JP2009512691A - Treatment of Clostridium difficile-related diarrhea - Google Patents

Abstract

Clostridium species, Staphylococcus species, Enterococcus species, and combinations thereof, comprising administering to a patient in need thereof an effective amount of a mixture comprising at least one of thiacumicin B, lipirmycin A4, and other macrocyclic compounds A method of treating a disease or disorder caused by the presence of a bacterium selected from the group consisting of:
[Selection figure] None

Description

Related Applications This application claims the benefit from US provisional application serial number 60 / 729,135 filed on October 21, 2005 and US provisional application serial number 60 / 749,641 filed on December 12, 2005. This application claims the benefit of US provisional application serial number 60 / 399,956, filed July 29, 2002, and claims the benefit of international application number PCT / US2003 / 021977, filed July 15, 2003. This is a continuation-in-part of US patent application serial number 10 / 520,863 filed on January 11, 2005. This application is a continuation of international application number PCT / US05 / 16750 filed on May 13, 2005, claiming the benefit of US provisional application serial number 60 / 570,687 filed on May 14, 2004 It is an application. This application is a continuation-in-part of International Application No. PCT / US05 / 02887 filed on January 31, 2005.

  The above referenced application is incorporated herein by reference in its entirety.

Background of the Invention The present invention relates to diseases caused by the presence of bacteria selected from the group consisting of Clostridium species, Staphylococcus species and Enterococcus species and combinations thereof, in particular Clostridium difficile ("C. difficile"), Clostridium Diseases caused by the presence of bacteria selected from the group consisting of perfringens (“C. perfringens”), Staphylococcus aureus (“S. aureus”) and combinations thereof, specifically diseases caused by the presence of C. difficile Related to the treatment. The disease can be colitis, pseudomembranous colitis or diarrhea.

2. Description of Related Fields Antibiotic-associated diarrhea (AAD) is caused by toxin-producing strains of S. aureus and Clostridium perfringens (C. perfringens), including C. difficile, methicillin-resistant Staphylococcus aureus (MRSA). ADD represents a major economic burden in a health care system that is conservatively estimated at $ 3-6 billion per year of excess hospital costs in the United States alone.

  ADD is an important issue in hospitals and long-term care facilities and communities. C. difficile is the most common cause of ADD in hospital settings, accounting for approximately 20% of cases with ADD and the majority of cases with antibiotic-associated colitis (AAC). The rising incidence of C. difficile-related diarrhea (CDAD) is due to frequent prescribing of broad-spectrum antibiotics to hospitalized patients [Wilcox et al., Lancet 1996, 348: 767-8].

  The most severe form of the disease is pseudomembranous colitis (PMC), histologically manifested as colitis with mucosal plaques, clinically manifested by severe diarrhea, abdominal cramps and systemic toxicity It is. Overall mortality from CDAD is low, but very high in patients with severe colitis or systemic toxicity symptoms. Recent studies have shown that mortality in patients with CDAD is very high compared to case-matched controls, even when death is not a direct cause of C. difficile.

  Diarrhea and colitis are caused by the production of one or more C. difficile toxins. The organism grows in the large intestine of patients who have been given broad spectrum antibiotics or are less frequently receiving cancer chemotherapy. CDAD is diagnosed in approximately 20% of hospital patients who develop diarrhea after treatment with such drugs.

  Current treatment of AAD or CDAD includes discontinuation of the relevant antibiotic or chemotherapeutic agent, nonspecific supportive measures, and treatment with direct antibiotics against C. difficile. The most common antibiotic treatment options include vancomycin and metronidazole. Treatment of CDAD with antibiotics is associated with clinical recurrence of the disease. The frequency of recurrence is reported to be 5-50%, with a recurrence rate of 20-30%, the most commonly quoted number. Recurrence occurs almost equally, regardless of the drug, dose, or duration of the primary treatment with any of the above antibiotics. A major challenge in treatment is the management of patients with multiple relapses, where antibiotic control is a problem.

  Although vancomycin is the only FDA approved drug for this application, the two most commonly used specific therapies are vancomycin and metronidazole. However, vancomycin is not recommended as a first-line treatment for CDAD, mainly because it is the only antibiotic active against several serious life-threatening multidrug resistant bacteria. Therefore, in order to minimize the emergence of vancomycin-resistant Enterococcus (VRE) or vancomycin-resistant Staphylococcus aureus (VRSA), the medical community is not using this drug unless it is absolutely necessary.

  Metronidazole is recommended as an initial treatment because of the fear of vancomycin-resistant gut microflora (especially enterococci) and fear of sputum. Despite reports that C. difficile resistance is> 6% in some countries, metronidazole is almost as effective as vancomycin, is fairly cheap, and is used either orally or intravenously be able to. Metronidazole is associated with notable side effects, including nausea, neuropathy, leukopenia, seizures and addictive responses to alcohol. Furthermore, it is not safe for use in children or pregnant women.

  Although both drugs are effective in treating infections, increasing rates of treatment failure in early responding patients and recurrence of diarrhea at approximately 20% are deficiencies in standard treatment. Treatment with metronidazole has been reported to be a significant risk factor for VRE colonization and infection. Furthermore, current therapies are rather cumbersome and require qids of up to 500 mg for 10-14 days. Therefore, there is a need for better treatment for CDAD cases and other AAD and AAC cases.

  Therefore, developing a bactericidal drug that has a low tendency to develop resistance and has reduced cross-resistance or non-cross-resistance and / or long-term post-antibiotic effects against existing antimicrobial agents There is a need for that.

およびそれらの組合せからなる群から選択される大環式化合物(macrocycle)をさらに含む。式XIVの化合物が存在するとき該混合物は約0.1〜約5％の式XIVの化合物を含む。 SUMMARY OF THE INVENTION The present invention relates to a disease caused by the presence of a bacterium selected from the group consisting of Clostridium species, Staphylococcus species, Enterococcus species, and combinations thereof, comprising administering an effective amount of a mixture to a patient in need thereof. Or provide a method of treating a disorder. The mixture comprises an effective amount of tiacumicin B, as well as the following compound:

And a macrocycle selected from the group consisting of combinations thereof. When the compound of formula XIV is present, the mixture comprises from about 0.1 to about 5% of the compound of formula XIV.

  Preferably, the mixture comprises at least 90% by weight tiacumicin B. More preferably, the mixture comprises at least 95% by weight tiacumicin B.

  Preferably, the mixture further comprises at least 1 wt%, more preferably from about 2 wt% to about 5 wt% macrocyclic compound.

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

の少なくとも１つを含む。 Preferably, when lipirmycin A4 is present, the mixture also contains the following compound:

At least one of the following.

  Preferably, the mixture exhibits an HPLC profile substantially as shown in FIG.

  Preferably, the disease or disorder treated according to the present invention relates to C. difficile, C. perfringens, S. aureus and combinations thereof. More preferably, the disease or disorder treated according to the present invention relates to C. difficile.

  Preferably, the disease to be treated according to the present invention is diarrhea or colitis, in particular diarrhea, more particularly CDAD.

Preferably, the mixture according to the invention has the following:
Produced by a method comprising culturing microorganisms in a nutrient medium comprising an adsorbent for adsorbing the mixture, accumulating the mixture in the nutrient medium; and isolating the mixture from the nutrient medium .

The nutrient medium preferably contains 0.5-15% by weight of adsorbent. The adsorbent is preferably an adsorbent resin. More preferably, the adsorption resin is selected from the group consisting of Amberlite® XAD16, XAD16HP, XAD2, XAD7HP, XAD1180, XAD1600, IRC50 and Duolite® XAD761. The microorganism is preferably Dactylosporangium aurantiacum subspecies hamdenensis. The nutrient medium, 0.2 wt% to 10 wt% of glucose, 0.02% to 0.5 wt% of K ₂ HPO _4, 0.02% to 0.5% by weight of MgSO ₄ · 7H ₂ O, 0.01 wt% to 0.3 wt% KCl, 0.1% to 2% by weight of CaCO _3, 0.05 wt% to 2 wt% of casamino acid, 0.05 wt% to 2 wt% of yeast extract, and 0.5 wt% to 15 wt% of XAD-16 resin . The fermentation step is preferably performed at a temperature of about 25 to about 35 ° C. and a pH of about 6.0 to about 8.0.

  Preferably, the disease to be treated according to the present invention relates to the use of antibiotics or cancer chemotherapy or antiviral therapy.

  According to one preferred embodiment, the mixture comprises an amount of about 50 mg to 1000 mg, more preferably 100 mg to 400 mg, especially 200 mg, 1 to 3 times a day, more preferably once or twice a day. In particular twice a day for 3-15 days, in particular for about 10 days. Oral administration is preferred.

  The treatment of the present invention is associated with enterotoxigenic strains of C. difficile, S. aureus, C. perfringens without compromising systemic antibiotics and without increasing intestinal vancomycin-resistant enterococci (VRE) Can enable effective treatment of diarrhea. The present invention also reduces the presence of VRE in the intestine.

  Other objects and features of the present invention will become apparent from the following detailed description considered in conjunction with the accompanying drawings. It should be understood, however, that the drawings are intended for illustrative purposes only and not as a definition of the limitations of the invention, which should serve as a reference to the appended claims. It is further understood that unless otherwise indicated, the drawings are not necessarily to scale, but are merely intended to conceptually describe the structures and methods described herein. Should.

Brief Description of the Drawings In the drawings,

  FIG. 1 shows the Phase 1B-MD medication schedule.

  FIG. 2 shows the total number of bacteroids after treatment. Two-tailed signed rank test. For a total number <3 log 10, a value of 2.9 was used.

  FIG. 3 shows the effect of vancomycin treatment vs. B. fragilis group.

  FIG. 4 shows a quantitative decrease in the total number of C. difficile growth after treatment with MCC.

  FIG. 5 is a typical HPLC profile of a mixture that can be used in the method of the present invention.

DETAILED DESCRIPTION OF CURRENTLY PREFERRED EMBODIMENTS Some abbreviations or term definitions used in the present invention are given as follows:
AAD = Antibiotic-related diarrhea
ATCC = American Type Culture Collection
¹³ C = carbon 13
CO ₂ = carbon dioxide
N ₂ = Nitrogen
H ₂ = hydrogen
TAPS = N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid
MOPS = 3- (N-morpholino) propanesulfonic acid
CDAD = Clostridium difficile related diarrhea
CLSI = Clinical and Laboratory Standards Institute, former NCCLS
ED ₅₀ = effective dose that produces a 50% response
HPLC = high performance liquid chromatography
IR = infrared spectroscopy
LLOQ = lower limit of quantification
MCC = macrocyclic compound-containing composition
MIC = minimum inhibitory concentration
MIC ₅₀ = minimum inhibitory concentration to prevent 50% of the bacterial strains tested
MIC ₉₀ = minimum inhibitory concentration to inhibit 90% of the bacterial strains tested
MRSA = methicillin resistant Staphylococcus aureus
NCCLS = National Committee for Clinical Laboratory Standards, now CLSI
PMC = pseudomembranous colitis
VRE = vancomycin-resistant enterococci
VRSA = Vancomycin resistant Staphylococcus aureus

  The term “antibiotic-related condition” occurs when antibiotic treatment disrupts the balance of the gut microbiota, and pathogens such as enterotoxin-producing strains of C. difficile, S. aureus and C. perfringens The state that can breed. These microorganisms cause diarrhea, pseudomembranous colitis, and colitis and are manifested by fever in diarrhea, urgency, abdominal cramps, tenesmus, and other symptoms. When severe, diarrhea causes dehydration and medical complications associated with dehydration.

  The term “MCC” means a formulation (eg, at least 90%, preferably 95% to 98% by HPLC analysis) that contains mainly tiacumicin B relative to the total antibiotic. The MCC also includes a small amount (eg, at least 1%, preferably 2% to 5%) of at least one of thiacumicin B related compounds, ie, ripiermycin A4 and the compounds of formulas III-XIV described above. PCT application PCT / US03 / 21977 having the international publication number WO 2004/014295 A2 provides a method of making a mixture comprising thiacumicin B. The entire contents of this PCT application are incorporated herein by reference. However, MCCs intended exclusively for non-human use may contain less than 80% thiacumicin B (relative to total antibiotics by HPLC analysis).

  The term “excipient” means an inert substance added to a pharmacological composition to further facilitate administration of a compound. Examples of excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars and various types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

  The term “halogen” means F, Cl, Br and I.

  The term “macrocyclic compound” means an organic molecule having a macrocyclic structure, usually containing 10 or more atoms.

  The term “18-membered macrocyclic compound” means an organic molecule having a ring structure containing 18 atoms.

  The term “membered ring” can encompass any cyclic structure, including the above carbocycles and heterocycles. The term “membered” is intended to indicate the number of skeletal atoms that constitute the ring. Thus, for example, pyridine, pyran and thiopyran are 6-membered rings and pyrrole, furan and thiophene are 5-membered rings.

  The term “MIC” or “minimum inhibitory concentration” means the minimum concentration of antibiotic required to inhibit the growth of bacterial isolates in vitro. A common method for determining the MIC of an antibiotic is to prepare several tubes containing serial dilutions of the antibiotic and then inoculate it with a bacterial isolate of interest. After incubation at the appropriate atmosphere and temperature, the MIC of the antibiotic can be determined from the tube with the lowest concentration that does not show turbidity (growth).

The term “MIC ₅₀ ” means the minimum concentration of antibiotic required to prevent 50% growth of the bacterial strain being tested within a given bacterial species.

The term “MIC ₉₀ ” means the minimum concentration of antibiotic required to prevent 90% growth of the bacterial strain being tested within a given bacterial species.

  The term “patient” means a human or animal in need of medical treatment. For the purposes of the present invention, human patients are usually housed in primary medical facilities such as hospitals or nursing homes. However, treatment of illnesses associated with the use of antibiotics or cancer chemotherapy or antiviral treatment can be performed on an outpatient basis, on discharge from a primary health facility, or at home treatment rather than a physician associated with the primary health facility. May be prescribed by a doctor for. Animals in need of medical treatment are usually under the control of a veterinarian.

  The term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable carrier or diluent.

The term “pharmaceutically acceptable salts” refers to salts derived from pharmaceutically acceptable inorganic and organic bases. Salts derived from appropriate bases include alkali metals (eg, sodium or potassium), alkaline earth metals (eg, magnesium), ammonium and N (C ₁ -C ₄ alkyl) ₄ ⁺ salts and the like. Some examples of them include sodium hydroxide, potassium hydroxide, choline hydroxide, sodium carbonate and the like.

  The term “pharmaceutical composition” refers to one or more thiacumicins described herein or physiologically acceptable salts thereof and other physiologically acceptable carriers and / or excipients such as excipients. It means a mixture with chemical components. The purpose of a pharmaceutical composition is to facilitate administration of a compound to a living body.

  The term “physiologically acceptable carrier” means a carrier or diluent that does not cause significant irritation to an organism and does not exclude the biological activity and properties of the administered compound.

  The terms “pseudomembranous colitis” or “enteritis” refer to the formation of pseudomembranous material (ie, a material composed of fibrin, mucosa, necrotic epithelial cells and leukocytes) due to inflammation of the mucosa of both the small and large intestines.

で示される18員の大環式化合物を含む全ての化合物のファミリーを意味する。 As used herein, the term “tiacumin” has the following formula I:

Means a family of all compounds, including the 18-membered macrocyclic compound represented by

で示される18員の大環式化合物を意味する。 As used herein, the term “tiacumin B” has the following formula II:

Means a 18-membered macrocyclic compound represented by

で示される18員の大環式化合物を意味する。 As used herein, the term “Ripirmycin A4” has the following formula XIV:

Means a 18-membered macrocyclic compound represented by

According to one embodiment of the present invention, low MCC levels were detected in plasma after multiple oral administrations, most of which were below the limit of quantification. In contrast, fecal levels in the two studies were very high, exceeding 10,000 times the MIC ₉₀ (0.125 μg / mL) for C. difficile.

  According to one embodiment of the invention, the recurrence of C. difficile-related diarrhea in a patient is effective in inhibiting the recurrence of C. difficile and does not affect the normal gut microbiota of the patient. And can be inhibited by administering MCC.

  According to one embodiment, the daily oral dose of MCC to CDAD is in the range of about 50 mg to about 1.0 g of active agent per day, preferably in the range of about 100 mg to about 600 mg per day. I will. Generally, treatment will continue for a period ranging from about 3 days to about 15 days. Greater or lesser drug amounts and treatment intervals can be used as needed. For example, according to the results of the clinical studies described herein below, a dose of about 100-400 mg of MCC per day over about 10 days is effective in treating CDAD with minimal clinical recurrence. Proved to be

  According to one embodiment of the invention, the mixture can be made by the following general method.

  MCC producing bacteria were cultured in vessels ranging from shake flasks to large “batch” fermenters. For production of large amounts of MCC, submerged aerobic fermentation in tanks is used. However, a small amount can be obtained by shaking flask culture. For tank fermentation, it is preferable to use a vegetative inoculum. A nutritional inoculum is produced by inoculating a small volume of media or lyophilized pellets with microbial spore forms, mycelial fragments to obtain a fresh and active growth culture of the microorganism. The nutrient inoculum is then transferred to a larger tank where, after a suitable incubation time, the MCC antibiotic is produced in a greatly improved yield. If foaming is a problem, it may be necessary to add a small amount of antifoam to a large amount of fermentation medium.

  The manufacture proceeds in a control medium with other additives / components to improve production. For production of MCC, a deep liquid, stirred culture method is used. Fermentation is performed at a temperature in the range of 25 ° C to 37 ° C. Carbon source consumption is carefully monitored and additional amounts of carbon source are added as needed. The pH of the fermentation is preferably maintained between about 6.0 and about 8.0. After fermentation inoculation, MCC is produced and accumulated in 3-15 days.

  It has been found that commercially available adsorption resins increase the yield and recovery efficiency of MCC during fermentation. The adsorbent is preferably present in the range of 0.5 to 15% by weight. MCC was recovered from the fermentation broth in an unusual yield (> 100 mg / L broth) by resin adsorption and eluted from the resin and mycelium by washing with various polar solvents.

  MCC was first captured from the broth during fermentation using an adsorbent resin such as Amberlite resin (XAD-16). At the end of the fermentation, the solid mass (including the adsorbent resin) is separated from the broth by sieving. The solid mass is eluted with ethyl acetate and then concentrated under reduced pressure.

At the end of the fermentation, the solid mass (including the adsorbent resin) is separated from the broth by sieving. MCC is eluted from the resin using ethyl acetate, methanol, acetonitrile or a mixture of two or more organic solvents. The extract is then concentrated under reduced pressure. This residue may be triturated with a low polarity solvent such as hexane, heptane, methylcyclohexane, or ethyl acetate / water; a two-phase solvent system such as ethyl acetate / aqueous sodium chloride solution; various ratios of methanol / hexane, acetonitrile Further purification by partitioning between / hexane or other mixtures and combinations of two or more solvents, or column chromatography eluting with a suitable organic solvent system. Current purification methods for MCC are medium pressure reverse phase (C-18) columns using 50: 50: 1 CH ₃ CN / H ₂ O / AcOH or 70: 30: 1 MeOH / H ₂ O / AcOH as eluents. Based on. Fractions containing the desired MCC were washed with saline and concentrated. The residue is dissolved in ethyl acetate, washed with water and the organic layer is evaporated to dryness to give a pale yellow foam which is washed again with isopropyl alcohol and dried under reduced pressure to give a white powder. Obtained. Combine fractions with purity> 88%. Concentrate the fraction to half of its original volume. The precipitate is filtered and the filter cake is washed with water. The solid was dried in high vacuum overnight to give a white powder that was analyzed by HPLC. Usually, the mixture contains at least one or more of thiacomycin B, lipearmycin A4 (0.1% to 5%), and the aforementioned macrocyclic compounds of formulas III to XIV in the range of 90% to 99% as main components.

  The following examples are provided for the purpose of describing specific embodiments of the present invention, in no way intended to limit the scope of the invention.

The mixtures used in the following examples are produced according to the production method described above. The following table shows the composition of some typical mixtures made in accordance with the present invention.

  HPLC analysis is performed according to the following method.

ここで、R_u＝分析品から得られるチアクミシンＢのピーク面積
R_s＝標準品から得られるチアクミシンＢのピーク面積
P＝水ファクターを含む、参照標準の純度
W_std＝標準品の重量（mg）
StdDil＝標準品の希釈量（mL）
W_sam＝試料の重量（mg）
WF＝試料の水ファクター。
希釈液に由来するピークは破棄し、個々の全関連物質のw/wパーセントを次の式で計算する：

ここで、R_i＝試料品から得られる関連物質のピーク面積
R_u＝試料品から得られるチアクミシンＢのピーク面積
RF_i＝関連物質のレスポンスファクター（全ての関連物質に対してRFi＝1.0）。 Mobile phase A: Add 2.0 mL of trifluoroacetic acid to 2 L of HPLC water, filter and degas.
Mobile phase B: Add 1.0 mL of trifluoroacetic acid to 2 L of acetonitrile, filter and degas.
Column: 4.6 × 150 mm column containing octylsilane chemically bonded to porous silica or ceramic particulates 3-10 μm in diameter (eg Zorbax Eclipse XDB-C8, 3.5 μm).
Detector: 230 nm
Flow rate: About 1.0 mL / min Injection volume: About 10 μL
Operation time: Approximately 10μL
Diluent: 100% acetonitrile Gradient program: Time (min)% Mobile phase A Mobile phase B
0 60 40
3.0 50 50
14.0 39 61
14.5 60 40
Note: The retention time of the mixture must be within 8-12 minutes.
Standard: Weigh accurately about 20 mg of mixture into a 100 mL volumetric flask, dissolve in diluent, and dilute to volume with diluent.
Sample: Weigh accurately about 20 mg of mixture into a 100 mL volumetric flask. Add about 60 mL of diluent and swirl to dissolve. Dilute to volume with diluent and mix.
System suitability: Chromatographic standards as directed in the procedure and record peak response. The relative standard deviation of the peak area of tiacumicin B for 5 repeated injections is NMT 2.0%, and the tailing factor of tiacumicin B area is NMT 2.0.
Procedure: Inject approximately 10 μL of diluent. Separately, inject equal volumes (approximately 10 μL) of standard and sample samples, record the chromatogram, and measure the detector response to the main peak.
Relative retention time:
Comparative Substance RT Ratio Compound of Formula II (Tiacumicin B) 1.0
Compound of formula III 0.71
Compound of formula IV 0.81
Compound of formula V 0.84
Compound of formula VI (Tiacumin F) 0.92
Compound of formula VII (Tiacumicin C) 0.95
Compound of formula VIII (Tiacumicin A) 1.10
Compound of formula IX 1.13
Compound of formula X 1.19
Compound of formula XI 1.24
Compound of formula XII 1.39
Compound of formula XIII 1.48
Compound of formula XIV (Ripirmycin A4) 0.89
Calculation: Calculate the analytical value using the following formula:

Here, the peak area of tiacumicin B obtained from R _u = analysis products
R _s = peak area of thiacumicin B obtained from standard product
P = purity of reference standard, including water factor
W _std = weight of standard product (mg)
StdDil = Standard product dilution (mL)
W _sam = sample weight (mg)
WF = sample water factor.
Discard the peaks from the diluent and calculate the w / w percent of all individual related substances with the following formula:

Where R _i = peak area of the related substance obtained from the sample
R _u = peak area of thiacumicin B obtained from the sample
RF _i = response factor of related substances (RFi = 1.0 for all related substances).

  Furthermore, a typical HPLC profile of a mixture according to the invention is shown in FIG. Compounds contained in the mixture, such as compounds of formula II-XIV, can be detected in the HPLC profile based on their RT ratio. Par-101 in FIG. 5 represents thiacumicin B having an RT ratio of 1.0.

  The mixture (50 mg) is then mixed with Avicel PH 102, FMC (microcrystalline cellulose) 100 mg in a size 1 capsule shell.

Example 1. Effects of inoculation, pH and cations on in vitro activity of MCC against Clostridium difficile MIC values measured for many antibiotics are such as pH, concentration of divalent cations such as calcium and magnesium, and bacterial density It is known to be affected by environment variables. The dependence of antibacterial activity on these factors is an important consideration, especially for antibiotics that target bacteria in the gut, where these factors can vary greatly in diet and pathology.

The sensitivity of MIC to these environmental variables can also be an important factor to consider when planning future in vitro testing methods. The Clinical and Laboratory Standards Institute, CLSI (formerly NCCLS) recommends the use of Brucella agar supplemented with vitamin K ₁ and hemin for determining the minimum inhibitory concentration (MIC) of anaerobic bacteria . However, the level of divalent cations in this medium is not standardized. Furthermore, the pH of the medium used under the anaerobic glove box can also change under different gas mixtures. In general, anaerobic bacteria are incubated in a mixture of nitrogen, hydrogen and carbon dioxide, and the presence of CO ₂ will acidify the medium and can be a significant source of variation. The size of the inoculum can also result in a variety of atmospheric conditions (H ₂ / CO ₂ generator, evacuation / replacement method, or anaerobic chamber) available for anaerobic susceptibility testing, making standardization difficult. The anaerobic conditions available in each laboratory determine the duration of microbial exposure to an aerobic atmosphere and the anaerobic equilibrium during operation on the workbench, and thus the viability of the culture and It will affect the experimental results.

  In this study, we investigated the effects of divalent cation calcium and magnesium levels, pH (5-10), inoculum density (over 3 orders of magnitude) on MIC, and lots of Brucella broth The effect of variation on MIC was also tested.

Materials and Methods Bacterial strains:

  Experimental strains of Clostridium difficile 9689, 700057, 43255, 17857 and Eubacterium lentum 43055 were obtained from the American Type Culture Collection (ATCC). All strains were streaked from refrigerated storage maintained at −78 ° C. in 10% glycerol before use to Brucella agar plates supplemented with vitamin K and hemin.

MIC test The current CLSI procedure (4) for anaerobic broth and agar dilution was used for MIC evaluation. Broth dilution is not a valid method for Clostridium's MIC test, but because of the potential inaccuracy of measuring the pH of solid agar after equilibration in an anaerobic chamber, both were used to evaluate the effect of pH. The method was used for comparison.

Influence of inoculum density on MIC values
The effect of inoculum density on the sensitivity of C. difficile to MCC and vancomycin was measured using the agar dilution method (4). The inoculum first makes a suspension of ~ 10 ⁸ cfu / mL and then serially dilutes the suspension by a factor of 10 to obtain a culture density in the range of 10 ⁵ to 10 ⁸ cfu / mL is prepared by, it gave 10 ² ~10 ⁵ cfu / spot of the spot density.

Effect of pH on MIC values:
The sensitivity of C. difficile to MCC was evaluated at a pH range of 6-8 using both the agar dilution method and the micro broth dilution method.

Using the agar dilution method, the MCC MIC was measured for C. difficile strains in a range of 6.2-8.0 in two separate experiments. To obtain the desired anaerobic pH for susceptibility testing, a buffer (100 mM NaH ₂ PO ₄ or TAPS [N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid]) is added to pH 7 respectively. And 8 to the medium. Even in a strong buffer, the pH shifted slightly after equilibration in anaerobic gas, and in some cases, the media was titered in the open air to the desired anaerobic pH. The actual pH was always confirmed after equilibration in the anaerobic chamber. Vancomycin used as a control was tested only at pH 7.

Using the broth microdilution method, MCC and vancomycin MIC values were measured in 3 independent series at pH ranging from 6 to 8 for C. difficile strains. In the first series, unbuffered Brucella broth was titrated in ambient air to obtain a pH in the range of 5-9. However, anaerobic equilibration of the medium in the glove box environment (10% H ₂ /5% CO ₂ /85% N ₂ ) lowered the pH of the medium, resulting in an anaerobic pH ranging from 5 to 7.5 (glove box (Tested with a portable pH meter with a flat bottom pH probe, calibrated with standard buffer outside and then transferred into). For the next experiment, buffer was added to the medium to suppress the pH shift caused by anaerobic equilibrium. In the second series, 10 mM buffer (NaH ₂ PO ₄ • H ₂ O pH 7.0, MOPS pH 8.0, or TAPS pH 9.0, pH value is Outside air) was added to the medium at a pH value greater than 6. In the third series, the buffer concentration was increased to 100 mM for pH treatment above 6 to obtain anaerobic pH in the range of 6-8.1.

Effect of divalent cation concentration on MIC value:
The agar dilution method was used to measure the effect of calcium and magnesium ion concentrations on the sensitivity of C. difficile strains to MCC. The level of divalent cations in Brucella broth obtained from the manufacturer was measured by the Laboratory Specialists, Inc. Additional amounts of divalent cations added (in the form of calcium chloride or magnesium chloride) to obtain calcium ion concentrations of 2.1, 3.0 and 5.7 mg / dL and magnesium ion concentrations of 3.3, 4.5 and 7.5 mg / dL It was.

Reproducibility of MCC MIC values using different commercially available lot media:
Using CLSI agar dilution method, the sensitivity of C. difficile to MCC was increased from BBL with different lots of vitamin K (Sigma lot # V-3501 and 0214010) and hemin (Sigma lot # 072K1221 and 034K7656) added Three different lots of Brucella agar (lots # 30768960, 211086 and 3167036) were also tested.

表2．MCCのインビトロ活性（μg/mL）対C. difficile ATCC 700057の異なる接種物密度（10²〜10⁵ CFU/スポット）

result
Effect of inoculum density on MIC values:
Tables 1 and 2 show the effect of inoculum density on MCC and vancomycin MICs for two strains of C. difficile (ATCC 9689 and ATCC 700057). The sensitivity of both C. difficile strains to MCC is 10 ⁵ to 10 ⁸ cfu / ml (10 ² to 10 ⁵⁾ , as indicated by the same MIC values obtained for all inoculum concentrations tested. CFU / spot) was not affected by the inoculum concentration. However, vancomycin MIC gradually increases with increasing inoculum concentration, showing a 4-fold increase in MIC at the highest inoculum density compared to the lowest inoculum density. These results indicate that inoculum density is not an important factor affecting the results of the C. difficile MCC susceptibility test.
table 1. In vitro activity of MCC (μg / mL) vs. different inoculum densities of C. difficile ATCC 9689 (10 ² to 10 ⁵ CFU / spot)

Table 2. In vitro activity of MCC (μg / mL) vs. different inoculum densities of C. difficile ATCC 700057 (10 ² to 10 ⁵ CFU / spot)

Effect of pH on MIC values:
Table 3 shows the effect of various pH values on the sensitivity of C. difficile to MCC as measured by the agar dilution method on two separate days. During the first run, the highest pH treatment (pH 7.9) for both C. difficile strains showed an 8-fold increase in MIC value compared to the lower pH treatment (pH 6.2 & pH 7.2). It was. When the validation run was repeated at the highest pH (pH 8.0), the MIC values for both strains remained high. No increase in MCC MIC was observed between pH 6.2 and pH 7 for either strain.

The increase in MIC value with pH did not correlate with the increase in growth. Thus, the effect of pH on the MIC appeared not only to be due to increased microbial viability at higher pH. Treatment at pH 7 had a lower density organic spot growth compared to pH 6.2 and pH 7.9 treatments.
Table 3. Effect of pH on agar dilution MIC values (buffered media)

表5．弱く緩衝された培地(10 mM)を用いたMICへのpHの影響

表6．強く緩衝された培地(100 mm)を用いたMICへのpHの影響

Tables 4, 5 and 6 represent MIC data from broth microdilution sensitivity methods performed on 3 independent days at pH ranging from 5 to 8.1. In the first series, where the medium was not buffered, for both C. difficile strains, the MCC MIC at pH 7.5 was 8 × the MIC at pH 5.9 (Table 4). MIC at pH 5 could not be measured. This is because microorganisms could not grow at this pH. Buffered (10 mM) pH 7.6 treatment showed 8- and 16-fold increases in MCC MIC over pH 6 treatment for each of C. difficile ATCC 9689 & ATCC 700057 (Table 5). ). In the third strong buffer (100 mM) series, similar results show that the highest pH treatment (pH 8.1) shows a 16-fold increase in MIC over the lowest pH treatment (pH 6) for both microorganisms. It was observed. Vancomycin showed a similar trend in all three experiments where the highest pH treatment yielded a MIC that was 4-8 times greater than the lowest pH treatment.
Table 4. Effect of pH on MIC using unbuffered medium.

Table 5. Effect of pH on MIC using weakly buffered medium (10 mM)

Table 6. Effect of pH on MIC using strongly buffered medium (100 mm)

  Analytical plates at all pH treatments were also visually examined for overall growth. In the first series where unbuffered broth was used, the overall culture turbidity increased with increasing pH. In the second series, where 10 mM buffered broth was used, the same trend was observed except that the culture turbidity was the same at pH 7.2 and pH 7.6. In the third series, the culture turbidity was more equivalent throughout the pH treatment except for the most turbid pH 7.5.

  Overall, through the two methods of susceptibility testing and changes in buffer salt concentrations, MCC and vancomycin MIC values increased with increasing pH for both strains of C. difficile.

表8．2価のカチオンの異なる濃度が追加されたBrucella寒天でのMCCのインビトロ活性

Effect of divalent cation concentration on MIC values:
Measurement of calcium and magnesium levels in commercial Brucella broth showed calcium and magnesium ion concentrations of 21 and 33 mg / L, respectively. Various additional amounts of divalent cations were added, and MCC MIC values for C. difficile strains were measured at three different calcium ion concentrations (21, 30 and 57 mg / L) and three different magnesium ion concentrations (33, 45 And 75 mg / L). The MIC value was the same for all types of media. In media with varying cation concentrations, C. difficile 9689 had a MIC value of 0.063 μg / ml and C. difficile 700057 had a MIC value of 0.125 μg / ml. As a control during the experiment, vancomycin tested as a control on added Brucella agar without any additional calcium or magnesium showed an expected MIC value of 1 μg / ml for all runs ( Tables 7 and 8).
Table 7. In vitro activity of MCC on Brucella agar supplemented with different concentrations of divalent cations

Table 8. In vitro activity of MCC on Brucella agar supplemented with different concentrations of divalent cations

MCC MIC values using different commercial lot media:
Three different lots of added Burcella agar were used on three independent days to compare the activity of MCC against the C. difficile strain. MIC analysis was controlled by testing the activity of the QC microorganism Eubacterium lentum against clindamycin within an acceptable range of CLSI (NCCLS), ie 0.06-0.25 μg / mL. Another control step for MIC analysis involves metronidazole and monitors its activity against C. difficile strains that have been shown in our laboratory to have MIC values ranging from 0.25 to 0.5 μg / mL Was that. As shown in Table 9, the activity of MCC against C. difficile was not affected by different lots of added Brucella agar. All controls showed activity within the established range.
Table 9. In vitro activity of MCC tested in three different lots of media

In contrast to the conclusion vancomycin, C. Activity of MCC is for difficile, inoculum concentration was unaffected in the range of 10 ² ~10 ⁵ cfu / spot.

  The sensitivity of C. difficile to MCC was not affected by cation concentrations (calcium ions ranging from 2.1 to 5.7 mg / dL and magnesium concentrations from 3.3 to 7.5 mg / dL) and various lots of commercially available media.

  The MIC values for both MCC and vancomycin increased with increasing pH in the pH range of 6-8. High MIC values at basic pH may be due to the deprotonated form of the phenolic hydroxy group of both compounds above their pKa, and where they are present in bacterial cells Forms charged species that are expected to be poorly permeable. In contrast, below pKa (7.22 versus MCC), the antibiotic will be almost protonated and therefore will more effectively permeate the cell membrane.

  In general, microbial density increased with increasing pH; the dependence of growth density, but not MIC, on pH decreased with the presence of buffering agents. Although microbial density clearly correlated with basicity in the absence of buffer, the MIC trend does not appear to be the result of the effect of pH on microbial density alone. This is observed in buffered experiments where the same relationship between MIC and pH was more comparable throughout pH treatment, although microbial density was probably due to differential effects of buffer type on microbial growth. Because it was done.

Example 2 Safety, pharmacokinetics and outcomes in healthy subjects and patients with Clostridium difficile-related diarrhea (ADAD) Phase 1B-MD
Summary This was an oral, multiple dose, double-blind, randomized placebo comparison, dose escalation study conducted at the University of Miami Division of Clinical Pharmacology in Miami, Florida. Richard Preston, MD was the study director of the trial. The tolerability and pharmacokinetics of multiple oral doses of MCC were evaluated in a total of 24 healthy volunteers. Oral doses of MCC evaluated (3 groups of 8 subjects each, 6 active and 2 placebo) at 150, 300 and 450 mg (in powder-filled capsules containing 50 mg of test drug), 10 consecutive Daily for a day after breakfast. Subjects were administered and monitored on a combined inpatient / outpatient basis. Subjects were again accommodated in the study facility on Day 0 and Day 9 of the 10 day dosing period and remained for up to 48 hours after each accommodation. Subjects were released on Day 2 and Day 11 after completion of scheduled events and treatments. During the outpatient period, subjects reported their taking to the research facility daily and stayed under observation for 3 hours.

  Serial blood, urine and fecal samples were taken at various time points / intervals during the multiple dose period. Plasma, urine and fecal MCC concentrations were measured for pharmacokinetic analysis. A follow-up study was scheduled on Day 17 of each study period before the subject exited the study. Subjects were closely monitored for the occurrence of any adverse events or abnormal laboratory findings throughout the treatment period and in follow-up studies. See Figure 1, Phase 1B-MD dosing schedule.

Phase 2A
Summary This was a dose determination study to select safe and effective doses for MCC. Subjects were randomly selected for 10 days, 100 mg / day (50 mg every 12 hours), 200 mg / day (100 mg every 12 hours) or 400 mg / day (200 mg every 12 hours) Followed by clinical evaluation. Subjects recorded all signs on a daily diary card. Particular attention was paid to the frequency and viscosity of stool, the presence of blood in the stool, and abdominal discomfort. Laboratory assessments were performed at screening for entry and at the end of treatment (Days 10-12) or withdrawal (whichever was earlier). Clinical observations and diary card evaluation were performed at the end of treatment (Days 10-12). Patient interviews were performed on Day 2, Day 17, and Day 52 procedures 9 days later. Analysis for Clostridium difficile toxin was performed for entry inclusion criteria. Both C. difficile toxin analysis and culture were performed on subjects who did not respond to MCC treatment and in clinical relapse events. Subjects who did not respond to treatment were also subjected to clinical, laboratory and microbiological evaluation upon exit. Pharmacokinetic plasma samples were taken 0.5 hours before dosing and 2 hours after dosing on the first and last day of dosing.

Important Inclusion Criteria Subjects included 1) diarrhea (changes in stool habits with more than 3 amorphous stools in 24 hours or more than 6 diarrhea-like or watery stools within 36 hours) and 2) during stool Patients with C. difficile-related diarrhea, defined by the presence of either C. difficile toxin A or B.

Important exclusion criteria Subjects include 1) severe or life-threatening CDAD, 2) life-threatening or severe CDAD-related disease, 3) vancomycin, metronidazole, bacitracin or related drugs (if any, investigator) If you feel clinical need to start treatment before knowing laboratory results for fecal toxins, treat with up to 24 hours with only 3 doses of metrolidazole and / or vancomycin Any drug used to treat CDAD; or other concurrent antibiotics; 4) history of ulcerative colitis or Crohn's disease and multiple recurrences of CDAD within the past 3 months (more than 1) Could not have) defined as recurrence. (Subjects with only one recurrence of CDAD were allowed to enroll.)
Schedule of events Table 10. Evaluation Procedure Schedule for Phase 2A Study

Endpoint At the end of treatment, the investigator determined whether the subject was cured or unsuccessful. In addition, time to resolution of diarrhea (defined as resolution to <3 diarrhea-like or watery stools per day) and complete elimination of symptoms of CDAD by treatment day 10 (complete elimination is diarrhea (Resolve to stool total ≤3 times per day, whether creepy or stiff; fever, elevated white blood cells, or lack of abdominal pain) was followed as the first endpoint and within 6 weeks after treatment Recurrence (recurrent diarrhea, defined as diarrhea-like / watery bowel movements with a positive toxin test, 3 or more times per day) was followed as the second endpoint.

Analytical safety population The safety population was to include all random subjects who received at least one dose of study drug and had available safety information.

Effective population Clinical success or failure was measured in patients treated per protocol. Populations analyzed for time to resolution of diarrhea and complete elimination of symptoms have received at least one dose of study drug at baseline, have a history of diarrhea, and more than 3 in 24 hours It meant improvement for the treatment population (mITT) consisting of all random subjects with diarrhea-like bowel movements and positive C. difficle toxin.

  Time to resolution of diarrhea was defined as the time (in days) from the first dose of study drug to resolution of diarrhea; the time to resolution of diarrhea was compared between the three treatment groups. The date of diarrhea stop was defined as the first day that <3 undefined stool (watery or diarrhea) occurred within 24 hours and continued for the period of treatment up to Day 10 of the study. Diarrhea resolution was assessed over 10-12 days using subject diary data.

Complete elimination of CDAD symptoms:
Complete elimination of symptoms of CDAD may be related to other relevant factors such as fever (≧ 37.7 ° C), abdominal pain (no response to diary) and increased WBC (normal coverage of WBC) by Day 10 of the trial. Defined as resolution to ≦ 3 defecations / day (recorded in patient's diary) with no signs / symptoms. If any variable was missing, this result was considered unknown.

Assessment of clinical recurrence:
Clinical recurrence was defined as ≧ 3 amorphous stool (diarrhea or watery) and positive for C. difficile toxin A or B within 6 weeks after treatment.

Results Registrants and Demographics

  The following sections summarize study population registrants and demographic characteristics in Phase 1B-MD and 2A trials. A total of 24 healthy individuals were enrolled in the Phase 1B-MD trial. Alternating male and female subjects were enrolled to equalize gender differences. Subjects ranged from 38 to 62 years of age (average 51.6 ± 7.5 years), 55.5 to 90 kg weight range (average 71.5 ± 9.2 kg) and 147 to 183 cm height range (average 164.8 ± 10.8 cm) Met.

  A total of 49 subjects were enrolled in the Phase 2B study. One subject withdrew consent and dropped out of the study prior to receiving any study drug and was not evaluated for either safety or efficacy. One subject (400 mg medication group) had> 6 bowel movements at 36 hours, but had <3 bowel movements in the previous 24 hours and could be evaluated for time to resolution of diarrhea Although not possible, it was evaluated for clinical response and safety analysis. Withdrawal of consent (single subject, 100 mg medication group), demand for additional antibiotics for pneumonia (single subject, 100 mg medication group), or inability to take study drug (single subject, 200 mg medication) Group), 3 patients were discontinued after 1 or 2 doses. Subject demographics are shown in Table 11.

Table 11. Demographics for the Phase 2A study summary; demographics are shown for 48 subjects in a population that can be evaluated for safety.

Efficacy In the clinical evaluation of successful or unsuccessful treatment at the end of treatment, 2 patients in the low-dose group (2/14), 2 patients in the medium-dose group (2/15), top 0 patients (0/16) in the medication group were considered unsuccessful by the investigator. Among subjects who were unsuccessful in treatment (n = 41), one subject in the 100 mg / day medication group (1/12) and one subject in the top medication group (1/16) CDAD relapsed with an overall recurrence rate of 2/41 (5%). Both recurrences occurred approximately one month after the end of treatment.

The time to resolution of diarrhea was defined by the patient's diary card as the time for the patient to resolve to less than 3 unshaped bowel movements per day. In the mITT population, the intermediate time to removal was 5.5 days, 3.5 days, and 3.0 days for the 100 mg / day, 200 mg / day, and 400 mg / day treatment groups of MCC, respectively. The mean time to resolution of diarrhea in days is 100 mg / day-6.3 ± 3.66 days in treated subjects, 200 mg / day-4.8 ± 3.56 days in treated subjects and 400 mg / day-3.6 ± 2.03 days in treated subjects Met. There was no statistically significant difference in time to resolution of diarrhea between the 100 mg / day and 200 mg / day treatment groups and between the 200 mg / day and 400 mg / day treatment groups; Differences between treatment groups at 400 mg / day reached statistical significance (Kaplan Meier estimate p = 0.0506 and Kruskal-Wallis test p = 0.0503).
Table 13. Time to resolution of diarrhea (mITT population), defined as time to resolve <3 undefined stool per day (by patient diary card)

By the 10th day of the study, ≤3 total bowels / day (whether regular or undefined as recorded on the patient's diary card), no fever, no increase in WBC count, Complete elimination of symptoms of CDAD is shown in Table 14 by the end of the treatment, defined as no abdominal pain (by responding to the patient's diary card). Complete removal was achieved in 37.5% of the 100 mg / day treatment group, 50.0% of the 200 mg / day treatment group and 86.7% of the 400 mg / day treatment group. It is noteworthy that the majority of patients who did not have complete removal by Day 10 were still treated successfully and the symptoms resolved by Day 17 and did not require further treatment. Three patients who dropped out of the study (one because of withdrawal of consent, one because of a request for excluded antibiotics and one because they cannot take oral medications) also had complete elimination. It is described as not.
Table 14. By Day 10 of the study, ≤3 total bowels / day (typical as noted on the patient's diary card) without other related signs / symptoms such as fever, abdominal pain and increased WBC Or complete elimination of CDAD symptoms by the end of treatment in the mITT population, defined as a solution to

  Only 2 subjects (1 in the 100 mg / day treatment group and 1 in the 400 mg / day treatment group) experienced clinical recurrence.

Safety In the Phase 1B-MD study, MCC was well tolerated by all subjects at all dosages. 14 adverse events were reported, 7 in the 150 mg group, 2 in the 450 mg group and 5 in the placebo group. Adverse events are summarized as follows: headache (2), dizziness (1), weakness (1), malaise (1), nasal congestion (1), difficulty swallowing (1), sore throat (1), Conjunctivitis (1), upper respiratory tract infection (2), rash (1) and psychogenic pruritus (1). Subjects undergoing MCC did not have adverse events that were considered related to the drug.

In the Phase 2A study, 4/16 (25.0%) subjects in the 100 mg / day treatment group, 4/16 (25.5%) subjects in the 200 mg / day treatment group and 400 mg / day as shown in Table 15. 1/16 (6.3%) subjects in the day treatment group reported at least one AE during the study. The most frequent AEs (3/16; 18.8% subjects) were reported in infections and parasitic infections in the body system of the 100 mg / day treatment group. There were 2/16 (12.5%) subjects who reported vascular disease in the 100 mg / day treatment group and 2/16 (12.5%) subjects who reported gastrointestinal disease in the 200 mg / day treatment group.
Table 15. Incidence of adverse events in the safety population of the 2A study, summarized by tissue organ class and preferred term

Five subjects were reported as having SAE during the study (Table 16). In the 100 mg / day treatment group, one subject had moderately severe diarrhea and the other subject had a severe exacerbation of congestive heart failure (CHF). In the 200 mg / day treatment group, one subject had severe staphylococcal sepsis and severe cerebral hemorrhage, the other subject had moderate severe gastrointestinal bleeding, and the third Subject had moderate severity chest pain. Subjects in the MCC 400 mg treatment group did not have SAE. All SAEs were considered independent of study drug.
Table 16. Incidence of serious adverse events in the safety population of the 2A study

Pharmacokinetic Plasma Concentration Data In the Phase 1B-MD study, after multiple oral doses, the plasma concentration of MCC was almost below the limit of quantification throughout the dose range.

  Detectable plasma concentrations were found only in 12 samples from 6 subjects.

  Of the 12 detectable concentrations, only 2 significantly exceeded LLOQ, while the others barely exceeded 5 ng / mL LLOQ.

  These two concentrations (11.1 and 48.0 ng / mL) were observed just before the 10th dosing of subject 021 Day 1, Hour 1 and Day 10, respectively.

  It should be noted that the 150 mg dose did not produce a detectable concentration.

  Due to low MCC plasma levels throughout the dose range, there were insufficient plasma data points beyond the LLOQ for pharmacokinetic analysis.

  In the Phase 2A study, after multiple oral doses, the plasma concentration of MCC was almost below the limit of quantification, but at a measurable concentration, there was a dose-dependent increase in the number of samples and the number of subjects. did.

  Detectable plasma concentrations of 2/15 (13.3%) subjects in the MCC 100 mg / day treatment group, 9/16 (56.3%) subjects in the MCC 200 mg / day treatment group, and MCC 400 mg / day treatment It was seen in 13/17 (76.5%) subjects in the group.

  Observable MCC concentrations range from 9.45 to 12.3 ng / mL in the MCC 100 mg / day treatment group, 5.12 to 93.7 ng / mL in the MCC 200 mg / day treatment group, and 5.32 to 84.9 ng in the MCC 400 mg / day treatment group / mL range.

  Of the detectable concentrations of MCC in all treatment groups, the majority (35/41; 85.4%) were less than 21 ng / mL.

  MCC concentrations above 50 ng / mL were observed in 1 subject each in the 200 mg / day and 400 mg / day dosing groups in only 2 subjects.

MCC Urinary Excretion Data All urinary MCC levels in the Phase 1B-MD study were below the limit of quantification (LLOQ = 5 ng / mL).

MCC Fecal Concentration Data Table 17 shows fecal concentrations normalized to a dose of 150 mg from the 1B-MD study; fecal MCC average 916.0 μg / g (138.4-1768.9 μg / g).
Table 17. MCC stool concentration normalized to a dose of 150 mg in Phase 1B-MD

  In the phase 2A study MCC 100 mg / day treatment group (n = 11 samples sufficient), fecal MCC averaged 255.6 μg / g (range: 81.9-558.3 μg / g) at the end of treatment . In the MCC 200 mg / day treatment group (n = 9 satisfactory samples), fecal MCC averaged 441.7 μg / g (range: 11.7-786.7 μg / g). In the MCC 400 mg / day treatment group (n = 13 satisfactory samples), fecal MCC averaged 1433.3 μg / g (range: 389.0-3974.8 μg / g).

Table 18. MCC stool concentration at the end of treatment in Phase 2A study

Conclusion In summary, this study shows that MCC is well tolerated after multiple oral doses up to 450 mg, achieving high levels at the site of action, and has promising results in the treatment of C. difficile-related diarrhea. Is shown.

The study also found the following: 1) There were no adverse events that occurred during treatment, either felt to be related to the drug in either study, 2) low after multiple dose oral administration MCC levels were detected in plasma and most of them were below the limit of quantification. As a result of the low plasma concentration, no intact MCC was detected in the collected urine of the 1B-MD study. 3) In contrast, stool levels in both studies were very high, with a MIC ₉₀ against C. difficile. 4) out of 45 subjects treated with a full course of therapy (2 subjects in the 50-mg q12hr dosing group and 2 subjects in the 100-mg q12h dosing group) Only) was considered unsuccessful before or at the end of 10 days of treatment. No success was observed with the 200-mg q12hr dosing (0/16), 5) only 2 subjects relapsed after successful treatment. Both relapsed approximately 1 month after the end of treatment.6) Although not statistically significant, the mean time to diarrhea cessation tends to suggest that higher dosages may be more effective. Indicated. The time to cessation of diarrhea was measured to be 5.5 days in the 50-mg q 12hr dosing group, 3.5 days in the 100-mg q 12hr dosing group, and 3.0 days in the 200-mg q 12hr dosing group.

Example 3 MCC is selectively effective against C. difficile in vivo and is the key to a lower relapse rate: it does not affect the major members of fecal anaerobic microflora.
To test the assumption that MCC is selectively active in vivo against C. difficile and can be relatively modest against fecal normal anaerobic microbiota Stool cultures were performed on continuous stool samples obtained from patients entering the phase 2A dose-variation clinical trial of MCC (currently planned MMC). Optimal antibiotic treatment of C. difficile diarrhea should extinguish proliferative pathogens and should not harm the main constituents of the normal microflora presumed to play a role in colonization resistance Absent.

Methods Patients (n = 32) were randomized and received 50, 100, or 200 mg MCC twice a day for 10 days. Twenty-four subjects had no prior treatment; eight subjects received one or two doses of standard treatment. As an ecological control, 7 additional patients were treated with vancomycin 125 mg qid for 10 days. Fresh stool samples were cultured at ^10-2 , ⁴ , ⁶ , ⁸ against C. difficile growth and spore types; stool filtrates were tested for toxin B by cell analysis. On study entry and day 10, aerobic and anaerobic stool microbiota cultures were tested for major microbiota shifts at 10 ^-3 , 5, ⁷ , ⁹ dilutions. Since the Bacteroides group of microorganisms is ubiquitous and can be cultured, this genus was chosen as an indicator of microbiota integrity.

The detailed method is as follows.
1) Single center trial in Calgary Health Region jurisdiction, population ~ 1 million
2) Random, open-label, dose-variation phase 2A trial comparing 10-day po MCC 50 mg, 100 mg or 200 mg Q 12-hour unit for treatment of CDAD
3) Following termination of study recruitment, independent ecological control patients who were otherwise eligible for the study were treated with vancomycin 125 mg QID for 10 days as treatment / ecological control.
4) Mild to moderate CDAD:> 3 at study entry but <12 diarrhea samples / 24 hours, positive C. difficile toxin assay, fever <39 ° C, WBC <30,000 / mm3, no vomiting, severe No abdominal discomfort
5) Primary CDAD or first onset of recurrent symptoms only
6) Simple treatment when possible. The protocol allowed up to 3 pre-medications of standard treatment, but this evaluation allowed a maximum of 2 medications of standard treatment. In this study population, 24 patients were simple treatments.
7) There is no concomitant parenteral antibiotic treatment for any condition.
8) Continuous stool sample: In addition to the first diagnostic sample, repeated collections of stool> 5 grams (usually 10-30 grams) are available at the time of study entry, day 4, 7, 10, 14, 21, 28 and after study entry. Held on the 42nd.
9) For this report, the results of day 0 and day 10 stool will be compared to the change in the total number of C. difficile and the total number of the main genus of the normal gut microbiota.
10) Use the newly excreted sample because the quantitative total number of C. difficile and fecal filtration concentration of C. difficile cytotoxin B by HeLa cell analysis is detrimental to the measurement of the quantitative total number of C. difficile. Measured.
11) Since the Bacteroides group of microorganisms is considered to be present in large numbers and evenly in the subject and is probably one of the main components of the normal microflora that confer “fixation resistance”, this group It was used as an indicator of suppression of fecal anaerobic microflora. Subsequent samples were processed to record the time of return of this group for patients who were unable to show a return of the Bacteroides group species in 10 days. If the sample was not processed immediately, an aliquot was frozen at −80 ° C. with 15% glycerol / Brain Heart Infusion Broth for later processing.
12) The medium and method for anaerobic microbiota culture is based on the Wadsworth-KTL Anaerobic Manual, 6th edition, 2002. The total number of C. difficile was determined by diluting samples of 10-2, ⁴ , ⁶ , ⁸ / fecal wet weight grams on CCFA agar. Spore count was determined by treating a fixed amount of stool with an equal volume of 100% ethyl alcohol x 1 hour, centrifuging, washing twice, and resuspension for quantitative counting.
13) normal flora cultures, MacConkey were incubated for 48 hours before the first test, BAP, m-Enerococcus agar, Lab M anaerobic blood agar, BAP, BBE, KVLB, using PEA agar 10 ^- Quantified by diluting ^3,5,7,9 and further incubation for up to 7 days.
14) For vancomycin ecological control, vancomycin fecal filtrate concentration was measured in triplicate by bioassay using C. perfringens as indicator bacterium.
15) The difference in total number of microorganisms was determined using Wilcoxon matched after Log ₁₀ conversion.

Results The mean log ₁₀ CFU ± SD (all MCC patients) of the total number of C. difficile proliferative forms at study entry was 6.8 ± 3.6, range 2-10.95; one who received 50 mg at day 10 All other patients had a quantitative total number of C. difficile that was reduced to <2 log ₁₀ / g stool. Vancomycin had a similar effect. The total number of Bacteroides group with normal total number> 11, when the test entry, in each one-third of the patients, was _{<3,3~8 & 8.5~10 log 10 CFU /} g. The shifts in the Bacteroides group are shown in Table 19.
Table 19. Log ₁₀ CFU of total number of Bacteroides group / mean stool wet weight g ± SD

  The following figure further illustrates the results of this test.

Conclusion Based on the total number of quantitative Bacteroides groups, patients with C. difficile diarrhea are about 1/3 in the 3 log10 CFU / g range, 1/3 in the total of 4-7 log10 CFU / g, higher In total there was a remainder (not in the normal range of 11-12 log10 DFU), which indefinitely impaired the normal microbiota at study entry. All dosages of MCC, like vancomycin, appear to reduce the total number of C. difficile. A dose-dependent decrease in the total number of Bacteroides with increasing MCC dosage was not observed. Vancomycin significantly reduces the total number of bacteroides during treatment, and most patients recover their total number, but a few continue to be deficient.

  Based on these data and clinical outcomes showing a high response rate with low recurrence rates, the 200 mg dose of MCC appears to be an appropriate dosage for further clinical trials.

  The invention is not limited by the embodiments described above which are presented by way of example only, but can be varied in various ways within the scope of protection defined by the appended patent claims.

It is a figure which shows a phase 1B-MD medication schedule. Figure 2 shows the total number of bacteroids after treatment (two-sided sign rank test; a value of 2.9 was used for a total number <3 log 10). FIG. 6 shows the effect of vancomycin treatment versus B. fragilis group. It is a figure which shows the quantity reduction | decrease of the total number of the growth of C. difficile after a treatment with MCC. 2 is a typical HPLC profile of a mixture that can be used in the method of the invention.

Claims (46)

An effective amount of thiacumicin B, as well as the following compounds:

And a macrocyclic compound selected from the group consisting of combinations thereof, wherein when the compound of formula XIV is present, the mixture comprises an effective amount of a mixture comprising from about 0.1% to about 5% by weight of the compound of formula XIV. A method of treating a disease or disorder caused by the presence of bacteria, comprising administering to a patient in need thereof. The method of claim 1, wherein the mixture comprises at least 90% by weight tiacumicin B. The method of claim 1, wherein the mixture comprises at least 95% by weight tiacumicin B. The method of claim 1, wherein the mixture comprises at least 98% by weight tiacumicin B. The method of claim 1, wherein the mixture further comprises a total of at least about 1% by weight of macrocyclic compounds. The method of claim 1, wherein the mixture further comprises a total of about 2% to about 5% of the macrocyclic compound. The method of claim 1, wherein the mixture exhibits an HPLC profile substantially as shown in FIG. The method of claim 1, wherein the mixture comprises from about 0.3 to about 5% by weight of a compound of formula XIV. The method of claim 1, wherein the mixture comprises from about 0.3 to about 3% by weight of the compound of formula XIV. The method of claim 1, wherein the mixture comprises from about 0.3 to about 1.5% by weight of the compound of formula XIV. The method of claim 1, wherein the mixture comprises about 1% by weight of a compound of formula XIV. The mixture is the following compound:

The method of claim 1, further comprising at least one of: 2. The method of claim 1, wherein the bacterium is selected from the group consisting of Clostridium species, Staphylococcus species, Enterococcus species, and combinations thereof. 2. The method of claim 1, wherein the bacterium is selected from the group consisting of C. difficile, C. perfringens, S. aureus and combinations thereof. 2. The method of claim 1, wherein the bacterium is C. difficile. 2. The method of claim 1, wherein the mixture does not substantially affect major members of the patient's anaerobic gastrointestinal microbiota. 2. The method of claim 1, wherein the rate of recurrence of the disorder or disease is substantially reduced. The method according to claim 1, wherein the disease is at least one of diarrhea and colitis. 2. The method of claim 1, wherein the disease is infectious diarrhea. 19. The method of claim 18, wherein the disease is Clostridium difficile-related diarrhea. By a method comprising culturing a microorganism in a nutrient medium comprising an adsorbent for adsorbing the mixture, accumulating the mixture in the nutrient medium; and isolating the mixture from the nutrient medium The method of claim 1, wherein the method is manufactured. The method of claim 21, wherein the nutrient medium comprises about 0.5 to about 15 wt% adsorbent. The method of claim 21, wherein the adsorbent is an adsorbent resin. 24. The method of claim 23, wherein the adsorption resin is selected from the group consisting of Amberlite (R) XAD16, XAD16HP, XAD2, XAD7HP, XAD1180, XAD1600, IRC50 and Duolite (R) XAD761. The method according to claim 21, wherein the microorganism is Dactylosporangium aurantiacum subspecies hamdenensis. 2. The method of claim 1, wherein the disease is associated with antibiotic use or cancer chemotherapy or antiviral therapy. 2. The method of claim 1, wherein the Staphylococcus species is a methicillin resistant Staphylococcus species. The method of claim 1, wherein the Staphylococcus species is methicillin resistant Staphylococcus aureus. 2. The method of claim 1, wherein the Enterococcus species is vancomycin resistant Enterococcus. The method of claim 1, wherein the mixture is administered in an amount of about 50 mg to 1000 mg, 1 to 3 times daily for 3 to 15 days. The method of claim 1, wherein the mixture is administered in an amount of about 100 mg to about 400 mg once or twice daily. The method of claim 1, wherein the mixture is administered in an amount of about 200 mg once a day. The method of claim 1, wherein the mixture is administered in an amount of about 200 mg twice a day. 2. The method of claim 1, wherein the plasma concentration of the mixture in the patient is administered to be below 5 ng / mL. The method of claim 1, wherein the concentration of the mixture is administered to be lower than 5 ng / mL in the patient's urine. Thiacmicin B, as well as the following compounds:

And a macrocyclic compound selected from the group consisting of combinations thereof and a pharmaceutical mixture comprising from about 0.1 to about 5% by weight of the compound of formula XIV, when the compound of formula XIV is present. 37. The mixture according to claim 36, comprising at least 90% by weight tiacumicin B. 37. The mixture of claim 36, comprising at least 95% by weight tiacumicin B. 37. The mixture of claim 36, further comprising a total of at least 1% by weight of a macrocyclic compound. 40. The mixture of claim 36, further comprising a total of at least about 2 wt% to about 5 wt% macrocyclic compound. 37. The mixture of claim 36, wherein the mixture exhibits an HPLC profile substantially as shown in FIG. 40. The mixture of claim 36, wherein the mixture comprises from about 0.3% to about 5% by weight of a compound of formula XIV. 40. The mixture of claim 36, wherein the mixture comprises from about 0.3% to about 3% by weight of a compound of formula XIV. 40. The mixture of claim 36, wherein the mixture comprises from about 0.3% to about 1.5% by weight of the compound of formula XIV. 37. The mixture of claim 36, wherein the mixture comprises about 1% by weight of a compound of formula XIV. The mixture is the following compound:

37. The mixture of claim 36, further comprising at least one of:

Images