WO2009145619A1

WO2009145619A1 - Antibiotic composition

Info

Publication number: WO2009145619A1
Application number: PCT/NL2009/050206
Authority: WO
Inventors: Peter Christian De Visser; Gerard Johannes Platenburg
Original assignee: Prosensa Holding Bv
Priority date: 2008-04-17
Filing date: 2009-04-17
Publication date: 2009-12-03

Abstract

The invention relates to an antibiotic composition comprising (i) an antibiotic sensitizer having formula X-Y, in which X is a hydrophobic group containing a backbone with at least 8 carbon atoms, wherein X may contain unsaturated bonds and additional substituents, and Y represents a redox-capable group, in any oxidation state; and (ii) an antibiotic, for use in inhibiting microbial, preferably bacterial, growth. Particularly preferred embodiments of the present invention are those wherein the sensitizer is selected from the group consisting of vitamin E, vitamin K and ubiquinone and their derivatives.In another preferred embodiment, the compositions according to the present invention comprises α-tocopherol and/or phylloquinone, and/or its subfamily members.

Description

ANTIBIOTIC COMPOSITION

FIELD OF THE INVENTION

The present invention is in the field of antimicrobial compositions, in particular in the field of antimicrobial compositions comprising an antibiotic and a compound that acts as an enhancer of the antimicrobial effect of the antibiotic. The invention is particularly useful in combating bacterial strains that are resistant to antibiotics.

BACKGROUND OF THE INVENTION During the last decades a dramatic increase in bacterial strains that are resistant to one or more antibiotics has been reported. This increase has led to the occurrence of bacterial infections that can hardly or not at all be treated with the existing spectrum of antibiotics, which is a particularly serious problem in connection with e.g. hospital- acquired infections and has led to an increase in bacterial infections with a fatal outcome. The emergence of antibiotic resistance appears to be a result of the incorrect use of antibiotics in human and veterinary medicine combined with efficient bacterial mutation machinery.

The bacterial cell wall is a feature where particular antibiotic resistance originates, as it prevents many antibiotics from reaching their targets inside the cell, and may contain antibiotic efflux pump systems. Furthermore, bacteria may produce antibiotic- hydrolyzing proteins (e.g. β-lactamases) that inactivate antibiotics. It is generally believed that if the bacterial membrane could only be rendered more permeable, the effect of antibiotics would be enhanced.

Many attempts have been made to find effective ways of permeabilizing the bacterial outer membrane. For instance, US 4,510,132 teaches the use of the polycationic polymyxin B and its derivatives to permeabilize the outer membrane of Gram-negative bacteria. US 6,165,997 discloses negatively charged phospholipids enhancing the activity of antimicrobials, but also having antimicrobial activity themselves. These compounds appear to act, at least partially, as cation chelators. Nakanishi (Igaku Kenyu 1993, 63, 95) reports on the antibacterial enhancing effect of administration of the vitamin C in bedsore patients. This effect is likely to be attributed to its antioxidant activity. However, vitamin C fails to produce results in our assay at desirably low level, as evidenced in the accompanying examples (vide infra), at a concentration of 20μM.

Byron et al. (Antimicrob. Agents Chemother. 2003, 47, 3357) found that millimolar concentrations of the sesquiterpenoid flavorant and aroma compounds nerolidol, farnesol, bisabolol, and apritone can nonspecifically enhance the permeability of bacterial cells to certain exogenous chemical compounds, including antimicrobial agents.

Jabra-Rizk et al. (Antimicrob. Agents Chemother. 2006, 50, 1463) describe that high concentrations of farnesol (150μM) sensitizes resistant Gram-positive S. aureus to antimicrobials, and enables enhancement of the antimicrobial efficacy of antibiotics in bio films; high concentration farnesol (lOOμM) combined with gentamicin (2.5 x MIC) decreased bacterial counts with a factor log 2.

These literature examples of sensitizing agents indicate that farnesol has limited sensitizing properties and applied in respectable amounts. Moreover, its effect on resistant bacteria appears limited. Hence, in the art a need exists to provide antimicrobial sensitizers which may be applied in low amounts and even in cases where resistance is reported.

Outside the field of sensitizers, WO-02/066006 discloses a sustained-release composition containing ceftiofur hydrochloride, tocopherol and a biocompatible oil. The tocopherol is used as an antioxidant, in preferred amounts ranging 10 - 50 wt%. US 6,479,540 discloses compositions of tocol-solubel therapeutics in which a tocol- soluble ion pair is formed. EP 535.446 discloses a pharmaceutical composition containing a chelating agent, a tocopherol and an antimicrobial agent. The tocopherol is present as an antioxidant, radical scavenging compound, and amounts to 0.5 - 15 wt% of the total composition. WO-2004/112805 discloses an antimicrobial composition comprising silver. Also, WO-2005/107740 discloses enhanced efficacy of tubercular drugs by combination with alpha-tocopherol. It maintains the Factor Alpha tx in a chemically reduced state, thus increasing Factor Alpha tx's antioxidant effect in vivo. None of the aforementioned publications discusses sensitizing properties.

SUMMARY OF THE INVENTION

It is an objective of the present invention to provide antimicrobial compositions. It is further an objective to provide antimicrobial compositions that are active towards microorganisms that are resistant to one or more antibiotics, in particular it is an objective to provide antimicrobial compositions that are active towards microorganisms that are resistant to one or more antibiotics comprising the one or more antibiotics to which the microorganisms are resistant. It is further an objective to provide antimicrobial compositions that have an enhanced activity of one or more antibiotics towards microorganisms as compared to the antibiotic(s) alone.

It was found that certain compounds having a hydrophobic alkyl chain, preferably containing at least 8 carbon atoms in linear fashion, either saturated or unsaturated, connected to a redox-capable group, were capable of rendering microorganisms susceptible to an antibiotic, whereas the antibiotic without these compounds was much less or not at all active against the microorganisms.

Conveniently, these compounds are non-toxic since they are naturally found in the host, and are presently contemplated not to cause problems with regard to allergy. In fact, these could be administered in any reasonable amount without negatively affecting the patient.

Although the inventors do no wish to be tied down to any theory, the compounds of the invention are thought to facilitate antibiotic uptake over the cell membranes of the microorganism. Due to their inherent lipophilicities, these compounds show an affinity for and partition with biological membranes, where their accumulation may impact substantially on the structural and functional properties of these membranes, thus making antimicrobial targets more accessible. Notably, the compounds of the invention are distinct from cholesterol and steroids. Due to its action, a compound according to the present invention may be referred to as an "antibiotic sensitizer", a well-known concept in the field of antibiotics.

In particular it is found that microorganisms, in particular bacteria, that are resistant to certain types of antibiotics, or at least not affected by antibiotics at acceptable dosages, now effectively could be stopped proliferating or in fact killed at acceptable concentrations of the antibiotic when this antibiotic is administered in the presence of a sensitizer in accordance with the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present compositions comprise a compound of formula (I):

X-Y (I),

in which:

X is a hydrophobic group containing a backbone with at least 8 carbon atoms, wherein X may contain unsaturated bonds and additional substituents, preferably isoprenyl units (either saturated, unsaturated or mixed) and Y represents a redox-capable group, preferably a quinone or phenolic moiety, in any oxidation state.

Preferably, X is an alkyl chain.

X preferably has a backbone of at least 8, more preferably at least 10 consecutive carbon atoms. More preferably, X contains at least 3 isoprenyl moieties, more preferably up to 10 isoprenyl moieties, either saturated or unsaturated, or combinations thereof. To guarantee its hydrophobic character, it is preferred that the backbone of X contains no heterogeneous atoms. More preferably, X contains at most 10 side-chain substituents (e.g. one per isoprenyl moiety), more preferably no heteroatoms.

The redox-capable group Y preferably contains one or more cyclic moieties, preferably at least one aromatic moiety. Y preferably contains at least two oxygen atoms. In its broadest aspect, the invention pertains to a composition comprising (i) a hydrophobic alkyl-containing compound as defined above, and (ii) an antibiotic, for use in inhibiting microbial growth. Throughout the specification and claims, the term (ii) "antibiotic" encompasses the use of multiple antibiotics, which may be applied for treating the same or different microorganisms. More specifically, the invention pertains to the above composition, wherein said hydrophobic alkyl-containing compound acts as a sensitizer, meaning that it renders microorganisms, in particular bacteria, susceptible to the action of the antibiotic or that it renders said microorganisms susceptible to the action of the antibiotic at a lower concentration or dosage of the antibiotic.

The compounds of the invention likely temporarily destabilize the microbial cell membrane and are considered essential for (human) life. Since they are naturally found in the host, they are presently not contemplated to be non-toxic nor to cause problems with regard to allergy. In fact, these could be administered in high doses without negatively affecting the patient.

Here below, the hydrophobic alkyl-containing compound of the invention will be addressed to as "the antibiotic sensitizer", thus referring to its use. It is not excluded that the compounds presently contemplated also exhibit antimicrobial activities themselves, without the presence of an antibiotic, albeit at far higher concentrations than opted for here. The current findings on "antibiotic sensitizing properties", i.e. the ability to permeabilize the, preferably bacterial, membrane to an antibiotic, brings additional advantages, including treating microorganisms having reduced susceptibility to antibiotics, or even microorganisms resistant to certain antibiotics. Although it is immediately clear from the remainder of the text and the term "antibiotic sensitizer", it is emphasized that the hydrophobic alkyl-containing compound is an active ingredient in the composition, included to enhance the activity of the antibiotic. This is reflected by the preferred (relative) amounts of the sensitizer.

By "inhibiting microbial growth" is understood to comprise the combat of infectious microbial disease, preferably bacterial disease, including treatment, prophylaxis, cytocidal, antimicrobial, preferably antibacterial, bacteriostatic and/or bacteriocidal effects. In one embodiment the sensitizer as described above contains at least 3 non-cyclized isoprenyl, saturated or unsaturated, or combinations thereof, and isomers and derivatives thereof, such as alcohol, acid, or amine forms and their addition salts, such as HCl, HBr, HF, H₃PO₄, H₂SO₄, citric acid, acetic acid, trifluoroacetic acid, lactic acid, isethionic acid, methanesulfonic acid or ethylenediamine tetraacetic acid addition salt or with cationic counter ions such as Mg²⁺, Ca²⁺, Na⁺, K⁺, Li⁺, NH₄ ⁺.

Formula (I) preferably encompasses compounds of all redox states selected from the group consisting of vitamin E (including, but not limited to α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, α-tocotrienol, β-tocotrienol, γ-tocotrienol, δ-tocotrienol and isomers and synthetic derivatives as tocopheryl acetate and succinate), vitamin K

(including, but not limited to, phylloquinone, menaquinone and isomers and synthetic derivatives), plastoquinone and ubiquinone (including, but not limited to coenzymes Qn, wherein n has the above-defined meaning; Q3, Q4, Q5, Q6, Q7, Q8, Q9 and QlO, isomers and synthetic derivatives thereof). "Plastoquinone and "ubiquinone" include their synthetic derivatives, such as alkylquinones.

Examples of compounds according to Formula (I) include, but are not limited to alkylascorbates, alkylsalicylates (e.g. anacardic acids such as ginkgolic acid), alkylresorcinols (e.g. binobol), alkylquinones (e.g. embelin, irisquinones, sorgoleone) and alkylcatechols (e.g. urushiols) in every redox state.

Particularly preferred embodiments of the present invention are those wherein the sensitizer is selected from the group consisting of vitamin E, vitamin K and ubiquinone and their derivatives. In another preferred embodiment, the compositions according to the present invention comprises α-tocopherol and/or phylloquinone, and/or its subfamily members.

In the most preferred embodiment, X is a hydrophobic group containing a backbone with at least 8 carbon atoms, wherein X may contain unsaturated bonds and additional substituents, and Y represents a redox-capable quinone or phenolic moeity, in any oxidation state, and wherein said compound (i) contains at least 3 non-cyclized isoprenyl units, saturated or unsaturated, or combinations thereof. Suitable examples, some of which are commercially available, but are not limited to lipophilic derivatives of carnosol, vitamin E, vitamin K, Gibb's reagent, gallic acid, flavonols, flavonoids, flavones, resveratrol, magnolol, ellagic acid, celastrol, lignans, BHT, catechols, chromanes, curcumins, carnosic acid, coenzyme Q, methoxatin and aporphines.

In the present compositions, preferably the sensitizer is present in an amount that is sufficient to enhance the effectiveness of the antibiotic.

By an effectiveness of an antibiotic is understood that the addition of the antibiotic to a culture medium inhibits growth of the inoculum such that the number of colony forming units (CFU) with the antibiotic is less than 30%, such as 20%, 15%, 10%, or 5% of the CFU without addition of the antibiotic. Preferably the addition of the antibiotic kills the inoculum such that the CFU is less than 70%, such as 60%, 50%, 40%, 30%, 20%, 10%, 5%, 2%, 1%, 0.1%, or 0.01% of the inoculum.

By an enhanced effect of an antibiotic is understood that either/both the minimum inhibitory concentration (MIC) or/and the minimum microbicidal, preferably bactericidal concentration (MBC) of the antibiotic without the sensitizer according to the present invention is decreased by at least 2-fold by the addition of said sensitizer. Preferably, the decrease is at least 2-fold, such as 4-fold, 10-fold or even more such as 20-fold, 50-fold or even 100-fold.

A person skilled in the art is able, based on routine experimentation, to determine what a suitable concentration of the sensitizer is, taking into account the particular sensitizer, the particular antibiotic and the extent to which enhanced effectiveness of the antibiotic is attained.

Administration of sensitizer(s) and antibiotic(s) in a certain composition can be done, independently from each other, systemically (such as i.p., s.c, i.m., i.v.) or topically. In one embodiment, the composition is suited for systemic administration. Furthermore, the sensitizer(s) can be administered before, after, or simultaneously with the antibiotic(s). A person skilled in the art is able to determine the best route of administration and time span between two components to obtain a maximum effect. It is preferred that the maximum time between administration of the first sensitizer and the antibiotic is less than 24 hours or more preferably, less than 10 hours.

As reported above, it is part of the invention that the amounts of sensitizer need not be high to enhance the effectiveness of the antibiotic(s). It is preferred that the molar ratio of sensitizer(s) of formula I to antibiotic(s) is attractively lower than 500:1, more preferably lower than 100:1, particularly at most 20:1.

Thus also the present compositions preferably comprise an antimicrobially, or antibiotically, effective amount of an antibiotic. From the above it follows that a skilled person is able, based on routine experimentation, to determine what a suitable concentration of the antibiotic is, taking into account the particular sensitizer, the particular antibiotic and the extent to which enhanced effectiveness of the antibiotic is attained. In one embodiment the composition of the invention includes the sensitizing compound is in an amount lower than its antimicrobially effective amount, i.e. in an amount lower than its own MIC (i.e. if no (other) antibiotic were present). More preferably, the amount of sensitizer is at least 2 times, preferably at least 5 times, more preferably at least 10 times lower than its MIC value. In one embodiment, it is preferred that the amount of sensitizer in the composition is at least 10^"3 its MIC value.

In one embodiment, the antibiotic consists of organic materials, thus excluding inorganic elements being an antibiotic or important part thereof.

In one embodiment the present composition comprises an antibiotic which is selected from the group consisting of β-lactams, (e.g. ampicillin, ceftazidime, meropenem), quinolones (e.g. norfloxacin, ciprofloxacin), glycopeptides (e.g. vancomycin), macrolides (e.g. erythromycin), oxazolidinones (e.g. linezolid), peptide antibiotics (e.g. magainin II, synercid), lipopeptides (e.g. polymyxins, bacitracin), nitroimidazoles (e.g. metronidazole), ansamycins (e.g. rifampin), azoles (e.g. fluconazole), D-cycloserine, lincosamides (e.g. clindamycin), mupirocin, streptogramins (e.g. dalfopristin, quinupristin), fosfomycin, aminoglycosides (e.g. gentamicin, tobramycin), sulfonamides (e.g. sulfomethoxazole), trimethoprim, tetracyclines (e.g. tigilcycline), novobiocin, chloramphenicol, platensimycin, monobactams and synthetic derivatives of these antibiotics.

More in particular, a (combination of) suitable antibiotic(s) to be used in combination with the sensitizer(s) is selected from the group consisting of glycopeptides (preferably vancomycin or teicoplanin), β-lactams, preferably penicillins, such as amdinocillin, ampicillin, amoxicillin, azlocillin, bacampicillin, benzathine penicillin G, carbenicillin, cloxacillin, cyclacillin, dicloxacillin, methicillin, mezlocillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, and ticarcillin; cephalosporins, such as the first generation drugs cefadroxil, cefazolin, cephalexin, cephalothin, cephapirin, and cephradine, the second generation drugs cefaclor, cefamandole, cefonicid, ceforanide, cefoxitin, and cefuroxime, or the third generation cephalosprins cefoperazone, cefotaxime, cefotetan, ceftazidime, ceftizoxime, ceftriaxone, and moxalactam; carbapenems such as imipenem; or monobactams such as aztreonam; further tetracyclines such as demeclo eye line, tigilcycline, doxycycline, methacycline, minocycline, and oxytetracycline; aminoglycosides such as amikacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, spectinomycin, streptomycin, and tobramycin; polymyxins such as colistin, colistimathate, and polymyxin B, and erythromycins and lincomycins and also sulfonamides such as sulfacytine, sulfadiazine, sulfisoxazole, sulfamethoxazole, sulfamethizole, and sulfapyridine; trimethoprim, quinolones, novobiocin, pyrimethamine, platensimycin and rifampin are also expected to have enhanced activity in the presence of the sensitizer in a composition according to the present invention.

The invention is particularly suitable in case the microorganism is to a large extent resistant to the antibiotic. The skilled person is aware of the meaning of the definition of "resistant", as can be found in guidelines produced by e.g. the Clinical and Laboratory Standards Institute (CLSI, formerly known as NCCLS) in the US, more in particular Performance Standards for antimicrobial susceptibility testing; NCCLS document MlOO, its contents incorporated by reference. In a preferred embodiment, the treatment involves inhibiting growth of a microorganism which is resistant to said antibiotic if it were administered in absence of the compound of the invention. The invention also concerns a composition of a sensitizer as defined above and an antibiotic together with a pharmaceutical acceptable carrier. Such a pharmaceutical composition may be in solid, semi-solid, liquid etc. form, which are for internal or external application such as a tablet, capsule, liquor, vapour, ointment, paste, spray etc. Systemic application is preferred. Formulation into a suitable form is well known to a person skilled in the art, see e.g., "Remington's Pharmaceutical Sciences" and "Encyclopedia of Pharmaceutical Technology".

In general the present compositions can be used to eradicate Gram negative and/or Gram positive bacteria from places where they are not desired. Thus the present invention also concerns the use of the present antimicrobial compositions for cleansing and/or sterilising of objects and areas. Preferably for this purpose the sensitizers as defined above and (an) antibiotic(s) and optionally further cleaning and/or sterilising agents are combined with a suitable carrier or diluent, such as water and/or (an) alcohol.

The present invention also concerns the use of sensitizers as defined above and (an) antibiotic(s) for the preparation of a medicament for the treatment of a microbial, preferably bacterial infection. In one embodiment the medicament is used for the treatment of infections by microorganisms, preferably bacteria, that are resistant or multi-resistant to certain specific antibiotics. Alternatively, the present invention also provides a method for treating microbial, preferably bacterial infections in a patient in need thereof, preferably a human being, the method comprising administering the composition of the invention to the patient. Systemic administration is preferred.

In one embodiment the present composition comprises an antibiotic that is effective against Gram-negative bacteria. Surprisingly, the sensitizing compounds of the present invention enable inhibiting Gram-negative bacteria growth using antibiotics otherwise intended to treat Gram-positive bacteria, and vice versa. Gram-positive and Gram- negative bacteria are differentiated by the Gram stain. Gram-positive species retain the primary stain (crystal violet) when treated with a decolourising agent (alcohol or acetone) whereas a Gram-negative bacterium loses the primary stain. The staining difference reflects the structural differences in the cell walls of Gram- negative and Gram-positive bacteria. The Gram-positive cell wall consists of a relatively thick peptidoglycan layer and teichoic acids whereas the Gram-negative cell wall consists of a relatively thin peptidoglycan layer, and an outer membrane consisting of a lipid bilayer containing phospholipids, lipopolysaccharide, lipoproteins and proteins.

In one embodiment the medicament is used for the treatment of infection by Gram- negative bacteria. In one embodiment the medicament is used for the treatment of infections by Escherichia spp, in particular E. coli, Haemophilus spp, in particular H. influenzae, Pseudomonas spp, in particular P. aeruginosa, Klebsiella, in particular K. pneumoniae, Enterobacter spp., Helicobacter spp, in particular Helicobacter pylori, Shigella spp, Salmonella spp, Yersinia spp, Campylobacter spp, Neisseria spp, Bordetella spp, Aeromonas spp, Burkholderia spp, Serratia spp, Vibrio spp, Proteus mirabilis, and Acinetobacter spp, in particular A. baumannii.

In one embodiment the medicament is used for the treatment of infection by Gram- positive bacteria. The medicament thus comprises an antibiotic that is effective against Gram-positive bacteria. In one embodiment the medicament is used for the treatment of infections by Staphylococcus spp, in particular methicillin-resistant Staphylococcus aureus, Streptococcus spp, Enterococcus spp, Listeria spp, Staphylococcus spp, Streptococcus spp, Clostridium spp, Bacillus spp, Enterococcus spp, Corynebacterium spp, Legionella spp, Mycobacterium spp.

The present invention also concerns the use of sensitizers of formula I as defined above and (an) antibiotic(s) for the preparation of a medicament for the treatment of a microbial, preferably bacterial infection, preferably in human beings, particularly to enhance the antimicrobial activity of the antibiotic(s). Alternatively, the present invention also provides a method for treating microbial, preferably bacterial infections in a patient in need thereof, preferably a human being, the method comprising administering the composition of the invention to the patient. Additionally, the invention also pertains to a kit of parts comprising: a) at least one sensitizer represented by formula I; and b) at least one antibiotic, intended for the treatment of a microbial, preferably bacterial infection, particularly to enhance the antimicrobial effect of the antibiotic.

The sensitizer and the antibiotic may comprise one or more additional features as defined above. The kit of parts is intended for sequential or simultaneous administration, wherein the administration routes for the sensitizer and the antibiotic may be the same or different. Therein, it is preferred to adapt the amount of sensitizer administered to enhance the effectiveness of the antibiotic. Means for achieving this are described above. In case of sequential administration, it is preferred that the time between administration of the sensitizer and the antibiotic(s) is less than 300 minutes.

EXAMPLES

Experimental procedure for assaying sensitizers

• SOLUTION An 8mM stock solution of natural essential hydrophobic compound as described above in ethanol was prepared for serial 2-fold dilution with ethanol.

• BACTERIA

The concentration of an overnight culture (16h, 37⁰C) of Ps eudomonas aeruginosa PAOl in 100% LB was determined by comparison with a calibration curve and diluted to IxIO⁵ CFLVmL with 100% LB. Likewise, cultures of the following clinical isolates (obtained at the Leiden University Medical Center) were prepared: Pseudomonas aeruginosa PA102582 (tobramycin-resistant) and PA5981 (ceftazidime-resistant).

• TEST

Using 96-well plates, concentrations of sensitizer (lμL of every ethanolic solution) were added to the wells containing 20μL P. aeruginosa (final concentration 1x10⁴ CFU/mL, OD55o=0.1) and antibiotic. The volume in the wells was adjusted to 200μL with 20% LB. As controls, 20% LB, bacteria+20% LB, bacteria+20% LB+antibiotic were included. In some cases the medium was changed to 100% LB (vide infra). After addition of sensitizer at t=0, the 96-well plate was covered (not airtight) and incubated at 37⁰C while shaking for 18h in a BioTek plate reader; OD550 was determined at least every lOmin for PAOl studies or once after 18h for clinical isolates. A minimum inhibitory concentration (MIC) value for a specific compound was determined as the lowest concentration at which, after 18h incubation, the OD550 value was comparable to that of the blank used.

Minimum bactericidal concentration (MBC) values were determined in a number of cases by transferring lOOμL of wells that showed no growth in the MIC determination assay above to a 15% agar/LB plate and culturing overnight at 37⁰C. The MBC value for a specific compound was determined as the lowest concentration at which no colonies were visible.

• Results

In the tables below, representative examples can be found for α-tocopherol and phylloquinone. The results for α-tocopherol and phylloquinone are considered representative for compounds having structures analogue thereto.

Table 1. Effect of sensitizers on P. aeruginosa PAOl in 20% LB in presence of non- lethal concentrations of antibiotics.

n/e - no effect

From Table 1 can be deduced that ampicillin is indeed inactive in the concentration applied (50μg/mL). Upon addition of a sensitizer however, the combination is able to inhibit bacterial growth of PAOl . To evaluate lowering activities of sensitizers on the MIC values of antibiotics, a range of concentrations were applied in combination with a fixed concentration sensitizer. For results, see Table 2 below.

Table 2. Sensitizing effect of non- inhibitory concentrations of sensitizers on P. aeruginosa PAOl in 20% LB in presence of non- lethal concentrations of antibiotics.

MIC

Sensitizer Cone. (μM) Antibiotic Range (μg/mL) (μg/mL) penicillin G 256-0.125 >256 α-tocopherol 1.25 penicillin G 256-0.125 8 ampicillin 200-0.098 100 α-tocopherol 1.25 ampicillin 200-0.098 3.125 α-tocopherol 10 ampicillin 200-0.098 0.78 phylloquinone 20 ampicillin 32-0.5 2 vancomycin 200-0.098 200 α-tocopherol 2.5 vancomycin 200-0.098 50 linezolid 200-0.098 >200 α-tocopherol 80 linezolid 200-0.098 6.25 α-tocopherol 10 linezolid 200-0.098 100 tobramycin 16-0.0075 0.50 α-tocopherol 1.25 tobramycin 16-0.0075 0.25

Especially interesting in the results depicted in Table 2 is the ability of the sensitizers to render PAOl more vulnerable to the action of the antibiotics vancomycin and linezolid, as these two antibiotics are prescribed only for treatment of Gram-positive bacteria due to their inactivity against Gram-negative bacteria. Interestingly, the hydrophilic antioxidant ascorbic acid did not show sensitizing activity at high concentration (80μM) in combination with tobramycin, whereas the lipophilic antioxidant tocopherol did lower the MIC value of tobramycin.

Table 3. Sensitizing effect of non- inhibitory concentrations of α-tocopherol on the MIC value of tobramycin against tobramycin-resistant P. aeruginosa PAl 02582 in 20% LB.

Sensitizer Cone. (μM) Antibiotic Range (μg/mL) MIC (μg/mL) tobramycin 256-0.125 64 α-tocopherol 1.25 tobramycin 256-0.125 32 Although apparently a small effect of this sensitizer, it is obvious that even only small concentrations of antibiotically inactive sensitizers can lower the MIC value of tobramycin against tobramycin-resistant PA102582, a clinical isolate, by a factor 2.

Table 4. Sensitizing effect of non- inhibitory concentrations of sensitizers on PA5981, a ceftazidime-resistant species (20% LB).

Sensitizer Range (μM) Antibiotic Cone. (μg/mL) MIC (μM) ceftazidime 50 n/e α-tocopherol 160-0.31 - - >160 α-tocopherol 160-0.31 ceftazidime 50 0.62 n/e - no effect

Table 5. Sensitizing effect of non- inhibitory concentrations of α-tocopherol on the MIC value of ceftazidime against PA5981 (20% LB).

Sensitizer Cone. (μM) Antibiotic Range (μg/mL) MIC (μg/mL) ceftazidime 200-0.098 25 α-tocopherol 1.25 ceftazidime 200-0.098 6.25

From Tables 4 and 5 it is apparent that α-tocopherol can render PA5981 vulnerable again to the action of ceftazidime already at low concentrations.

Table 6. Sensitizing effect of non- inhibitory concentrations of sensitizers on the MIC value of antibiotics against PAOl in 100% LB.

Sensitizer Cone. (μM) Antibiotic Range (μg/mL) MIC (μg/mL) α-tocopherol 20 - - n/e gentamicin 8-0.125 8 α-tocopherol 20 gentamicin 8-0.125 4 ampicillin 32-0.5 >32 α-tocopherol 10 ampicillin 32-0.5 4 n/e - no effect

Enriching the growth medium from 20% to 100% LB (Table 6) shows that sensitizing activity is only slightly lower than in 20% medium. Besides effect on the MIC value of antibiotics, said sensitizers can also (or, exclusively) have an effect on the MBC value of antibiotics. In Table 7, results are given on effects of α-tocopherol on the MBC value.

Table 7. Effect of sensitizer on MBC value (from Table 6 MIC results).

Sensitizer Cone. (μM) Antibiotic Range (μg/mL) MBC (μg/mL) ampicillin 32-0.5 >32 α-tocopherol 20 ampicillin 32-0.5 16

In the same manner as described above also methicillin-resistant Staphylococcus aureus (MRSA) is tested and is found to be sensitive towards methicillin when this is administered together with said sensitizers. Likewise, vancomycin-resistant Enterococcus faecalis (VRE) can be killed with vancomycin and other glycopeptides in presence of said sensitizers. Also, lower antibiotic dosages are found to be necessary for inhibiting growth of Acinetobacter spp, Escherichia spp, Helicobacter spp, Klebsiella spp, Staphylococcus spp, Streptococcus spp, Clostridium spp, Bacillus spp, Enterococcus spp, Corynebacterium spp, Legionella spp, Mycobacterium spp, Listeria spp in presence of said sensitizers.

Claims

1. An antibiotic composition comprising:

(i) a compound having formula (I): X-Y (I), in which:

X is a hydrophobic group containing a backbone with at least 8 carbon atoms, wherein X may contain unsaturated bonds and additional substituents, and Y represents a redox-capable quinone or phenolic moeity, in any oxidation state, wherein said compound (i) contains at least 3 non-cyclized isoprenyl units, saturated or unsaturated, or combinations thereof; and (ii) an antibiotic, for use in inhibiting microbial, preferably bacterial, growth.

2. The composition according to claim 1, wherein said compound acts as an antibiotic sensitizer.

3. The composition according to any one of the preceding claims, wherein said compound is selected from the group consisting of vitamin E, vitamin K and ubiquinone, and their isomers and (synthetic) derivatives.

4. The composition according to any one of the preceding claims, wherein the molar ratio of said compound to said antibiotic is lower than 500: 1.

5. The composition according to any one of the preceding claims, wherein said compound is present in an amount lower than its antimicrobially effective amount.

6. The composition according to any one of the preceding claims, wherein the micororganism is resistant to said antibiotic if it were administered in absence of said compound.

7. The composition according to any one of the preceding claims, for treating microbial disease.

8. The composition according to any one of the preceding claims, wherein said composition is in a form suitable for pulmonary delivery, optionally comprising one or more suitable carriers and/or diluents.

9. Use of an antimicrobial composition as a cleaning and/or sterilizing agent, wherein said antimicrobial composition comprises: (i) a compound having formula (I):

X-Y (I), in which:

X is a hydrophobic group containing a backbone with at least 8 carbon atoms, wherein X may contain unsaturated bonds and additional substituents, and

Y represents a redox-capable quinone or phenolic moiety, in any oxidation state, wherein said compound (i) contains at least 3 non-cyclized isoprenyl units, saturated or unsaturated, or combinations thereof; and (ii) an antibiotic,

(iii) optionally a carrier or diluent.

10. Use according to claim 9, wherein said compound (i) acts as an antibiotic sensitizer.

11. Kit of parts comprising: a) at least one compound represented by formula (I), X and Y having their meanings as defined in claim 1 ; and b) at least one antibiotic, for use in inhibiting microbial, preferably bacterial, growth.