Aminoglycoside derivatives and nano-assemblies thereof, including those with quorum sensing inhibitory function

Abstract

The present invention relates to conjugates of aminoglycosides and terpenoids, in particular sesquiterpenoids. Furthermore, the present invention relates to nano-assemblies formed by the inventive conjugates and to a method for producing the conjugates and/or the nano-assemblies. The present invention also relates to the inventive conjugates and nano-assemblies for use in therapy, in particular for use in the treatment of infectious diseases. Particularly preferred embodiments of the present invention relate to farnesylated aminoglycosides and nano-assemblies thereof, in which farnesol and its derivatives do not only function as carrier for the aminoglycosides but do themselves have pharmaceutical activity upon cleavage of the conjugate, in particular quorum sensing inhibitory activity.

Claims

1. A conjugate comprising an aminoglycoside moiety and at least one terpenyl moiety, wherein the terpenyl moiety has at most 20 carbon atoms, and wherein the aminoglycoside moiety and the terpenyl moiety are linked via a pH-sensitive link that is not stable at a pH<5.0, wherein the term “not stable” indicates that at least 80% of the aminoglycoside is released from the conjugate after incubation at pH<5.0 for at most at 8 hours at a temperature of 37° C.

2. The conjugate according to claim 1, wherein the aminoglycoside moiety and the terpenyl moiety are covalently linked.

3. The conjugate according to claim 1, wherein the aminoglycoside moiety and the terpenyl moiety are covalently linked via an imine group.

4. The conjugate according to claim 1, wherein the aminoglycoside moiety and the terpenyl moiety are linked via electrostatic interactions.

5. The conjugate according to claim 1, wherein the conjugate is cleavable into a pharmaceutically active aminoglycoside and a pharmaceutically active terpenoid.

6. The conjugate according to claim 1, wherein the terpenyl moiety is a farnesyl moiety.

7. The conjugate according to claim 1, wherein the conjugate is selected from the group consisting of (i) tobramycin-farnesyl conjugates of the following formula ##STR00101## wherein F.sub.1, F.sub.2, F.sub.3, F.sub.4, F.sub.5 are —H.sub.2 or Fi and wherein at least one of F.sub.1, F.sub.2, F.sub.3, F.sub.4, F.sub.5 is Fi, (ii) kanamycin A-farnesyl conjugates of the following formula ##STR00102## wherein F.sub.1, F.sub.2, F.sub.3, F.sub.4 are —H.sub.2 or Fi and wherein at least one of F.sub.1, F.sub.2, F.sub.3, F.sub.4 is Fi, (iii) kanamycin B-farnesyl conjugates of the following formula ##STR00103## wherein F.sub.1, F.sub.2, F.sub.3, F.sub.4, F.sub.5 are —H.sub.2 or Fi and wherein at least one of F.sub.1, F.sub.2, F.sub.3, F.sub.4, F.sub.5 is Fi, (iv) kanamycin C-farnesyl conjugates of the following formula ##STR00104## wherein F.sub.1, F.sub.2, F.sub.3, F.sub.4 are —H.sub.2 or Fi and wherein at least one of F.sub.1, F.sub.2, F.sub.3, F.sub.4 is Fi, (v) 6′-OH kanamycin A-farnesyl conjugates of the following formula ##STR00105## wherein F.sub.1, F.sub.2, F.sub.3 are —H.sub.2 or Fi and wherein at least one of F.sub.1, F.sub.2, F.sub.3 is Fi, (vi) dibekacin-farnesyl conjugates of the following formula ##STR00106## wherein F.sub.1, F.sub.2, F.sub.3, F.sub.4, F.sub.5 are —H.sub.2 or Fi and wherein at least one of F.sub.1, F.sub.2, F.sub.3, F.sub.4, F.sub.5 is Fi, (vii) amikacin-farnesyl conjugates of the following formula ##STR00107## wherein F.sub.1, F.sub.2, F.sub.3, F.sub.4 are —H.sub.2 or Fi and wherein at least one of F.sub.1, F.sub.2, F.sub.3, F.sub.4 is Fi, (viii) arbekacin-farnesyl conjugates of the following formula ##STR00108## wherein F.sub.1, F.sub.2, F.sub.3, F.sub.4, F.sub.5 are —H.sub.2 or Fi and wherein at least one of F.sub.1, F.sub.2, F.sub.3, F.sub.4, F.sub.5 is Fi, (ix) gentamicin C1-farnesyl conjugates of the following formula ##STR00109## wherein F.sub.2, F.sub.3, F.sub.5 are —H.sub.2 or Fi, wherein F.sub.1, F.sub.4 are —H or Fen and wherein at least one of F.sub.2, F.sub.3, F.sub.5 is Fi, (x) gentamicin C2-farnesyl conjugates of the following formula ##STR00110## wherein F.sub.1, F.sub.2, F.sub.3, F.sub.5 are —H.sub.2 or Fi, wherein F.sub.4 is —H or Fen and wherein at least one of F.sub.1, F.sub.2, F.sub.3, F.sub.5 is Fi, (xi) gentamicin C1A-farnesyl conjugates of the following formula ##STR00111## wherein F.sub.1, F.sub.2, F.sub.3, F.sub.4 are —H.sub.2 or Fi, wherein F.sub.5 is —H or Fen and wherein at least one of F.sub.1, F.sub.2, F.sub.3, F.sub.4 is Fi, (xii) geneticin (G418)-farnesyl conjugates of the following formula ##STR00112## wherein F.sub.1, F.sub.2, F.sub.3 are —H.sub.2 or Fi, wherein F.sub.4 is —H or Fen and wherein at least one of F.sub.1, F.sub.2, F.sub.3 is Fi, (xiii) netilmicin-farnesyl conjugates of the following formula ##STR00113## wherein F.sub.1, F.sub.2, F.sub.3 are —H.sub.2 or Fi, wherein F.sub.4, F.sub.5 are —H or Fen and wherein at least one of F.sub.1, F.sub.2, F.sub.3 is Fi, (xiv) sisomicin-farnesyl conjugates of the following formula ##STR00114## wherein F.sub.1, F.sub.2, F.sub.3, F.sub.4 are —H.sub.2 or Fi, wherein F.sub.5 is —H or Fen and wherein at least one of F.sub.1, F.sub.2, F.sub.3, F.sub.4 is Fi, (xv) verdamicin-farnesyl conjugates of the following formula ##STR00115## wherein F.sub.1, F.sub.2, F.sub.3 are —H.sub.2 or Fi, wherein F.sub.4, F.sub.5 are —H or Fen and wherein at least one of F.sub.1, F.sub.2, F.sub.3 is Fi, (xvi) plazomicin-farnesyl conjugates of the following formula ##STR00116## wherein F.sub.1, F.sub.2, F.sub.3 are —H.sub.2 or Fi, wherein F.sub.4, F.sub.5 are —H or Fen and wherein at least one of F.sub.1, F.sub.2, F.sub.3 is Fi, (xvii) isepamicin-farnesyl conjugates of the following formula ##STR00117## wherein F.sub.1, F.sub.2, F.sub.3 are —H.sub.2 or Fi, wherein F.sub.4 is —H or Fen and wherein at least one of F.sub.1, F.sub.2, F.sub.3 is Fi, (xviii) neomycin B-farnesyl conjugates of the following formula ##STR00118## wherein F.sub.1, F.sub.2, F.sub.3, F.sub.4, F.sub.5 are —H.sub.2 or Fi and wherein at least one of F.sub.1, F.sub.2, F.sub.3, F.sub.4, F.sub.5 is Fi, (xix) neomycin C-farnesyl conjugates of the following formula ##STR00119## wherein F.sub.1, F.sub.2, F.sub.3, F.sub.4, F.sub.5 are —H.sub.2 or Fi and wherein at least one of F.sub.1, F.sub.2, F.sub.3, F.sub.4, F.sub.5 is Fi, (xx) neomycin E-farnesyl conjugates of the following formula ##STR00120## wherein F.sub.1, F.sub.2, F.sub.3, F.sub.4 are —H.sub.2 or Fi and wherein at least one of F.sub.1, F.sub.2, F.sub.3, F.sub.4 is Fi, (xxi) lividomycin B-farnesyl conjugates of the following formula ##STR00121## wherein F.sub.1, F.sub.2, F.sub.3, F.sub.4 are —H.sub.2 or Fi and wherein at least one of F.sub.1, F.sub.2, F.sub.3, F.sub.4 is Fi, (xxii) lividomycin A-farnesyl conjugates of the following formula ##STR00122## wherein F.sub.1, F.sub.2, F.sub.3, F.sub.4 are —H.sub.2 or Fi and wherein at least one of F.sub.1, F.sub.2, F.sub.3, F.sub.4 is Fi, (xxiii) butirosin B/A-farnesyl conjugates of the following formula ##STR00123## wherein F.sub.1, F.sub.2, F.sub.3 are —H.sub.2 or Fi and wherein at least one of F.sub.1, F.sub.2, F.sub.3 is Fi, (xxiv) ribostamycin-farnesyl conjugates of the following formula ##STR00124## wherein F.sub.1, F.sub.2, F.sub.3 are —H.sub.2 or Fi and wherein at least one of F.sub.1, F.sub.2, F.sub.3 is Fi, (xxv) streptomycin-farnesyl conjugates of the following formula ##STR00125## wherein F.sub.1, F.sub.2 are —H.sub.2 or Fi, wherein F.sub.3 is —H or Fen and wherein at least one of F.sub.1, F.sub.2 is Fi, (xxvi) 5′-hydroxystreptomycin-farnesyl conjugates of the following formula ##STR00126## wherein F.sub.1, F.sub.2 are —H.sub.2 or Fi, wherein F.sub.3 is —H or Fen and wherein at least one of F.sub.1, F.sub.2 is Fi, (xxvii) bluensomycin-farnesyl conjugates of the following formula ##STR00127## wherein F.sub.1, F.sub.2 are —H.sub.2 or Fi, wherein F.sub.3 is —H or Fen and wherein at least one of F.sub.1, F.sub.2 is Fi, (xxviii) istamycin A-farnesyl conjugates of the following formula ##STR00128## wherein F.sub.1, F.sub.2, F.sub.3 are —H.sub.2 or Fi, wherein F.sub.4 is —H or Fen and wherein at least one of F.sub.1, F.sub.2, F.sub.3 is Fi, (xxix) istamycin B-farnesyl conjugates of the following formula ##STR00129## wherein F.sub.1, F.sub.2, F.sub.3 are —H.sub.2 or Fi, wherein F.sub.4 is —H or Fen and wherein at least one of F.sub.1, F.sub.2, F.sub.3 is Fi, (xxx) istamycin C-farnesyl conjugates of the following formula ##STR00130## wherein F.sub.1, F.sub.2, F.sub.3 are —H.sub.2 or Fi, wherein F.sub.4 is —H or Fen and wherein at least one of Fi, F.sub.2, F.sub.3 is Fi, (xxxi) fortimicin A (fortimicin B)-farnesyl conjugates of the following formula ##STR00131## wherein F.sub.1, F.sub.2, F.sub.3, F.sub.4 are —H.sub.2 or Fi and wherein at least one of F.sub.1, F.sub.2, F.sub.3, F.sub.4 is Fi, and (xxxii) apramycin-farnesyl conjugates of the following formula ##STR00132## wherein F.sub.1, F.sub.2, F.sub.3, F.sub.4 are —H.sub.2 or Fi, wherein F.sub.5 is —H or Fen and wherein at least one of F.sub.1, F.sub.2, F.sub.3, F.sub.4 is Fi, wherein Fi is a farnesyl moiety of the following formula ##STR00133## and wherein Fen is a farnesyl moiety of the following formula ##STR00134##

8. The conjugate according to claim 1, wherein the aminoglycoside is selected from the group consisting of tobramycin, amikacin, plazomicin, neomycin, gentamicin, kanamycin, netilmicin, sisomicin and dibekacin.

9. A method of treating an infectious disease in a subject, comprising administering a therapeutically effective amount of one or more of the conjugates of claim 1 to a subject having an infectious disease.

10. The method according to claim 9, wherein the infectious disease is responsive to the pharmaceutical activity of the aminoglycoside and/or to the pharmaceutical activity of the terpenoid.

11. A pharmaceutical composition comprising the conjugate of claim 1.

12. A method for preparing the conjugate of claim 1, the method comprising the following steps: a) mixing an aminoglycoside with a terpenoid in a solvent, b) incubating the mixture, c) isolating the conjugate.

13. The method according to claim 12, wherein the terpenoid is selected from the group consisting of farnesol and derivatives thereof.

14. A nano-assembly comprising the conjugate of claim 1.

15. The conjugate according to claim 1, wherein the conjugate is selected from the group consisting of ##STR00135## ##STR00136## ##STR00137## ##STR00138## ##STR00139## ##STR00140## ##STR00141## ##STR00142## ##STR00143##

Description

DESCRIPTION OF FIGURES

(1) FIG. 1 schematically shows the preparation of a conjugate of the present invention comprising a tobramycin moiety and a farnesyl moiety. Notably, the preparation of the conjugate is essentially a single step preparation.

(2) FIG. 2 schematically shows covalent linkage of farnesal to an aminoglycoside. When farnesal is bound to a primary amine group on the aminoglycoside, the farnesyl moiety is linked to the aminoglycoside moiety via an imine group (top). When farnesal is bound to a secondary amine group on the aminoglycoside, the farnesyl moiety is linked to the aminoglycoside moiety via an enamine group (bottom).

(3) FIG. 3 schematically shows the preparation of a conjugate of the present invention comprising a tobramycin moiety and a farnesyl moiety. Notably, the preparation of the conjugate is essentially a single step preparation. The tobramycin moiety and the farnesyl moiety of the conjugate are linked via electrostatic interactions.

(4) FIG. 4 shows farnesyl moieties comprising acid functional groups.

(5) FIG. 5 shows representative TEM images of nano-assemblies comprising a kanamycin A moiety and a farnesyl moiety. The nano-assemblies were stained by phosphotungstic acid hydrate. The images were taken with a magnification of 6000×. The scale bar indicates 0.2 μm.

(6) FIG. 6 schematically shows the preparation of a conjugate of the present invention comprising a kanamycin A moiety and a farnesyl moiety. Notably, the preparation of the conjugate is essentially a single step preparation.

(7) FIG. 7 shows stability of the nano-assembly of the present invention at different pH values. Release of tobramycin (y-axis) is plotted against the duration of incubation (x-axis).

(8) FIG. 8 schematically shows drug release from a conjugate of the present invention upon cleavage of the imine bond linking the aminoglycoside moiety and the farnesyl moiety.

(9) FIG. 9 shows percentages of inhibition of released Pyocyanin molecules in comparison to PA14 wild type control when applying Farnesal 18 μg/mL diluted in DMSO, Farnesol 18 μg/mL diluted in DMSO, Farnesal 18 μg/mL in the nano-assembly dispersed in MilliQ water, Farnesol 18 μg/mL loaded in the nano-assembly dispersed in MilliQ water, and Farnesyl hydrogen sulfate 10 μg/mL nano-assembly dispersed in MilliQ water. All values represent the average of three independent experiments with practical triplicate (n=9). The error bars indicate the standard deviation.

(10) FIG. 10 shows MBEC assay results of 24 hours old PA14 wild type biofilm treated with free Tobramycin for 24 hours. Each dot represents the results of one experiment. 9 independent experiments have been performed for each condition. The mean results are indicated as a horizontal bar. The error bars show the standard deviation. The dotted horizontal line shows the OD650 of PPGAS medium that serves as the standard for determining complete eradication.

(11) FIG. 11 shows MBEC assay results of 24 hours old PA14 wild type biofilm treated with nano-assemblies of conjugates of Tobramycin and Farnesal for 24 hours. Each dot represents the results of one experiment. 9 independent experiments have been performed for each condition. The mean results are indicated as a horizontal bar. The error bars show the standard deviation. The dotted horizontal line shows the OD650 of PPGAS medium that serves as the standard for determining complete eradication.

(12) FIG. 12 shows MBEC assay results of 24 hours old PA14 wild type biofilm treated for 24 hours with nano-assemblies of conjugates of Tobramycin and Farnesal further loaded with Farnesol. Each dot represents the results of one experiment. 9 independent experiments have been performed for each condition. The mean results are indicated as a horizontal bar. The error bars show the standard deviation. The dotted horizontal line shows the OD650 of PPGAS medium that serves as the standard for determining complete eradication.

(13) FIG. 13 shows MBEC assay results of 24 hours old PA14 wild type biofilm treated for 24 hours with nano-assemblies of conjugates of Tobramycin and Farnesal further loaded with Farnesyl Hydrogen Sulfate. Each dot represents the results of one experiment. 9 independent experiments have been performed for each condition. The mean results are indicated as a horizontal bar. The error bars show the standard deviation. The dotted horizontal line shows the OD650 of PPGAS medium that serves as the standard for determining complete eradication.

(14) FIG. 14 shows schematically the formation of Farnesyl hydrogen sulfate nano-assemblies in aqueous media.

EXAMPLES

Example 1: Preparation of the Conjugate

(15) In the following, preparation of inventive conjugates will be exemplarily described. The described conjugates comprise an aminoglycoside (kanamycin A or tobramycin) moiety and a farnesyl moiety. However, it should be noted that the present invention is not restricted to these specific conjugates. Rather, the aminoglycoside moiety may be any aminoglycoside moiety in accordance with the present invention. Likewise, the farnesyl moiety may be replaced by any terpenyl moiety.

(16) The preparation of the conjugate is schematically illustrated in FIGS. 1 (conjugate of tobramycin and farnesal) and 6 (conjugate of kanamycin A and farnesal). It can be seen that the aminoglycoside moiety and the farnesal moiety are linked via an imine group.

(17) Preparation of an Aminoglycoside Solution and a Farnesal Solution

(18) Aminoglycoside (kanamycin A or tobramycin) was dissolved in water. The pH of the aminoglycoside solution was adjusted to values of about 6.5 to 7.0. Then ethanol was slowly added into the solution until the volume ratio of water to ethanol reached about 1:0.8.

(19) A farnesal solution was prepared in a separate flask using ethanol as a solvent.

(20) Formation of the Conjugate

(21) The farnesal solution was slowly added into the aminoglycoside solution until the molar ratio of aminoglycoside to farnesal was 1:1.1. The resulting solution was vigorously stirred for 3 hours. Then the pH was adjusted to values of about 7.0 to 7.8 and the solution was incubated for another 12 hours.

(22) During the incubation a conjugate of the present invention was formed. The conjugate comprised an aminoglycoside moiety and a farnesyl moiety as shown in FIG. 1.

(23) Isolation and Storage of the Conjugate

(24) Subsequent to the formation of the conjugate described above the ethanol was removed by a vacuum rotary evaporator. Trace amounts of ethanol as well as non-conjugated aminoglycosides were removed by membrane dialysis in water. Subsequently, water was removed by freeze-drying and the sample was stored at −20° C. as closed and dried conditions.

Example 2: Preparation of the Nano-Assembly without Storage of the Conjugate

(25) The conjugate of the present invention was formed as described in example 1 above. Subsequent to formation of the conjugate during incubation of the mixture of tobramycin and farnesal, ethanol was removed by a vacuum rotary evaporator as described in example 1 above. Trace amounts of ethanol as well as non-conjugated aminoglycosides were removed by membrane dialysis in purified water (MilliQ water). The present inventors found that the nano-assembly of the present invention formed spontaneously during removal of ethanol.

Example 3: Preparation of the Nano-Assembly Subsequent to Storage of the Conjugate

(26) The nano-assembly of the present invention can also be formed using the conjugate prepared and stored as described in example 1 as starting material. For this purpose, the respective conjugate was dissolved in a mixture of water and ethanol. The ratio (v/v) of water to ethanol was 1:1. Subsequently, ethanol was removed by a vacuum rotary evaporator as described in example 1 above. Trace amounts of ethanol as well as non-conjugated aminoglycosides were removed by membrane dialysis in purified water (MilliQ water). The present inventors found that the nano-assembly of the present invention formed spontaneously during removal of ethanol.

Example 4: Confirmation of Conjugation

(27) Nano-assemblies of examples 2 and 3 were analyzed.

(28) Successful formation of conjugates comprising an aminoglycoside moiety and a farnesyl moiety and formation of nano-assemblies thereof were confirmed by dynamic light scattering (DLS) measurements. A dramatic increase in zeta-potential was observed. Zeta-potential of pure farnesal was −33.8±0.5 mV, while the zeta potential of the nano-assemblies was found to be 29.6±0.5 mV. The increase in zeta-potential demonstrates successful conjugation of aminoglycoside and farnesal.

Example 5: Size of Nano-Assemblies

(29) Nano-assemblies of examples 2 and 3 were analyzed by transmission electron microscopy and were found to have a mean diameter of about 200 nm. FIG. 5 shows a representative TEM image. DLS experiments confirmed the mean diameter to be about 270 nm.

(30) DLS experiments also confirmed homogeneous size distribution of the nano-assemblies. In fact, the polydispersity index (PDI) was determined to be 0.060±0.001, indicating a homogeneous distribution. PDI was calculated according to the following formula:

(31) $\mathrm{PDI}={\left(\frac{\mathrm{size}\mathrm{standard}\mathrm{deviation}\left(\mathrm{nm}\right)}{\mathrm{mean}\mathrm{size}\left(\mathrm{nm}\right)}\right)}^2$

Example 6: pH-Dependent Drug Release

(32) In the obtained conjugates, the aminoglycoside moiety and the farnesyl moiety are connected via an imine bond. In order to check whether the imine bond might be cleaved in a pH-dependent manner, the tobramycin-containing nano-assemblies of examples 2 and 3 were incubated at different pH-values and the release of tobramycin was monitored at different time points by fluorescent activating method. Briefly, the product fluorescence of tobramycin was measured at 344/450 nm (Ex/Em) by Tecan microplate reader. The reagent consisted of 0.2 g of O-Phthalaldehyde dissolved in 1 mL methanol, 19 mL boric acid 0.4 M at pH 10.4 and 0.4 mL of 2-mercaptoethanol 14.3 M. 2 mL of the resulting mixture was then diluted with 16 mL methanol before use.

(33) The nano-assemblies were either incubated in saline (150 mM NaCl, pH 7.2) or in an acetate buffer consisting of sodium acetate and acetic acid (pH 4.5, 150 mM) at 37° C. and the release of tobramycin was checked after 2, 4, 6, 8 and 24 hours. The results of the experiment are shown in FIG. 7.

(34) As can be seen clearly, tobramycin was almost completely released from the nano-assembly after 2 hours of incubation at pH 4.5. In contrast, the nano-assembly is stable in saline without release of drugs. In other words, there is no burst release because release is not based on a concentration gradient. Hence, the nano-assembly of the present invention does not only prevent burst release but also enhances locally sustained release at acidic pH environment. This is a huge advantage for controlled drug release.

(35) In the present experiment, only release of the aminoglycoside moiety of the conjugate of the present invention was monitored. However, it should be noted that pH-dependent cleavage of the imine bond does not only release the aminoglycoside moiety but also the terpenoid moiety. The drug release upon cleavage of the imine bond is schematically shown in FIG. 8.

Example 7: Minimum Inhibitory Concentration (MIC)

(36) The minimum inhibitory concentration (MIC) of the nano-assemblies of the tobramycin containing nano-assemblies against E. coli was tested and compared to known MIC values of tobramycin. The term “MIC-90” refers to the minimum inhibitory concentration required to inhibit the growth of 90% of the bacteria.

(37) Dienstag and Neu (Antimicrobial Agents and Chemotherapy, 1972, vol. 1, p. 41-45) reported the MIC-90 of tobramycin against E. coli to be 6.25 μg/ml. The present inventors found the MIC-90 of the nano-assemblies of the present invention against E. coli to be substantially lower, namely about 0.125 μg/ml. In other words, the MIC-90 of the nano-assemblies of the present invention against E. coli is lower by a factor of 50 as compared to tobramycin alone.

(38) Notably, the nano-assemblies of the present invention comprise a terpenoid moiety in addition to the aminoglycoside moiety. Thus, considering the molecular of weight of tobramycin (467.515 g/mol) and of farnesal (220.356 g/mol), the tobramycin content of 0.125 μg nano-assemblies is about 0.125*(467.515/(467.515+220.356))=0.083 μg. Hence, in relation to the content of tobramycin, the MIC-90 against E. coli is even at 0.083 μg/ml, which corresponds to a reduction of about 75-fold as compared to the known MIC-90 of tobramycin alone.

Example 8: Loading Additional Substances into the Nano-Assemblies

(39) Nano-assemblies of the present invention loaded with different additional substances were produced.

(40) Farnesol-Loaded Nano-Assemblies

(41) 3 mg of a tobramycin-farnesyl conjugate of example 1 as well as 1 mg of farnesol were dissolved in a mixture of water and ethanol, wherein the ratio (v/v) of water to ethanol was 1:1. Subsequently, ethanol was evaporated. The nano-assembly formed spontaneously upon evaporation of ethanol. Farnesol was co-precipitated into the forming nano-assembly. Subsequently, trace amounts of ethanol were removed by membrane dialysis in purified water (MilliQ water). Loading of farnesol into the nano-assembly is schematically shown in the following scheme:

(42) ##STR00097##

(43) The farnesol loaded nano-assemblies had a mean diameter of 576.5±19.3 nm, a PDI of 0.116±0.064 and a zeta-potential of 33.2±1.68 mV. Thus, the mean diameter of the loaded nano-assemblies is substantially increased in comparison to unloaded nano-assemblies (see example 5).

(44) The loading of farnesol was quantified by liquid chromatography-mass spectrometry (LC-MS). Encapsulation efficiency was determined to be 93.5%, and loading rate was determined to be 23.76%. Thus, loading of farnesol into the nano-assembly was very efficient.

(45) The encapsulation efficiency was calculated according to the following formula:

(46) $\mathrm{Encapsulation}\mathrm{Efficiency}=\frac{\begin{array}{c}\mathrm{Actual}\mathrm{weight}\mathrm{of}\mathrm{Farnesol}\mathrm{loaded}\\ \mathrm{in}\mathrm{the}\mathrm{nanoassembly}\end{array}}{\mathrm{Initial}\mathrm{weight}\mathrm{of}\mathrm{Farnesol}\mathrm{used}\mathrm{to}\mathrm{load}}\times 100\%$

(47) The loading rate was calculated according to the following formula:

(48) $\mathrm{Loading}\mathrm{rate}=\frac{\begin{array}{c}\mathrm{Actual}\mathrm{weight}\mathrm{of}\mathrm{Farnesol}\mathrm{loaded}\\ \mathrm{in}\mathrm{the}\mathrm{nanoassembly}\end{array}}{\begin{array}{c}\mathrm{Actual}\mathrm{weight}\mathrm{of}\mathrm{Farnesol}\mathrm{loaded}\mathrm{in}\mathrm{the}\mathrm{nanoassembly}+\\ \mathrm{Weight}\mathrm{of}\mathrm{conjugate}\end{array}}\times 100\%$
Farnesal-Loaded Nano-Assemblies

(49) Farnesal may be loaded into the nano-assemblies as described for farnesol-loading above. However, more preferably, farnesal may be used in excess during conjugate formation in order to obtain farnesal-loaded nano-assemblies as described in the following.

(50) A conjugate comprising an aminoglycoside moiety and a farnesyl moiety can be formed using a farnesal solution and an aminoglycoside solution as described in example 1. Furthermore, nano-assemblies of such conjugates can be formed as described in examples 2 and 3.

(51) Example 1 describes that for formation of the conjugates the farnesal solution was slowly added into the aminoglycoside solution until the molar ratio of aminoglycoside to farnesal was 1:1.1. Higher amounts of farnesal can be used for preparing nano-assemblies of conjugates of the invention loaded with additional farnesal.

(52) For example, the molar ratio of aminoglycoside to farnesal may be 1:x (with x>1). When x is smaller or equal to the total number of amine functional groups in the aminoglycoside (e.g Tobramycin with 5 primary amine groups, or Kanamycin A with 4 primary amine groups, or Plazomicin with 3 primary amine groups, and 2 secondary amine groups), there will be covalent linkages formed between aminoglycoside and farnesal via the chemical reaction between aldehyde and amine groups.

(53) However, when x exceeds the total number of amine functional groups in the aminoglycoside, the un-reacted farnesal is loaded into the nano-assembly upon nano-assembly formation. Such farnesal-loaded nano-assemblies are formed when the excess amount of farnesal is not removed from the resulting conjugate prior to nano-assembly formation. Such farnesal-loaded nano-assemblies are particularly useful when the amount of quorum sensing inhibitor in the nano-assembly is needed to be increased for further application (e.g biofilm eradication).

(54) Farnesal-loaded nano-assemblies were prepared by using farnesal in excess over the total number of amine functional groups in the aminoglycoside during conjugate formation and subsequently forming the nano-assembly as described in example 2 without removing the excess amount of farnesal from the resulting conjugate prior to nano-assembly formation.

(55) Nano-Assemblies Loaded with Farnesyl Hydrogen Sulfate

(56) Preparing Farnesyl Hydrogen Sulfate Nano-Assemblies

(57) A newly synthesized quorum sensing inhibitor, Farnesyl hydrogen sulfate is a product of a reaction between Farnesol and sulfur trioxide triethylamine complex in dimethylformamide (DMF).

(58) Farnesyl hydrogen sulfate forms nano-assemblies when being dispersed in aqueous media due to the strong hydrophilicity of hydrogen sulfate functional group. Formation of Farnesyl hydrogen sulfate nano-assemblies is schematically shown in FIG. 14.

(59) The formation of Farnesyl hydrogen sulfate nano-assemblies in aqueous media was done as follows: Farnesyl hydrogen sulfate was solubilized in ethanol. 0.2 mL of the solution of Farnesyl hydrogen sulfate in ethanol (10 mg/mL) were then dropped into 1 mL MilliQ water, and the nano-assembly of Farnesyl hydrogen sulfate was formed spontaneously. Ethanol was then evaporated, and the trace amount of ethanol was further removed by membrane dialysis in MilliQ water.

(60) The nano-assemblies had a mean diameter of 145.8±1.4 nm, a PDI 0.053±0.014, and a zeta-potential of −33.7±0.9 mV after completely removing ethanol. The morphology was confirmed by Cryo Transmission Electron Microscopy (not shown). These Farnesyl hydrogen sulfate nano-assemblies were used in the Pyocyanin assay (example 10).

(61) Loading Farnesyl Hydrogen Sulfate into Nano-Assemblies of the Invention

(62) Farnesyl hydrogen sulfate and the Tobramycin-Farnesyl conjugate of Example 1 were dissolved in a mixture of water:ethanol ratio of 1:1 (v:v). The nano-assembly was formed spontaneously when ethanol was evaporated. Farnesyl hydrogen sulfate is in the core side together with Farnesyl moieties when ethanol is removed. Thus, Farnesyl hydrogen sulfate loaded nano-assemblies are obtained. Trace amount of ethanol were removed by membrane dialysis in MilliQ water. The formation of the Farnesyl hydrogen sulfate loaded nano-assemblies is schematically shown below.

(63) ##STR00098##

(64) The Farnesyl hydrogen sulfate loaded Tobramycin-Farnesyl nano-assemblies had a mean diameter of 378.5±8.3 nm, PDI of 0.103±0.024, and a zeta-potential of 27.2±2.98 mV. Thus, the mean diameter of the loaded nano-assemblies is increased in comparison to unloaded nano-assemblies (see example 5). However, the mean diameter of the Farnesyl hydrogen sulfate loaded nano-assemblies is substantially smaller as compared to the mean diameter of the Farnesol-loaded nano-assemblies.

(65) Due to the self-assembling properties of Farnesyl hydrogen sulfate and the similar hydrophobicity of Farnesyl moieties, the encapsulation efficiency was maximized to 100%, and the loading rate was controlled as the feeding ratio of the two compounds.

(66) Nano-Assemblies Loaded with Quinolone Derivative (Another Quorum Sensing Inhibitor)

(67) 3 mg of a tobramycin-farnesyl conjugate of example 1 as well as 50 μg of 2-heptyl-6-nitro-4-oxo-1,4-dihydroquinoline-3-carboxamide were dissolved in a mixture of water and ethanol, wherein the ratio (v/v) of water to ethanol was 1:1. Subsequently, ethanol was evaporated. The nano-assembly formed spontaneously upon evaporation of ethanol. 2-heptyl-6-nitro-4-oxo-1,4-dihydroquinoline-3-carboxamide was co-precipitated into the forming nano-assembly. Subsequently, trace amounts of ethanol were removed by membrane dialysis in water. Loading of 2-heptyl-6-nitro-4-oxo-1,4-dihydroquinoline-3-carboxamide into the nano-assembly is schematically shown in the following scheme:

(68) ##STR00099##

(69) The 2-heptyl-6-nitro-4-oxo-1,4-dihydroquinoline-3-carboxamide loaded nano-assemblies had a mean diameter of 294±3.9 nm, a PDI of 0.137±0.041 and a zeta-potential of 34.8±0.404 mV. Thus, the mean diameter of the loaded nano-assemblies is smaller as compared to the farnesol-loaded nano-assemblies.

(70) The loading of 2-heptyl-6-nitro-4-oxo-1,4-dihydroquinoline-3-carboxamide was quantified by liquid chromatography-mass spectrometry (LC-MS). Encapsulation efficiency was determined to be 94%, and loading rate was determined to be 1.54%. Thus, loading of 2-heptyl-6-nitro-4-oxo-1,4-dihydroquinoline-3-carboxamide into the nano-assembly was very efficient.

(71) Encapsulation efficiency and loading rate were calculated based on the formulas indicated above for loading of farnesol.

(72) Nano-Assemblies Loaded with Nile Red

(73) 3 mg of a tobramycin-farnesyl conjugate of example 1 as well as 6 μg of Nile red were dissolved in a mixture of water and ethanol, wherein the ratio (v/v) of water to ethanol was 1:1. Subsequently, ethanol was evaporated. The nano-assembly formed spontaneously upon evaporation of ethanol. Nile red was co-precipitated into the forming nano-assembly. Subsequently, trace amounts of ethanol were removed by membrane dialysis in water. Loading of Nile red into the nano-assembly is schematically shown in the following scheme:

(74) ##STR00100##

(75) The Nile red loaded nano-assemblies had a mean diameter of 270 nm, a PDI of 0.080±0.001 and a zeta-potential of 25.9±0.603 mV. Thus, the mean diameter of the loaded nano-assemblies is smaller as compared to the farnesol-loaded nano-assemblies.

(76) The loading of Nile red was quantified by fluorescence intensity of the Nile red dye. Encapsulation efficiency was determined to be 97%, and loading rate was determined to be 0.16%. Thus, loading of Nile red into the nano-assembly was very efficient.

(77) Encapsulation efficiency and loading rate were calculated based on the formulas indicated above for loading of farnesol.

Example 9: MIC Assay Against Pseudomonas aeruginosa

(78) The minimum inhibitory concentration (MIC) of the of the tobramycin containing nano-assemblies of example 2 against the P. aeruginosa strain PA14 (wild type) was tested and compared to known MIC values of tobramycin. The term “MIC-90” refers to the minimum inhibitory concentration required to inhibit the growth of 90% of the bacteria.

(79) The MIC-90 of Tobramycin against PA14 was found to be in the range 6.25-12.5 μg/mL, while the MIC values of the nano-assemblies of the present invention comprising Farnesyl moieties and Tobramycin with the molar ratio Farnesyl moieties:Tobramycin 1:1, and 4:1 were found to be 3.125 μg/mL, and 1.562 μg/mL, respectively.

(80) Thus, the amount of Tobramycin in the nano-assemblies with the molar ratio Farnesyl moieties:Tobramycin 1:1, and 4:1 was 2.075 μg/mL (calculated following the equation (see also example 7 above): 3.125*(467.515/(467.515+220.356))=2.075 μg/mL), and 0.541 μg/mL (calculated following the equation: 1.562*(467.515/(467.515+4*220.356))=0.541 μg/mL, which show the improvement by 3-6 fold, and 11.5-23 fold compared to the free Tobramycin.

Example 10: Pyocyanin Assay on Pseudomonas aeruginosa

(81) Pyocyanin assay was used to quantify the amount of Pyocyanin molecule released from PA14 bacteria (wild type) which were grown aerobically for 16 hours. The Pyocyanin assay was performed as follows:

(82) A single colony of PA14 was removed from agar plates after 16 hours of growth at 37° C. and transferred into 25 mL Erlenmeyer flasks with 10 mL of PPGAS (proteose peptone glucose ammonium salt medium which is composed of (1 g/L NH.sub.4Cl, 1.5 g/L KCl, 19 g/L Tris-HCl, 10 g/L peptone, glucose 5 g/L and 0.1 g/L MgSO.sub.4.Math.7H.sub.2O, the medium was adjusted to pH 7.4, and sterilized before use). Following 16 hours of aerobic growth with shaking at 200 rpm and 37° C., cultures were centrifuged at 7.450 g, washed twice with 10 mL of fresh PPGAS medium, and resuspended to a final volume of 5 mL solution. Cultures were then diluted to a final OD600 of 0.02 and distributed into 24 well-plates, 1.5 mL each well.

(83) Compounds Farnesal and Farnesol were diluted in DMSO, then added to wells containing PA14 bacteria, in 1:100 dilutions with a final DMSO concentration of 1% (v/v), so that the final concentration of Farnesal was 18 μg/mL, and of Farnesol was 18 μg/mL.

(84) The nano-assemblies containing Farnesal dispersed in MilliQ water (see example 8), or nano-assemblies containing Farnesol dispersed in MilliQ water (see example 8) were added to wells containing PA14 bacteria, in 1:100 dilutions with a final MilliQ water concentration of 1% (v/v). The final concentration of total Farnesal (=loaded Farnesal+Farnesyl moieties of the conjugates forming the nano-assemblies) was 18 μg/mL for the Farnesal-loaded nano-assemblies. The final concentration of total Farnesol (=loaded Farnesol) was 18 μg/mL for the Farnesol-loaded nano-assemblies. Notably, tobramycin is generated upon cleavage of the tobramycin-farnesyl conjugates forming the nano-assemblies both in the case of Farnesal-loaded nano-assemblies and in the case of Farnesol-loaded nano-assemblies. However, the amount of tobramycin was so low that it was excluded that the observed effects were based on tobramycin. Rather, the effects were caused by Farnesal and Farnesol, respectively. Notably, Farnesal is generated upon cleavage of the tobramycin-farnesyl conjugates forming the nano-assemblies in case of the Farnesol-loaded nano-assemblies as well. However, the amount of Farnesal was so low as compared to the amount of Farnesol that it was excluded that the observed effects were based on Farnesal. Rather, the effects of the Farnesol-loaded nano-assemblies were based on Farnesol.

(85) The nano-assemblies of Farnesyl hydrogen sulfate dispersed in MilliQ water (see example 8) were added to wells containing PA14 bacteria, in 1:100 dilutions with a final MilliQ water concentration of 1% (v/v), and the final concentration of total Farnesyl hydrogen sulfate was 10 μg/mL. The Farnesyl hydrogen sulfate nano-assemblies did not contain any moieties potentially generating aminoglycosides or Farnesal. Thus, the observed effects were based on Farnesyl hydrogen sulfate.

(86) Untreated cultures of PA14 were also incubated to serve as controls.

(87) All cultures were incubated for an additional 16 h under aerobic conditions as mentioned above. Pyocyanin was extracted by adding 900 μL of chloroform to 900 μL of 16 h culture and subsequently re-extracted with 250 μL of 0.2 M HCl from the organic phase. OD520 was measured in the aqueous phase. Pyocyanin formation values were normalized to a corresponding OD600 of the respective sample.

(88) OD600 and OD520 are absorbance values of the solution at the wave-length 600 nm and 520 nm, respectively.

(89) The results are presented as percentage (%) inhibition of released Pyocyanin molecules in comparison to PA14 wt control. The results are shown in FIG. 9.

(90) Pyocyanin molecules are important agents for the communication of bacteria and the formation of biofilms. Pyocyanin molecules play an essential role in resistance of bacteria. As a result, quorum sensing inhibitory molecules are developed to inhibit the production of Pyocyanin molecules, which would help preventing the fast resistance of bacteria, and at the same time improving infection treatment.

(91) Some Farnesyl derivatives are natural quorum sensing inhibitors which would be preferable in pharmaceutical application. They, however, are highly hydrophobic compounds; hence, its water-insolubility seriously limits their applying potential, as well as the pharmacological efficacy.

(92) As can be seen in FIG. 9, the free Farnesyl derivatives (Farnesal, and Farnesol) had to be diluted in DMSO before applying into bacteria culture. Although the inhibition of Pyocyanin production by those two compounds was shown, the efficacy was not significant with the percentage of inhibition lower than 20% at 18 μg/mL concentration in both cases. (Detailed results: Farnesal: 5.85±3.10% inhibition, Farnesol: 12.29±7.35%)

(93) Notably, the nano-assemblies loaded with Farnesal or Farnesol dispersed in MilliQ water showed significant improvement in Pyocyanin inhibition in comparison to corresponding free compound at the same concentration. The inhibitory efficacy of Farnesal loaded nano-assembly was 53.35±19.53%, and of Farnesol loaded nano-assembly was 53.80±11.68%.

(94) Notably, Farnesyl hydrogen sulfate forms the nano-assembly by itself in MilliQ water, and shows remarkable inhibition in Pyocyanin production even at lower concentration of 10 μg/mL. The percentage inhibition was 37.62±8.54%.

(95) In summary, the results of the Pyocyanin assay show that the conjugates of the present invention have remarkable quorum sensing inhibitory properties and are superior to free Farnesal and Farnsesol.

Example 11: MBEC Assay Against Pseudomonas aeruginosa

(96) Minimum Biofilm Eradicating Concentration (MBEC) assay was used to determine the minimum concentration of active agents at which a 24 hours old PA14 biofilm is completely eradicated. The MBEC assay was performed as follows:

(97) A single colony of PA14 was removed from agar plates after 16 hours of growth at 37° C. and transferred into 25 mL Erlenmeyer flasks with 10 mL of PPGAS (proteose peptone glucose ammonium salt medium which is composed of (1 g/L NH.sub.4Cl, 1.5 g/L KCl, 19 g/L Tris-HCl, 10 g/L peptone, glucose 5 g/L and 0.1 g/L MgSO.sub.4.Math.7H.sub.2O, the medium was adjusted to pH 7.4, and sterilized before use). Following 16 hours of aerobic growth with shaking at 200 rpm and 37° C., cultures were centrifuged at 7.450 g, washed twice with 10 mL of fresh PPGAS medium, and resuspended to a final volume of 5 mL solution. Cultures were then diluted to a final OD600 of 3.0 and distributed into 96 well-plates, 0.2 mL each well.

(98) The 96 well-plates were then kept at 37° C. without shaking for 24 hours to allow the forming of biofilm. Afterwards, the planktonic bacteria were removed, and the 24 hours old biofilm was washed twice with PBS, then fed with fresh PPGAS medium, and ready for the assay.

(99) For the assay, free Tobramycin was solubilized in MilliQ water at different concentrations, then added to wells containing PA14 bacteria biofilm, in 1:20 dilutions, so that the final concentration of Tobramycin was ranged from 6.25 to 100 μg/mL.

(100) The nano-assemblies of conjugates of Tobramycin and Farnesal dispersed in MilliQ water were prepared with various feeding concentrations of Tobramycin, then added to wells containing PA14 bacteria biofilm, in 1:20 dilutions, so that the final concentration of Tobramycin was ranged from 6.25 to 100 μg/mL, while concentration of Farnesal was kept constant.

(101) Similarly, the Farnesol-loaded nano-assemblies of example 8 dispersed in MilliQ water were prepared with various feeding concentrations of Tobramycin, then added to wells containing PA14 bacteria biofilm, in 1:20 dilutions, so that the final concentration of Tobramycin was ranged from 6.25 to 100 μg/mL, while concentrations of Farnesal and Farnesol were kept constant.

(102) Similarly, the nano-assemblies composed of Tobramycin, Farnesal and Farnesyl hydrogen sulfate dispersed in MilliQ water (see example 8) were prepared with various feeding concentrations of Tobramycin, then added to wells containing PA14 bacteria biofilm, in 1:20 dilutions, so that the final concentration of Tobramycin was ranged from 6.25 to 100 μg/mL, while concentrations of Farnesal and Farnesyl hydrogen sulfate were kept constant.

(103) Untreated biofilm of PA14 were also incubated to serve as controls.

(104) All cultures were incubated for an additional 24 hours at 37° C. without shaking. Then, all samples were washed twice with PBS, added 200 mL fresh PPGAS and sonicated to disperse the biofilm. From each well of the challenged plates, 100 mL of dispersed biofilm was transferred to new 96 well-plates which are further incubated for 24 hours at 37° C. without shaking. All cultures were then measured OD650, and the biofilm is considered to be completely eradicated once the resulting OD650 is equal or lower than OD650 of PPGAS medium.

(105) OD600 and OD650 are absorbance values of the solution at the wave-length 600 nm and 650 nm, respectively.

(106) The results are shown in FIGS. 10 to 13.

(107) As shown in Example 9, free Tobramycin concentration at 6.25 to 12.5 μg/mL was the minimum concentration against planktonic PA14. However, once bacteria are protected in biofilm, the concentration of Tobramycin needed to completely eradicate bacterial infection was 100 μg/mL or higher, as shown in FIG. 10. The fact of higher Tobramycin dose needed to eliminate biofilm is known and was verified here. The need for higher dose is a challenge in biofilm-associated infections and shortcomings in the delivery of the needed amount can promote bacterial antibiotic resistance development.

(108) The present invention comprises the complementary treatment strategy of combining Tobramycin and Farnesyl quorum sensing inhibitors. Moreover, the conjugates of the two active compounds were also able to self-assembly into nano-assemblies. The nano-assemblies remarkably improve the efficacy against PA14 biofilm in comparison to the use of just free Tobramycin. As shown in FIGS. 11, 12 and 13, the Tobramycin needed for the complete biofilm eradication was at 12.5 μg/mL which is 8-fold lower than that of free Tobramycin.

(109) In summary, the results of the MBEC assay show that the conjugates of the present invention have remarkable biofilm eradicating properties and are superior to free Tobramycin.