.Finish (10-12 days) in treatment and carry out clinical observation and the assessment of daily record card.At the 2nd day to the 9th day, the 17th day with the 52nd day treatment on carry out the patient and meet with.For getting into the participation standard, carry out the mensuration of clostridium difficile toxin.For the experimenter who MCC treatment is not made a response with in the situation of clinical recurrence, carry out clostridium difficile toxin and measure and cultivate.Also when the experimenter who treatment is not made a response withdraws from, carry out clinical, laboratory and microbiology assessment.In first day and the last day of administration, after preceding 0.5 hour of administration and administration, taked the pharmacokinetics plasma sample in 2 hours.

Crucial participation standard. the experimenter is the patient who suffers from the diarrhoea relevant with difficile toxins; It is through following definition: the 1) diarrhoea (change of intestines custom; Wherein in 24 hours, have 3 or the ight soil of moulding not more frequently; Or in 36 hours, have surpass 6 times loose or watery stool) and 2) in ight soil, have the toxin A or a B of difficile toxins.

Crucial exclusion standard experimenter can not suffer from 1) serious or life-threatening CDAD, 2) and incoherent life-threatening of CDAD or serious disease, 3) use simultaneously: vancomyein, metronidazole, bacitracin, or related drugs.(if before the laboratory result of knowing the ight soil toxin, the investigator thinks the clinical begin treatment that is necessary, use so metronidazole and/or vancomyein how by 24 hours, but the treatment that is no more than 3 dosage is allowed); Be used to treat any medicine of CDAD; Or other microbiotic, 4) in the past three months, the history of the repeatedly recurrence of ulcerative colitis or segmental enteritis and CDAD (orientate as and surpass once recurrence).(experimenter who allows the recurrence with a CDAD participates in).

The period planning table of incident

The period planning table of the appraisal procedure during table 10. was studied in the 2A stage

^aIn first day and the last day of administration, using preceding 0.5 hour and using the back blood sample of taking to be used for pharmacokinetics in 2 hours

Terminal point. when treatment finished, the investigator confirmed whether the experimenter has been cured or failure.In addition, the time that diarrhoea is disappeared (be defined as disappear time loose or watery stool) to every day＜3 and the alleviating fully of the symptom of the 10th day CDAD of treatment (always alleviate fully is to disappear to every day≤3 time just to count, no matter be loose or closely; And the white cell or the stomachache that do not have fever, raise) as initial terminal point, and after treatment the recurrence (recurrence of diarrhoea is defined as every day 3 times or more times loose/watery stool, has positive toxotest) in 6 weeks as secondary endpoints.

Analyze

Secure groups:

Secure groups comprises to be accepted to study the single administration at least of medicine and have all experimenters at random that obtain security information.

Effective population:

In according to the patient of method treatment, confirm clinical success or failure.Being used to analyze the disappear colony of time of alleviating fully with symptom of diarrhoea is the colony (mITT) of improvement purpose treatment; It is made up of all experimenters at random; Said experimenter accepts at least one Research on dose medicine; Have the diarrhoea history, in 24 hours, have 3 times or just loose more frequently and at the positive clostridium difficile toxin of baseline.

The time that diarrhoea disappearsBe defined as from first time administration research medicine to time (representing) that diarrhoea disappears with fate; The time of in three treatment groups, relatively suffering from diarrhoea and disappearing.Closing day of will suffering from diarrhoea is defined as in 24 hours shapeless just (water sample or loose) that take place＜3 times first day, and the time length of treatment is extended to the 10th day.Use daily entry data of the present invention to come disappearing the 10th and the 12nd day assessment diarrhoea.

Alleviating fully of CDAD symptom:

Alleviating fully of the symptom of CDAD is defined as the defecation (like what in patient's daily record, write down) that disappears to every day≤3 time in the 10th day of research; And the relevant sign/symptom that does not have other is like fever (>=37.7 ℃), the WBC (the normal scope of experiment of WBC) of stomachache (in daily record, not have reaction) and rising.If there is any variable not mentioned, think that then this result is unknown.

The clinical recurrence rate:

The clinical recurrence rate was defined as after treatment in 6 weeks >=positive of 3 times shapeless just (loose or water sample) and Clostridium difficile toxin A or B is just.

The result

Participate in and demography

Following part is summed up participation and the complicate statistics characteristic of research colony in 1B-MD stage and 2A test.24 health volunteers participate in the 1B-MD conceptual phase altogether.Alternative masculinity and femininity experimenter participates in to be provided at the equity between the sex.The subject age scope is in 38-62 year (average 51.6 ± 7.5 years old), and body weight 55.5-90kg (average 71.5 ± 9.2kg), height 147-183cm (average 164.8 ± 10.8cm).

In the 2B conceptual phase, totally 49 experimenters participate in.Before accepting any research medicine, experimenter disagrees with and from research, withdraws from, therefore can not be by assessment security or effect.An experimenter (400mg administration group) had in 36 hours＞6 defecations, but at preceding 24 hours＜3 times defecations, and can not assess the time of disappearing of suffering from diarrhoea and still can assess clinical response and safety analysis.Behind administration 1 or 2 dosage, three patients are owing to disagree with (1 experimenter, 100mg administration group), and the microbiotic (1 experimenter, 100mg administration group) to pneumonia that need be other maybe can not be obeyed research medicine (1 experimenter, 200mg administration group) and stopped.Experimenter's complicate statistics is listed in the table 11.

Table 11. carries out the concise and to the point complicate statistics that the 2A stage studies; Be presented at 48 experimenters' in the colony that can assess security complicate statistics.

Attention: value representation experimenter's numeral, only if point out in addition

^aComprising of other: East India, India

^bThe body-mass index that calculates is defined as (body weight is a unit with kg)/(highly, with rice being unit) ²

Effect

Clinical assessment is treated successfully or when failing when treatment finishes; The investigator thinks has 2 patients (2/14) in low dosage administration group, 2 patients (2/15) being arranged in median dose administration group and in maximum dose level administration group, do not have patient (0/16) is that treatment is failed.In the successful experimenter of treatment (n=41), had in the dosed administration group at 100mg/ days and to recur CDAD (1/12) among the experimenter, in maximum dose level administration group, there is an experimenter to recur CDAD (1/16), recurrence rate is 2/41 (5%) altogether.This two examples recurrence all is to finish the back in treatment to take place in about 1 month.

^aIn the patient of clinical success, be evaluated at the recurrence of the positive diarrhoea of treatment back 6 all intracellular toxins

The timing definition that diarrhoea is disappeared is the diary card according to the patient, and the patient is disappeared and is less than 3 times the shapeless time just to every day.In mITT colony, the intermediate value time that alleviates, 200mg/ days was respectively 5.5 days with 400mg/ days treatment groups, 3.5 days and 3.0 days for MCC 100mg/ days.The average fate time that diarrhoea disappears is 6.3 ± 3.66 days in the experimenter of treatment in 100mg/ days, in the experimenter of treatment in 200mg/ days, is 4.8 ± 3.56 days, in the experimenter of treatment in 400mg/ days, is 3.6 ± 2.03 days.Between 100mg/ days and 200mg/ days treatment groups and between 200mg/ days and 400mg/ days treatment groups, there is not significant difference on the statistics in the time that diarrhoea disappears; Yet the difference between 100mg/ days and 400mg/ days treatment groups has significance,statistical (p=0.0506 KaplanMeier estimates to check with p=0.0503Kruskal-Wallis).

The time (mITT colony) that table 13. diarrhoea disappears, be defined as and disappear every day to＜3 shapeless times (according to patient's daily record card) just

^aBy the 10th day, its time loose experimenter just of not disappearing that suffers from diarrhoea to every day＜3

^bKaplan-Meier estimates

^cP value available from broad sense Wilcoxon check

The symptom of CDAD alleviates fully when in table 14, being shown to the treatment end; It was defined as the 10th day that studies; Total the defecation number of every day≤3 time is (no matter be shaped or shapeless; Such as in patient's daily record card record), and the WBC counting or the stomachache (according to the reaction of patient's daily record card) that do not have fever, raise.37.5% 100mg/ days treatment groups, 50.0% 400mg/ days treatment groups and 86.7% 400mg/ days treatment groups obtain to alleviate completely.It should be noted that the Most patients that did not alleviate fully in the 10th day remains treats successfully, and it was to the 17th day resolution of symptoms and do not need further treatment.Three patients that from research, withdraw from (disagree with for one, the microbiotic that needs are special, one can not oral pharmaceutical) also are listed in the row that do not alleviate fully.

Table 14. is in mITT colony; During to the treatment end; Alleviating fully of CDAD symptom, it is defined as the 10th day of research, and total defecation number of every day≤3 time (is shaped or shapeless; Such as on patient's daily record card record), the relevant sign/symptom that does not have other is like fever, stomachache and the WBC that raises

Have only 2 experimenters (1 experimenter in 100mg/ days treatment groups, 1 experimenter in 400mg/ days treatment groups) to experience clinical recurrence.

Security

In studying in the 1B-MD stage, MCC on all dosage by all experimenter's well tolerable.Reported 14 routine adverse events, 7 examples are in the 150mg group, and 2 examples are in the 450mg group, and 5 examples are in placebo.Adverse events is summed up as follows: headache (2), dizzy (1), weak (1), tired (1), nasal congestion (1), dysphagia (1), pharyngitis (1), conjunctivitis (1), upper respiratory tract infection (2), fash (1) and itch (1).Accept not have among the patient of MCC the patient to have the relevant spinoff of medicine.

In studying in the 2A stage; Such as in table 15 demonstration; In the treatment group 4/16 (25.0%) experimenter was arranged at 100mg/ days, 4/16 (25.0%) experimenter was arranged in the treatment group at 200mg/ days, had 1/16 (6.3%) experimenter to be in the news under study for action in the treatment group at 400mg/ days and have at least one AE.In the infection of 100mg/ days treatment groups with infect the highest frequency (3/16 of having reported AEs in the system; 18.8% experimenter).There was 2/16 (12.5%) experimenter report to suffer from vascular disease at 100mg/ days in the treatment group, had 2/16 (12.5%) experimenter report to suffer from gastrointestinal illness at 200mg/ days in the treatment group.

The incidence of the spinoff of table 15. in the secure groups of 2A research is summed up through system's organ classification and preferred term

Attention: per-cent is the ratio of experimenter in such

5 experimenters it is reported to have SAEs (table 16) under study for action.In 100mg/ days treatment groups, an experimenter suffers from the diarrhoea of medium seriousness, and another experimenter suffers from the congestive heart failure (CHF) of severe exacerbation.In 200mg/ days treatment groups, an experimenter has serious staphylococcal septicemia and serious hematencephalon, and another experimenter has medium serious gastrointestinal hemorrhage, and third experimenter has medium serious pectoralgia.In MCC 400mg treatment group, there is not the experimenter to have SAE.Think that all SAEs are uncorrelated with the research medicine.

The incidence of the serious side effects of table 16. in the security colony of 2A research

^aThe date that the date of last administration research medicine deducts the research of administration for the first time medicine adds 1.

^bFollowing Calculation and Study fate: the date that the date of beginning deducts the administration first time of research medicine adds 1.

^cAssessment based on the investigator.

^dThe experimenter is dead.

Pharmacokinetics

Plasma concentration data

In studying in the 1B-MD stage, give use with multi-dose oral after, the plasma concns of MCC is usually less than the quantitative boundary of dosage range.

Only in from 12 samples of 6 experimenters, find detectable plasma concns.

In said 12 detectable concentration, only find 2 apparently higher than LLOQ, and other the LLOQ that seldom surpasses 5ng/mL.

Respectively at the 1st day, the 1st hour and only before the 10th day the 10th dosage, in experimenter 021, observe two kinds of concentration (11.1 and 48.0ng/mL).

It should be noted that 150mg dosage does not produce detectable concentration.

Because the low MCC blood plasma level in concentration range for the pharmacokinetics analysis, does not surpass the sufficient blood plasma data point of LLOQ.

In studying in the 2A stage, after multi-dose oral was used, the plasma concns major part of MCC was lower than the quantification boundary, but in many samples and many experimenters, had the increase of dose-dependently, had measurable plasma concns.

In 2/15 (13.3%) experimenter in MCC 100mg/ days treatment groups, in 9/16 (56.3%) experimenter in MCC200mg/ days treatment groups, in 13/17 (76.5%) experimenter of MCC 400mg/ days treatment groups, find detectable plasma concns.

Observable MCC concentration scope in MCC 100mg/ days treatment groups is 9.45 to 12.3ng/mL, MCC 200mg/ days treatment groups 5.12 to 93.7ng/mL, and in MCC400mg/ days treatment groups 5.32 to 84.9ng/mL.

In the detectable MCC concentration of all treatment groups, most of (35/41; 85.4%) below 21ng/mL.

Only in 2 experimenters, observe MCC concentration, in 200mg/ days administration groups and at 400mg/ days, respectively have one in the administration group above 50ng/mL.

The uropoiesis of MCC is drained data

The MCC level of urine all is lower than and quantizes boundary (LLOQ=5ng/mL) in studying in the 1B-MD stage.

The excrement concentration data of MCC

Table 17 shows that it is normalized to 150mg dosage from the excrement concentration of 1B-MD research; Excrement MCC average out to 916.0 μ g/g (138.4-1768.9 μ g/g).

MCC excrement concentration during table 17 was studied in the 1B-MD stage, it is standardized as 150mg dosage

Study for the 2A stage, MCC 100mg/ days treatment groups (n=11 sample, competent), when treatment finishes, excrement MCC average out to 255.6 μ g/g (scope: 81.9-558.3 μ g/g).In MCC200mg/ days treatment group (n=9 sample, competent), excrement MCC average out to 441.7 μ g/g (scope: 11.7-786.7 μ g/g).In MCC 400mg/ days treatment group (n=13 sample, competent), excrement MCC average out to 1433.3 μ g/g (scope: 389.0-3974.8 μ g/g).

When treatment finished during table 18. was studied in the 2A stage, the excrement concentration of MCC

Conclusion

Generally speaking, this research be presented at many behind the repeatedly oral dosage of 450mg MCC obtained high level at action site, and in the relevant diarrhoea of treatment difficile toxins, show promising result by well tolerable.

This research also finds 1) in arbitrary research, not have the spinoff of treatment-appearance possibly be that medicine is relevant, 2) after multi-dose oral is used, in blood plasma, detect low MCC level, its major part is lower than the quantification boundary.Because low plasma concns does not detect complete MCC in the urine that 1B-MD research is collected.3) opposite, the liquid dung in two researchs is flat very high, surpasses the MIC to difficile toxins ₉₀(0.125 μ g/mL) 10; 000 times; 4) in 45 experimenters that carry out the full course of treatment treatment, before treatment or when treatment finished in 10 days, have only 4 experimenters to be considered to failure; 2 experimenters are arranged in 50-mg q12hr dose groups, in 100-mg q12hr dose groups, have 2 experimenters to be considered to fail.The record (0/16) that in 200-mg q12hr dosage, does not have failure, 5) after successfully treating, have only 2 experimenters to observe recurrence.About the treatment end two recurrences, 6 were arranged in back one month) although be not that statistics is significant, a kind of trend of intermediate value time display that diarrhoea stops, it shows that higher dosage maybe be more effective.Time of stopping of diarrhoea confirms it is 5.5 days for 50-mg q 12hr dose groups, is 3.5 days and is 3.0 days for 200-mg q 12hr dose groups for 100-mg q 12hr dose groups.

Embodiment 3.MCC is optionally effective to difficile toxins in vivo, and does not influence anaerobism caprophyl crowd's main member: this is crucial for lower recurrence rate.

Have optionally active in vivo to difficile toxins in order to test MCC; And for normal anaerobism caprophyl crowd can be the hypothesis of relative deficiency (sparing); On serial excrement sample, carry out quantitative excrement and cultivate, said excrement sample is available from the patient of the 2A stage dosage range clinical experiment (being called MCC now) that gets into MCC.The optimization antibiotic therapy of difficile toxins diarrhoea should be eliminated the nourishing body form of pathogenic agent, but keeps the staple that (spare) possibly be responsible for the normal microflora of colonization resistance.

Method

Patient (n=32) accepts semidiurnal 50,100 or the MCC of 200mg at random, continues to accept 10 days.24 patients did not receive treatment in the past; 8 patients accept the standard care of 1 or 2 dosage.As the ecology contrast, 7 other patients were treated 10 days for four times on the one with vancomyein 125mg.Cultivate 10 ^-2,4,6,8Fresh excrement sample, to obtain difficile toxins nourishing body and spore form; Cytotoxin B through raji cell assay Raji test excrement filtrating.When beginning research and the 10th day, inspection dilution 10 ^-3,5,7,9The main flora of aerobic and anaerobism caprophyl crowd culture change.Because bacteroid all living creatures thing ubiquity and be educable belongs to the indicator of selecting as the microorganism species integrity with this.

Detailed method shows below:

1) the single center research in Calgary healthy district accumulation area (Calgary Health Region catchment area), colony～100 ten thousand

2) randomized open mark, administration in 2A stage research range, the per 12 hours more oral 50mg that are used for the CDAD treatment, 100mg or 200mg continue 10 days.

3) after end was raised in experiment, the patient with the independent ecological control group of four times on the one treatments of vancomyein 125mg treated 10 days, and as the contrast of treatment/ecology, said patient is qualified for this experiment.

4) light to moderate CDAD: in when beginning research,＞3 diarrhoea sample/24 hour still＜12, WBC＜30,000/mm are had a fever＜39 ℃ in positive clostridium difficile toxin test ³, there is not vomiting, there is not serious abdominal discomfort

5) initial CDAD or only recurrence outbreak for the first time.

6) if possible, do not carry out treatment in the past.This scheme allow many to 3 at preceding standard care dosage, but for this assessment, allow the standard care of maximum 2 dosage.In this research colony, 24 patients did not carry out treatment in the past.

7) do not carry out parenteral antibiotic therapy simultaneously for any illness.

8) serial excrement sample: except the diagnosis sample of beginning, in when beginning research, in research beginning the last the 4th day, the 7th day, the 10th day, the 14th day, the 21st day, the 28th day with the repeated collection sample that obtained the excrement of＞5 grams (normally 10-30 restrains) on the 42nd day

9) for this report, the difficile toxins counting of comparison the 0th day and the 10th day excrement and the main quantity result's who normally settles down the flora genus variation.

10) confirm the excrement filter liquor concentration of difficile toxins quantitative counting and difficile toxins cytotoxin B with the fresh sample that goes down to posterity through the HeLa raji cell assay Raji because freezing be disadvantageous for the quantitative counting of confirming difficile toxins.

11), and possibly be one of the staple of giving the normal microflora of " settling down resistance " because think bacteroid all living creatures thing ubiquity and be high counting in the experimenter, with this group as the index that suppresses anaerobism caprophyl crowd.For the patient of recovery that can not type of demonstration flora crowd species at 10 days, the sample of processing subsequent is to write down the time of this group recovery.If sample is not handled immediately, aliquots containig is chilled in 15% glycerine/brain heart infusion nutrient solution (Brain Heart Infusion Broth) at-80 ℃, be used to further handle.

12) substratum cultivated of anaerobic bacteria flora and method be based on Wadsworth-KTL anaerobism handbook, and the 6th edition, 2002.Through on cycloserine cefoxitin fructose egg yolk agar with diluted sample to 10 ^-2,4,6,8/Gram excrement weight in wet base is confirmed the difficile toxins counting.Through with the aliquots containig of isopyknic 100% Ethanol Treatment excrement 1 hour, centrifugal, wash 2 times and resuspension is confirmed spore quantity with quantitative counting.

13) use MacConkey, BAP, the m-enterococcosel agar, Lab M anaerobism blood agar, BAP, BBE, KVLB, PEA agar is with 10 ^-3,5,7,9Dilute, carried out before the initial inspection incubation 48 hours, and incubation quantizes normal flora culture over 7 days again.

14) for the ecological contrast of vancomyein, use clostridium perfringens, confirm vancomyein excrement filter liquor concentration in triplicate through biological assay as indicator organism.

15) use the wilcoxon coupling, at log ₁₀Transform the back and confirm the difference in the microorganism count.The result

When the research beginning, the average log of difficile toxins ₁₀CFU ± SD nourishing body counting (all MCC patients) is 6.8 ± 3.6, scope 2-10.95; At the 10th day, except accepting the patient of 50mg, every other patient's difficile toxins quantitative counting minimizing＜2log ₁₀/ gm excrement.Vancomyein is effective similarly.In when beginning research, in every group of patient of 1/3, bacteroid crowd counting is＜3,3-8, and 8.5-10log ₁₀CFU/gm, wherein normal counting be＞11.Variation in the bacteroid crowd is presented in the table 19.

The log of table 19. bacteroid crowd counting/gram excrement weight in wet base ₁₀MV ± SD of CFU

^*Wilcoxon matched pairs signed rank test (wilcoxon matched pairs signed-ranks test), two tails;

^*Counting＜3log ₁₀=2.90

Following accompanying drawing also illustrates the result from this research.

Conclusion

Based on quantitative bacteroid crowd counting; The patient who suffers from difficile toxins diarrhoea is when the research beginning; Has impaired changeably normal microflora; Wherein about 1/3 in the 3log10CFU/gm scope, 1/3 in 4-7log10CFU counting, and remainingly has a higher counting (not in normal 11-12log10CFU scope).As if the dosage of all MCC reduces the quantity of difficile toxins, as vancomyein.The dosage of not observing with MCC increases, and the dose-dependently of bacteroid counting reduces.Vancomyein seriously reduces the bacteroid counting in therapeutic process, although and their counting of Most patients recovery, only a few still lacks in the time that prolongs.

As if based on these data and the clinical effectiveness that show high responsiveness and low recurrence rate, the 200mg dosage of MCC is the dosage that is fit to that carries out further clinical study.

The invention is not restricted to above-mentioned embodiment, said embodiment only exists as the purpose of giving an example, but its can in protection domain, make amendment in every way, said protection domain is limited accompanying Patent right requirement.

Claims (10)

1. the compound of an isolating formula IX (OP-1435):

2. pharmaceutical composition, it comprises the compound of claim 1.

3. the pharmaceutical composition of claim 2, it also comprises the compound (platform colludes mycin B) of formula II:

4. the pharmaceutical composition of claim 3, it also comprises the compound (lipiarmycin A4) of formula XIV:

5. the application that each compsn is used to prepare medicine among the claim 2-4, said medicine are used for disease or the illness that treatment is caused by the existence of difficile toxins (Clostridium difficile).

6. the application of claim 5, wherein said compsn are in 3-15 days, and with the amount of about 50mg to about 1000mg, 1-3 time use every day.

7. the application of claim 6, wherein said compsn with about 100mg to the amount of about 400mg once a day or twice ground use.

8. the application of claim 7, wherein said compsn be with the amount of about 200mg, once a day or twice ground use.

9. the application of claim 5, thus wherein said compsn use by this way and make in the patient, the plasma concns of the compound of formula II is lower than 5ng/mL.

10. the application of claim 5, thus wherein said compsn use by this way and make in the patient, the urine concentration of the compound of formula II is lower than 5ng/mL.

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