ANTIMICROBIAL PHARMACEUTICAL COMPOSITIONS WITH MULTIPLE DRUG RESISTANCE INHIBITING PROPERTIES

Abstract

The invention provides a method for treating a microbial infection in an infected subject comprising administering to the subject an antimicrobial composition comprising a therapeutically effective amount of one or more lipophilic cation, and, optionally, another one or more antimicrobial compound or antimicrobial agent. The invention further provides a pharmaceutical composition with antimicrobial properties comprising a therapeutically effective amount of one or more lipophilic cation and another one or more antimicrobial compound or antimicrobial agent.

Claims

1. A method for treating a microbial infection in an infected subject comprising administering to the subject an antimicrobial composition comprising a therapeutically effective amount of one or more lipophilic cation of structural formula 1: ##STR00014## wherein A is hydrogen atom (H) or an antioxidant moiety having the following structure: ##STR00015## and/or reduced (quinole) form thereof, wherein m is an integer from 0 to 3; each Y is methyl; L is a linker group which is a straight or branched hydrocarbon chain which can be optionally substituted by one or more substituents and optionally contains one or more double or triple bonds; and B is a lipophilic cation, and a pharmacologically acceptable anion.

2. The method according to claim 1 wherein the one or more lipophilic cation is one or more of SkQ1, SkQ3, SkQ4, SkQ5, SkQR1, SkQRB1, SkQB1, SkQBP1, SkQT, SkQThy, SkQB, C.sub.nTPP (wherein n is from 5 to 12), C.sub.nR1 (wherein n is from 4 to 12), C.sub.nBerb (wherein n is from 4 to 12), and C.sub.nPalm (wherein n is from 4 to 12).

3. The method according to claim 1 or 2, wherein the microbial infection is caused by bacteria.

4. The method according to claim 3 wherein the microbial infection is caused by Gram-negative bacteria.

5. The method according to claim 3 wherein the disease is caused by Gram-positive bacteria.

6. The method according to claim 3 wherein the disease is caused by Gram-negative bacteria and Gram-positive bacteria.

7. The method according to claim 1 or 2, wherein the disease is caused by fungi.

8. The method according to claim 1 or 2, wherein the disease is caused by bacteria and fungi.

9. The method according to claim 1, 2, 3, 4, 5, 6, 7, or 8, further comprising administering to the subject another one or more antimicrobial compound or one or more antimicrobial agent.

10. The method according to claim 9, wherein the additional one or more antimicrobial compound is one or more antibacterial antibiotic.

11. The method according to claim 9, wherein the one or more additional antimicrobial compound is one or more antifungal compound.

12. The method according to claim 9, wherein the additional one or more antimicrobial agent is one or more antiseptic.

13. The method according to claim 9, wherein the additional one or more antimicrobial compound is one or more MDR pump inhibitor.

14. The method according to claim 9, wherein the additional one or more antimicrobial compound is one or more MDR pump substrate.

15. A pharmaceutical composition with antimicrobial properties comprising a therapeutically effective amount of one or more lipophilic cation of structural formula 1: ##STR00016## wherein A is hydrogen atom (H) or an antioxidant moiety having the following structure: ##STR00017## and/or reduced (quinole) form thereof wherein m is an integer from 0 to 3; each Y is methyl; L is a linker group which is straight or branched hydrocarbon chain which can be optionally substituted by one or more substituents and optionally contains one or more double or triple bonds; and B is a lipophilic cation, and a pharmacologically acceptable anion, and one or more additional antimicrobial compound or one or more antimicrobial agent.

16. The pharmaceutical composition according to claim 15 wherein the one or more lipophilic cation is one or more of SkQ1, SkQ3, SkQ4, SkQ5, SkQR1, SkQRB1, SkQB1, SkQBP1, SkQT, SkQThy, SkQB, C.sub.nTPP (wherein n is from 5 to 12), C.sub.nR1 (wherein n is from 4 to 12), C.sub.nBerb (wherein n is from 4 to 12), and C.sub.nPalm (wherein n is from 4 to 12).

17. The pharmaceutical composition according to claim 15 or 16, wherein the additional one or more antimicrobial compound is one or more antibacterial antibiotic.

18. The pharmaceutical composition according to claim 15 or 16, wherein the additional one or more antimicrobial substance is one or more antifungal compound.

19. The pharmaceutical composition according to claim 15 or 16, wherein the additional one or more antimicrobial agent is one or more antiseptic.

20. The pharmaceutical composition according to claim 15 or 16, wherein the additional one or more antimicrobial substance is one or more MDR pump inhibitor.

21. The pharmaceutical composition according to claim 15 or 16, wherein the additional one or more antimicrobial compound is one or more MDR pump substrate.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1 shows that expression levels of Pdr5 affect resistances to C.sub.12TPP. FIG. 1A show the chemical structure of dodecyltriphenylphosphonium C.sub.12TPP and its plastoquinone derivative (SkQ1) used in this study. FIG. 1B shows the results of glucose-grown cells treated with indicated concentration of C.sub.12TPP or SkQ1. FIG. 1C shows the results of galactose-grown cells treated with indicated concentration of C.sub.12TPP or SkQ1. *P<0.05 compared to untreated WT according to Wilcoxon signed-ranked unpaired test.

[0010] FIG. 2 shows that C.sub.12TPP or energy deprivation enhances Rhodamine 6G accumulation in yeast cells. FIG. 2A shows that R6G staining of yeast cells is enhanced by low concentrations of C.sub.12TPP but not FCCP. Exponentially grown yeast cells were stained with 500 nM R6G in the presence of indicated concentrations of C.sub.12TPP or FCCP. FIG. 2B shows that addition of glucose results in a decrease of total fluorescence. Weak signals are observed from R6G retained in mitochondria (enhanced contrast). (Representative photographs; bars, 5 m).

[0011] FIG. 3 shows that C.sub.12TPP and SkQ1 prevent R6G efflux from yeast cells. FIG. 3A shows indirect measurements of glucose-induced R6G efflux from yeast cells. FIG. 3B shows that glucose-induced R6G efflux is negligible from AD1-8 (MDR-negative) cells. FIGS. 3C and D show that FCCP (2 M), C.sub.12TPP (2 M) or SkQ1 (2 M) inhibits glucose-induced R6G efflux from yeast cells. FIG. 3E shows quantification of the results. The ordinate corresponds to the ratio of slopes in a and b. FIG. 3F shows that FCCP (2 M), C.sub.12TPP (2 M) or SkQ1 (2 M) inhibits glucose-induced R6G efflux from yeast cells pretreated with 10 mM NaN.sub.3. *P<0.05, **P<0.01 compared with untreated WT according to Wilcoxon signed-ranked unpaired test.

[0012] FIG. 4 shows that C.sub.12TPP enhances the effects of Pdr5 substrates cycloheximide D and clotrimazole. FIG. 4A shows duplication times of yeast cells grown in the presence of ethanol (mock control), C.sub.12TPP (1 M), Cycloheximide D (ChD, 0.05 M), or both chemicals. FIG. 4B shows that C.sub.12TPP (1 M) increases the inhibitory effect of Clotrimazole (CltrA, 60 M). *P<0.05 compared with untreated WT according to Wilcoxon signed-ranked paired test.

[0013] FIG. 5 shows that overexpression of PDR5 gene increases active efflux of C12R1 from yeast cells. To measure the relative rate of C12R1 efflux, the fluorometric assay as in Knorre et al. 2014 (http://dx.doi.org/10.1016/j.bbrc.2014.07.017) was used, except that, instead R6G energy deprived cells were stained with 10 M C12R1. The control laboratory strain W303-1A (WT) and the strain PGAL-PDR5 were grown on galactose containing media (conditions of PGAL overexpression). To repress PDR5 gene cells were grown on rich medium containing glucose (conditions of PGAL repression).

[0014] FIG. 6 shows that C12TPP prevents C12R1 efflux from yeast cells. The rate of C12R1 efflux (the slope of fluorescence increase on FIG. 1) was quantified in cells grown either on YPD (yeast peptone dextrose; conditions of PGAL repression), or YPGAL (yeast peptone galactose; conditions of PGAL overexpression). The measurements was conducted with supplementation of 10 M C12TPP where indicated.

[0015] FIG. 7 shows that C.sub.12R1 facilitates antifungal effect of clotrimazole (Cltr) and benzalkonium chloride (BnzCl). Yeast cells were grown in liquid YPD medium with indicated chemicals for 2 hours, then cells were plated on solid YPD and the number of emerged colony forming units (CFU) were calculated after 2 days. 100% corresponds to CFU in yeast suspension before addition of chemicals, control bar corresponds to the number of CFU after 2 hours of incubation without chemicals.

[0016] FIG. 8 shows growth curves of B. subtilis and E. coli in the presence and absence of SkQ1, as measured by absorption at 600 nanometers. Inhibitors added at zero timepoint in the range of concentration of 1-100 M.

[0017] FIG. 9 shows effects of different C.sub.nTPP concentrations on growth of B. subtilis and E. coli. Zero point indicates MIC for C.sub.nTPP.

[0018] FIG. 10 shows the effects of different compounds of formula 1 on growth of bacteria of B. subtilis in the LB media measured by absorption at 600 nanometers. Inhibitors were added at zero timepoint in the range of concentration of 1-100 M.

[0019] FIG. 11 shows antibacterial activity of SkQ1 on growth of bacteria To1C and AcrA and AcrB. Inhibitors were added at zero timepoint.

[0020] FIG. 12 shows antibacterial activity of C.sub.nTPP on growth of bacteria To1C strain. Inhibitors added at zero timepoint.

[0021] FIG. 13 shows accumulation of ethidium bromide in presence of SkQ1. For emulation of cell membrane leakage, bacterial cells were resuspended in deionized water. Ethidium bromide was added (20 g/ml) and the change of fluorescence intensity was recorded on Fluorat-02-Panorama. For detecting of the accumulation of ethidium bromide, SkQ1 was added to a concentration of 50 M.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] The invention relates to pharmaceutical compositions having antimicrobial properties and methods of their use to treat microbial infections. The invention further relates to such pharmaceutical compositions having multiple drug resistance-inhibiting properties. The invention provides pharmaceutical formulations of antibiotics that also have MDR-inhibiting properties, and methods for their use as antibacterial and antifungal agents. The present inventors have surprisingly discovered that lipophilic cationic compounds previously shown to have protective properties for cells actually have antibiotic properties for bacteria and fungi, while simultaneously having MDR-inhibiting properties.

[0023] Generally such lipophilic cation compounds have the structural formula 1:

##STR00003##

wherein A is hydrogen atom (H) or an antioxidant moiety having the following structure:

##STR00004##

and/or reduced (i.e. quinole) form thereof
wherein m is an integer from 0 to 3; each Y is methyl;
L is a linker group which is straight or branched hydrocarbon chain which can be optionally substituted by one or more substituents and optionally contains one or more double or triple bonds; and
B is a lipophilic cation. Such compounds are charged (cations), thus they are present in the form of salt with any pharmacologically acceptable anion (counterion).

[0024] The methods according to the invention comprise administering to a subject having a bacterial or fungal infection a pharmaceutically effective amount of one or more lipophilic cationic compound according to the invention, optionally with another one or more antimicrobial compound or one or more antimicrobial agent.

[0025] Compounds useful in the invention include, without limitation, the following exemplary compounds: SkQ1, SkQ3, SkQ4, SkQ5, SkQR1, SkQRB, SkQB1, SkQBP1, SkQT, SkQThy, SkQB, C.sub.nTPP (wherein n is from 5 to 12), C.sub.nR1 (wherein n is from 4 to 12), C.sub.nBerb (wherein n is from 4 to 12), and C.sub.nPalm (wherein n is from 4 to 12). Structural formulae of representative compounds are presented below.

##STR00005## ##STR00006##

SkQT (mixture of isomers that differ by the position of decyl linker, possible positions are indicated with arrows)

##STR00007##

[0026] Among these SkQT isomers there is SkQT-para:

##STR00008##

SkQThy (mixture of isomers that differ by the position of decyl linker, possible positions are indicated with arrows)

##STR00009##

Compounds useful in the invention also include reduced (quinole) forms of above mentioned compounds. In these reduced compounds the quinone moiety:

##STR00010##

is reduced, i.e. replaced with quinole moiety:

##STR00011##

For example in case of SkQ1 the reduced form SkQ1 H.sub.2 has the following structure:

##STR00012##

Also as an example, in case of SkQ3 the reduced form SkQ3H.sub.2 has the following structure:

##STR00013##

[0027] An important feature of the compounds of general formula 1 as anti-microbial agents is the positive charge of these compounds. The precise structure of the positively charged moiety is not particularly important, as long as the moiety contains sufficient hydrophobic character to spread out the positive charge, such that the overall compound is lipophilic. Once such compounds are expelled from a microbe cell by MDR or other protection system, the compounds are able to penetrate back into the cell due to electric charge on the outer membrane of the microbe (with negative charge inside the cell). This feature makes compounds of general formula 1 recyclable anti-microbial agents.

[0028] In our experiments we have unexpectedly found that different compounds of the general formula 1 provide antimicrobial (i.e. anti-bacteria or anti-fungal) effect. Partially, this effect can be explained by the ability of these compounds to suppress MDR systems in bacteria or fungi. These findings provide a new area of application for the compounds of general formula 1. This area of application is the treatment of different infectious diseases caused by fungi, bacteria, or some viruses. As demonstrated by our experimental examples, antimicrobial properties of compounds of general formula 1 allow this treatment to be performed by a compound of general formula 1 alone, i.e. in monotherapy. Another set of our experimental examples demonstrated that compounds of general formula 1 are able to suppres MDR system in bacteria or fungi. Thus another aspect of our invention is application of compounds of general formula 1 in combination with another antimicrobial agent. This agent can be an antibiotic (anti-bacterial agent), an antimycotic (anti-fungi agent) or an antiseptic.

[0029] Previous studies of lipophilic cations have demonstrated that these compounds have anti-inflammatory properties (WO2012167236, WO2014116591, WO2011162633, Zinovkin R. A. et al, Aging (Albany, N.Y.). 2014 ;6(8):661-74). Thus another aspect of this invention is the use of the benefit of compounds of general formula 1 as an agent providing both anti-microbial and anti-inflammatory effects. Yet another aspect of the invention is a method of treatment of a patient who would benefit from simultaneous suppression of infection and inflammation.

[0030] Within the scope of this invention, the treatment of infection can be systemic or local. Systemic treatments include, without limitation, oral administration, intravenous injection, nasal administration, rectal administration of a compound of general formula 1 to the patient. Local treatments include, without limitation, administration in a form of eye-drops, gels, ointments, lotions, sprays, bandages.

[0031] Experimental examples and examples of compositions are presented below solely to illustrate the applicability of invention and cannot be regarded as examples limiting the scope of claims of the present invention. The following abbreviations are used in the examples:

[0032] SkQ1plastoquinonildecyltriphenylphosphonium.

[0033] C.sub.12TPPdodecyltriphenylphosphonium

[0034] R6Grhodamine 6G

[0035] MDRmultiple drug resistance

[0036] FCCPcyanide p-trifluoro-methoxyphenyl hydrazone

[0037] ABCATP binding cassette

[0038] MICminimal inhibitory concentration.

Examples of Compositions

[0039] In general the compositions provided by the invention includes effective amount of membrane penetrating cation alone and/or with another anti-bacterial or ant-fungal agent. Below are several non-limiting examples of possible compositions:

[0040] (1) Compositions comprising an effective amount of one or more membrane penetrating cation according to the invention and an effective amount of one or more ergosterol synthesis inhibitor, including, without limitation, one or more of Ketoconazole or Itraconazole, Fluconazole, Voriconazole, Posoconazole, Ravuconazole, Bifonazole, Butoconazole, Clomidazole, Clotrimazole, Croconazole, Econazole, Fenticonazole, Isoconazole, Miconazole, Neticonazole, Omoconazole, Oxiconazole, Sertaconazole, Sulconazole, Tioconazole, Terconazole, and Hexaconazole.

[0041] (2) Compositions comprising an effective amount of one or more membrane penetrating cation according to the invention and an effective amount of one or more ergosterol membrane disrupter, including, without limitation, one or more of amphotericin B, Hamycin, Natamycin, and Nystatin.

[0042] (3) Compositions comprising an effective amount of one or more membrane penetrating cation according to the invention and effective amount of one or more squalene epoxidase inhibitor, including, without limitation, one or more of morpholines, Amorolfine, Terbinafine, and Naftifine.

[0043] (4) Compositions comprising an effective amount of one or more membrane penetrating cation according to the invention and an effective amount of one or more -glucan synthase inhibitor, including, without limitation, one or more of Anidulafungin, Caspofungin, and Micafungin.

[0044] (5) Compositions comprising an effective amount of one or more membrane penetrating cation according to the invention and an effective amount of one or more chitin synthesis inhibitor, including, without limitation, nikkomycin and polyoxins (6) Compositions comprising an effective amount of one or more membrane penetrating cation according to the invention and an effective amount of one or more fungi-specific protein synthesis inhibitor, including, without limitation, sordarins.

[0045] (7) Compositions comprising an effective amount of one or more membrane penetrating cation according to the invention and an effective amount of one or more thymidylate synthase inhibitor, including, without limitation, Flucytosine.

[0046] (8) Compositions comprising an effective amount of one or more membrane penetrating cation according to the invention and an effective amount of one or more fungi specific mitotic inhibitor, including, without limitation, Griseofulvin.

[0047] (9) Compositions comprising an effective amount of one or more membrane penetrating cation according to the invention and an effective amount of one or more antimicotic, including, without limitation, one or more of Bromochlorosalicylanilide, Methylrosaniline, Tribromometacresol, Undecylenic acid, Polynoxylin, Chlorophetanol, Chlorphenesin, Ticlatone, Sulbentine, Ethylparaben, Haloprogin, Salicylic acid, Selenium sulfide, Ciclopirox, Amorolfine, Dimazole, Tolnaftate, Tolciclate, and Taurolidine.

EXPERIMENTAL EXAMPLES

Experimental Example 1

Anti-Fungi Effect of Compounds of General Formula 1

[0048] In this experiment we used yeast Saccharomyces cerevisiae as a model fungi. We have found that ABC-transporter Pdr5p protects the cells from the anti-fungi effects of C.sub.12TPP, a compound of general formula 1. In agreement with this, we show that C.sub.12TPP augments the toxic effects of Pdr5p substrates cycloheximide D and clotrimazole and also inhibits rhodamine 6G efflux. This demonstrates that C.sub.12TPP is an effective competitive inhibitor of MDR. An important feature of C.sub.12TPP and other penetrating lipophilic cations is their ability to reenter cell due to electric charge on the outer membrane of the cell. Thus, C.sub.12TPP and other penetrating lipophilic cations act as recyclable MDR inhibitors demonstrating high efficiency.

[0049] Strains and Growth Conditions

[0050] In this example, we used W303-1A S. cerevisiae strains and its derivatives: AD1-8 with deletions of eight MDR genes [W303-1A, yor1::hisG, snq2::hisG, pdr5:shisG, pdr10::hisG, pdr11::hisG, ycf1::hisG, pdr3::hisG, pdr15::hisG][4], P.sub.GAL-PDR5 [W303-1A HIS3::P.sub.GAL-PDR5], P.sub.GAL-SNQ2 [W303-1A HIS3::P.sub.GAL-SNQ2], and P.sub.GAL-M1H1 [W303-1AHIS3::P.sub.GAL-YOR1](this study). Cells were grown in YPD medium (2% glucose, 1% bactopeptone, 1% yeast extract), or in YPGal (2% galactose, 1% bactopepton, 1% yeast extract). For genetic screening and maintenance of strains with conditionally expressed genes synthetic drop-out media YNB-Leu or YNB-His were used according to Sherman 2000 [5]. The growth rates were measured by increase of light scattering (=550 nm) in liquid yeast culture.

[0051] Microscopy

[0052] Cells stained with R6G were visualized with an upright Olympus BX2 microscope and U-MNG2 filter set (excitation 530-550 nm, 570 nm beamsplitter filter, emission >590 nm).

[0053] Genetic Screening.

[0054] Yeast mutants of W303 strain of S. cerevisiae carrying multicopy plasmid YEp13 with inserts of 8-10 Kb at BamHI restriction site have been constructed by transformation. Three cycles of enrichment of mutant collection for C.sub.12TPP resistant strains were performed. During each cycle the mutants at logarithmic stage of growth on YNB-LEU media were treated with 18 M C.sub.12TPP for 3 hours, then washed, diluted and grown overnight on fresh solid YNB-LEU. After the third cycle cells were transferred on solid YNB-LEU media. C.sub.12TPP resistance of separated colonies was compared with a wild type. To identify the genes carried by the multicopy plasmid, the genomic DNAs of the selected strains were transformed in E. coli. Loci of insertion were determined by sequencing the selected YEp13-insertion plasmids with primers YEp13-DIR 5-cgctatatgcgttgatgc YEp13-REV 5-cctgccaccatacccacg.

[0055] Rhodamine 6G Efflux

[0056] To measure the relative rate of rhodamine 6G efflux we used fluorometric assay implemented by Kolaczkowsky et al. [6], with few modifications. Cells were grown overnight in 40 ml in liquid YPD to density of 0.5-1*10.sup.7 cells/ml, washed twice with cold sterile water and resuspended in 10 ml phosphate buffer saline supplemented with 5 mM 2-deoxyglucose and 2.5 mM 2,4-dinitrophenol. Cell suspension was incubated for 45 minutes on a rotatory shaker, then the inhibitors were removed by two cycles of centrifugation/resuspension in cold water. The energy deprived cells were resuspended in 10 ml of PBS and then stained with R6G (10 M) for 30 minutes. Then cell suspension was pelleted, resuspended in equal volume of PBS and stored on ice for 1-5 hours. The measurements of efflux were performed with FluoroMax-3 fluorometer system with exication wavelength set to 480 nm, and emission wavelength set to 560 nm. The R6G efflux was initiated by addition of 0.1% glucose, cell density in fluorometric cuvette was 10.sup.6 cells/ml.

[0057] Survival Assay.

[0058] Exponentially grown cells were taken and treated with indicated amounts of C.sub.12TPP or SKQ1 for 3 hours. Then cell suspensions were plated on solid YPD medium and incubated for 48 hours, the number of formed colonies were counted. 100% refers to the number of colony forming units (CFU) in yeast suspension at the beginning of experiment.

[0059] Statistics

[0060] Wilcoxon signed-ranked tests (n=3-11) and the slopes of the fluorometric curves were calculated using R software package. Error bars on figures represent standard errors of the mean.

[0061] Results and Discussion

[0062] To see whether C.sub.12TPP can inhibit MDR pump activity we first decided to find the particular pump which extrudes it from S. cerevisiae cells. We performed genetic screening using yeast S. cerevisiae with a yeast genome library on a multicopy plasmid. As a result it was found that a plasmid with chromosome II fragment (coordinates from 216287 to 222344) harboring two genes, LDB7 and PDR3, provided a significant increase of resistance to 20 M C.sub.12TPP. Pdr3p is a transcription factor responsible for upregulation of a set of ABC-transporters including three unspecific MDR pump genes: PDR5, SNQ2 and YOR1 [7-9]. These data strongly suggested that C.sub.12TPP is a substrate of one of these pumps. To find a specific pump responsible for C.sub.12TPP detoxication we produced a set of mutant strains with corresponding genes under the control of galactose inducible, glucose repressible GAL promoters.

[0063] It appeared that expression of PDR5 is critical for resistance of cells to C.sub.12TPP, whereas repression of SNQ2 or YOR1 did not show a statistically significant effect (FIG. 1B). Accordingly, overexpression of PDR5 provided a moderate protection to high concentrations of C.sub.12TPP (FIG. 1C). We did not observe any effect of MDR overexpression in the case of SkQ1. Possibly, basal levels of expression of the pumps that extrude SkQ.sub.1 are sufficient to prevent its toxicity.

[0064] One possible explanation for these results is that C.sub.12TPP is extruded by Pdr5 from yeast cells, and that C.sub.12TPP competes with other PDR5 substrates. Indeed, low concentrations of C.sub.12TPP enhance the staining of yeast cells with positively charged fluorescent dye rhodamine 6G (FIG. 2A) which is likely to be a Pdr5p substrate [6]. Accordingly, in energy deprived cells R6G accumulates in high concentration, and after the addition of glucose the fluorescence inside cells is decreased and remains mostly in polarized mitochondria (FIG. 2B). We measured the efflux of R6G from intact yeast cells using fluorometric method. The efflux was visualized by fluorescent spectroscopy: rhodamine 6G is self-quenched in cells and therefore the R6G release results in a detectable increase of total fluorescence of the dye (FIG. 3A). Importantly, R6G release is completely abrogated in MDR-negative cells lacking all major PDR genes (FIG. 3B).

[0065] The addition of uncoupler FCCP was able to partially prevent R6G efflux (FIG. 3C), while C.sub.12TPP and SkQ1 appeared to be much more efficient in this respect (FIGS. 3D, E). Apparently, this effect of lipophilic cations could be either due to a direct inhibition of MDR pumps, or a result of mitochondrial uncoupling causing a decrease of ATP concentration. However, the latter is seemed unlikely because FCCP is a much more potent uncoupler than C.sub.12TPP [10]. Moreover, this possible effect of C.sub.12TPP can be ruled out because it was also able to inhibit R6G efflux in cells pretreated with an excess of NaN.sub.3 (FIG. 3F). NaN.sub.3 inhibits both respiratory chain [11] and mitochondrial ATP synthase [12]. At the same time, in contrast to another ATP synthase inhibitor, oligomycin, it is not as efficient in inhibiting MDR activity in yeast [6]. Therefore, in the presence of 10 mM NaN.sub.3, the contribution of mitochondria in ATP supply appears to be minimal and the effects of C.sub.12TPP and SkQ1 i are mainly due to the repression of MDR.

[0066] Is it possible to induce uptake of other PDR5 substrates by addition of C.sub.12TPP? To test this we measured the growth rate of yeast cells in the presence of protein synthesis inhibitor cycloheximide D (ChD) which is a well known substrate of Pdr5 [13]. It was found that 1 M of C.sub.12TPP significantly enhances the inhibitory effect of ChD (FIG. 4A). Moreover, C.sub.12TPP augments the action of antifungal clotrimazole (FIG. 4B), which was also previously reported to be a substrate of yeast MDR-pumps [14]. Importantly, we did not detect any significant stimulation of the toxicity for another antifungalamphotorecine B, which acts in the plasma membrane and is not attributed to ABC pumps substrates.

[0067] To conclude, together with the previous observations our data show that C.sub.12TPP inhibits the multidrug resistance and suggest that this inhibition is due to a futile cycle of its extrusion followed by returning back into the cells. Therefore it can be used to increase cellular uptake of other ABC-transporter substrates (including other amphiphilic compounds). Remarkably, earlier it was shown that C.sub.12TPP facilitates the transport of anionic molecules across the membranes: fluorescent dye fluoresceine [15], fatty acids [16] and anionic uncouplers [10]. Thus, C.sub.12TPP appears to be a universal plasma membrane permeabiliser. Therefore, our findings make it a promising supplement for anti-mycotic drugs to prevent their efflux from cells.

[0068] In the next experiment we have tested dodecylated rhodamine C.sub.12R1 (see Antonenko et al. 2012; doi: 10.1074/jbc.M110.212837). We found that this compound accumulated in energy deprived yeast cells in a similar way to rhodamine 6G. The addition of glucose to such cells promoted the efflux of C.sub.12R1. The efflux was detected by decrease of self quenching in fluorometer cuvette by increase of fluorescent signal (excitation wavelength was 480 nm, emmision wavelength 560 nm). We found that efficient efflux could be observed only in cells overexpressing PDR5 genes (FIGS. 5, 6). This result indicates that, while being exported by Pdr5p multidrug efflux pump, C.sub.12R1 could be withdrawn from cell only under condition of PDR5 overexpression. Next, we have shown that C.sub.12TPP prevents efflux of C.sub.12R1. This confirms that C.sub.12TPP and C.sub.12R1 are substrates of MDR pump Pdr5p.

[0069] Next we tested the ability of C.sub.12R1 to increase sensitivity of fungi cell to common antifungals. It was found that in non-toxic concentrations C.sub.12R1 increase the toxicity of antifungal clotrimazole and benzalkonium chloride (FIG. 7).

Experimental Example 2

Anti-Bacterial Effect of Compounds of General Formula 1

[0070] Multidrug efflux transporters cause serious problems in the treatment of bacterial infections and cancer chemotherapy. In gram-negative bacteria, such as E. coli, transporters belonging to the resistance-nodulation-cell division (RND) family are particularly effective in generating resistance because they form a tripartite complex with periplasmic proteins and an outer membrane protein channel [Du D. et al., (2014) Structure of the AcrAB-To1C multidrug efflux pump. Nature, 509, 512-515]. The AcrAB-To1C efflux pump is able to transport great number of compounds with little chemical similarity, thus conferring resistance to a broad spectrum of antibiotics [Pos K. M. (2009) Drug transport mechanism of the AcrB efflux pump. Biochem. Biophys. Acta, 1794, 782-793; Seeger M. A. et al., (2008) The AcrB Efflux Pump: Conformational Cycling and Peristalsis Lead to Multidrug Resistance. Current Drug Targets, 2008, 9, 729-749; Sulavik M. C. et al., (2001) Antibiotic Susceptibility Profiles of Escherichia coli StrainsLacking Multidrug Efflux Pump Genes. Antimicrob. Agents Chemother., 45, 4, 1126-1136]. TheAcrAB-To1C system is composed of the RND transporter AcrB, membrane fusion protein AcrA, and multifunctional outer membrane channel To1C. To1C is the universal outer membrane channel for export of toxins and drug efflux [Andersen C., Hughes C., Koronakis V. (2001) Protein export and drug efflux through bacterial channel-tunnels. Curr Opin Cell Biol., 13, 412-416].

[0071] To1C interacts with a variety of inner membrane transporters and enables E. coli to expel structurally diverse molecules out of the cell. In E. coli, AcrB, AcrD, AcrEF, MdtABC, and MdtEF belong to the RND transporters and require To1C to function. [Horiyama T. and Nishino K. (2014) AcrB, AcrD, and MdtABC Multidrug Efflux Systems Are Involved in Enterobactin Export in Escherichia coli. PLoS ONE 9, 9, e10864].

Antibacterial Activity of 1 M SkQ1.

[0072] Growing in the liquid medium method was selected as the method to test antibacterial activity of SkQ1. Overnight bacterial cells cultures were diluted in fresh LB media. Bacterial cell culture (510.sup.6 cells/ml) were inoculated 200 l into 96-well plates (Eppendorf, Eppendorf AG, Hamburg, Germany) and SkQ1 or C.sub.nTPP were added to reach various final concentrations in the range from 0.5 to 200 M. Cells were left to grow for 21 hours at 37 C. Optical density at 600 nm was measured using an AnthosZenyth 3100 multimode reader (Anthos Labtec, Austria) during incubation.

[0073] We selected Ecsherishia coli cells as a Gram-negative model organism and Bacillus subtilis cells as a Gram-positive model organism. SkQ1 was selected as a model compound of formula 1 with antioxidant moiety. To select a proper concentration of a compound of formula 1 we performed growth inhibition assays for several SkQ1 concentrations (see FIGS. 8 A and B). It can be concluded from the results of this experiment that 1 M SkQ1 is the minimal inhibitory concentration (MIC) for B. subtilis cells. But this concentration did not inhibit significantly growth of E. coli cells. 50 M SkQ1 was found to be the MIC for E. coli cells. These results indicate that SkQ1 has strong antimicrobial activity against gram-positive bacteria cells and mild antimicrobial activity against gram-negative bacteria.

Antibacterial Activity of C.SUB.n.TPP.

[0074] We tested resistance of B. subtilis to triphenylphosphonium derivatives from selected ranges of concentration, such as, dodecyltriphenylphosphonium (C.sub.12TPP), decyltriphenylphosphonium (C.sub.10TPP), octyltriphenylphosphonium (C.sub.8TPP), and butyltriphenylphosphonium (C.sub.4TPP). We performed growth inhibition assays for several triphenylphosphonium derivative concentrations (see FIG. 9) and found the MIC for B. subtilis for all used triphenylphosphonium derivatives, except C.sub.4TPP. A gradual increase of the inhibiting concentration for C.sub.n-TPP with growing length of the hydrocarbonic tail was observed. Unexpectedly. MIC of C.sub.4TPP could not be reached in this experiment. These results indicate that C.sub.nTPPs, except C.sub.4TPP, have antimicrobial activity against gram-positive bacterial cells.

Antibacterial Activity of Other Compounds of General Formula 1.

[0075] In the following experiments we used different compounds of general formula 1 such as SkQ5, SkQT, SkQThy, SkQT-para, and SkQ3. We performed growth inhibition assays for several concentrations of the selected compounds (see FIGS. 10A and B) and found that all of the selected triphenylphosphonium derivatives have antimicrobial activity. These results indicate that all selected compounds of general formula 1 have antimicrobial activity based on the length of the hydrocarbon linker and antioxidant structure (i.e. variables A, in and n of the general formula 1).

Antibacterial Activity of SkQ1 against To1C-Required RND Efflux Pumps (MDR).

[0076] In this experiment we have tested whether To1C-dependent transporter is responsible for resistance of E. coli's cells to SkQ1. In the experiment we used single-gene knockout mutants from Keio collection [Baba T., et al., (2006)]. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2, 2006.0008.]. We found that To1C-deletion mutant has lost resistance to SkQ1, and demonstrated same sensitivity as B. subtilis, with minimal bactericidal concentration around 2 M. Similar results were observed for AcrA and AcrB deletion mutants (see FIG. 11). We used other knockout mutants of genes encoding proteins of transporters that require To1C for their function. Deletion of all of transporter genes, except AcrA and AcrB, did not affect resistance of E. coli cells (data not shown). In contrast, deletion of genes acrA or acrB dramatically changed the sensitivity of E. coli's cells to SkQ1. AcrA- or AcrB-deletion mutants showed similar sensitivity to that of the To1C-deletion mutant (Table 1). These results show that resistance of E. coli to SkQ1 can be a result of AcrAB-To1C transporters activity.

Antibacterial Activity of C.SUB.C.TPP Against To1C-Required RND Efflux Pumps.

[0077] We tested resistance of the To1C-deletion mutant to other compounds of general formula 1, such as, dodecyltriphenylphosphonium (C.sub.12TPP), decyltriphenylphosphonium (C.sub.10TPP), octyltriphenylphosphonium (C.sub.8TPP), butiltriphenylphosphonium (C.sub.4TPP). Concentrations of the compounds were selected on the basis of an assumption that the minimal concentration would be about the minimal bactericidal concentration of the compounds for B. subtilis (FIG. 12), and maximal concentration did not exceed half of minimal bactericidal concentration of the compounds for E. coli cells (data not shown). We observed that To1C-deletion mutant also lost resistance to compounds of general formula 1 (FIG. 12). To1C-deletion mutant had similar sensitivity to C.sub.nTPP as B. subtilis. These results indicates that all selected C.sub.nTPPs have antimicrobial activity against To1C-deletion mutant, showing that AcrAB-To1C transporter is responsible for C.sub.nTPP efflux from the cell.

TABLE-US-00001 IMPOSSIBILITY OF GROWTH 5 M SkQ1 30 M SkQ1 5 M C12-TPP 30 M C12-TPP E. coli NO NO NO NO TolC YES YES YES YES AcrA YES YES YES YES AcrB YES YES YES YES AcrD NO NO NO NO AcrE NO NO NO NO AcrF NO NO NO NO MdtA NO NO NO NO MdtB NO NO NO NO MdtC NO NO NO NO MdtE NO NO NO NO MdtF NO NO NO NO
Table 1. Results of growth inhibition assay for deletion mutants of genes encoding To1C-required efflux pumps proteins. Deletion strains JW5503 (devoid of the to1C gene), JW0452 (devoid of the acrA gene), JW0451 (devoid of the acrB gene), JW2454 (devoid of the acrD gene), JW3233 (devoid of the acrE gene), JW3234 (devoid of the acrF gene), JW5338 (devoid of the mdtA gene), JW2060 (devoid of the mdtB gene), JW2061 (devoid of the mdtC gene), JW3481 (devoid of the mdtE gene), and JW3482 (devoid of the mdtF gene) have been described in Baba T., et al., (2006). Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2, 2006.0008.

Ethidium Bromide Accumulation Experiment.

[0078] To confirm whether AcrAB-To1C is the appropriate transporter, we used a test based on the measurement of ethidium bromide leakage through intact bacterial membranes. If SkQ1 is a substrate for AcrAB-To1C transporter, then it is expected that ethidium bromide leakage through bacterial membranes will be comparable to ethidium bromide leakage during osmotic shock when SkQ1 concentration is about minimal bactericidal concentration for E. coli. Bacterial cells were prepared and resuspended in PBS. For emulation of cell membrane leakage bacterial cells were resuspended in deionized water (Milli-Q water purification system, EMD Millipore, Billerica, Mass.). Ethidium bromide was added (20 g/ml) and the change of fluorescence intensity was recorded on Fluorat-02-Panorama (Lumex Instruments, Russia) spectrofluorometer. For increasing the accumulation of ethidium bromide, SkQ1 was added to reach a concentration of 50 M. Usually, wild type E. coli cells have resistance to ethidium bromide up to 800 g/ml, therefore using concentration of ethidium bromide 20 g/ml, we did not expect to see ethidium bromide leakage through bacterial membranes except in case of adding exhausting concentration of substrate. We observed ethidium bromide accumulation when ethidium bromide concentration was 40-times less than MIC for E. coli cells that only can be the result of collateral action of SkQ1 and ethidium bromide as substrates for AcrAB-To1C transporter. As a result it was confirmed that SkQ1 is a substrate for AcrAB-To1C transporter, because we observed ethidium bromide leakage through bacterial membranes, comparable to ethidium bromide leakage during osmotic shock, when we added SkQ1 to reach minimal bactericidal concentration for E. coli (FIGS. 13A and B).

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