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WO2018187867A1 - Conjugués amphiphiles de tobramycine liés à un mimétique peptoïde à base de lysine par l'intermédiaire d'une attache - Google Patents

Conjugués amphiphiles de tobramycine liés à un mimétique peptoïde à base de lysine par l'intermédiaire d'une attache Download PDF

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WO2018187867A1
WO2018187867A1 PCT/CA2018/050439 CA2018050439W WO2018187867A1 WO 2018187867 A1 WO2018187867 A1 WO 2018187867A1 CA 2018050439 W CA2018050439 W CA 2018050439W WO 2018187867 A1 WO2018187867 A1 WO 2018187867A1
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compound
tether
tobramycin
lysine
aeruginosa
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Frank Schweizer
George G. Zhanel
Yinfeng LYU
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University of Manitoba
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University of Manitoba
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A61K47/552—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds one of the codrug's components being an antibiotic

Definitions

  • aeruginosa displays low membrane permeability which limits the penetration of most antibiotics into the cell and a highly efficient membrane-associated efflux system of broad substrate specificity that significantly reduces bioaccumulation of drugs within its cytosol. 7 ' 8
  • the reduced intracellular concentration further promotes the activation of secondary adaptive resistance mechanisms (such as overexpression of efflux pump proteins and a variety of sensor kinases) that renders it completely refractory to treatment. 9
  • resistance often emerges when antibiotics are administered as monotherapy, 10 - 11 thus combination therapy being the preferred choice in the treatment of complicated infections.
  • 12 ' 13 Although debatable, the argument for combination drug treatment is premised on the large-scale genetic interaction networks between targets.
  • a compound comprising tobramycin connected by a tether to a lysine-based peptoid mimic.
  • a method of treating a bacterial infection in an individual in need of such treatment comprising administering to said individual an effective amount of a compound comprising tobramycin connected by a tether to a lysine-based peptoid mimic.
  • a method of preparing a medicament for treatment of a bacterial infection comprising admixing a compound comprising tobramycin connected by a tether to a lysine-based peptoid mimic and a suitable excipient.
  • a compound comprising tobramycin connected by a tether to a lysine-based peptoid mimic for treating a bacterial infection.
  • a compound comprising tobramycin connected by a tether to a lysine-based peptoid mimic for treating a bacterial infection.
  • Figure 2 Absolute MIC of minocycline or rifampicin alone or in combination with 4 pg/mL of compound 1- 4 against P. aeruginosa PAO1.
  • Figure 3 FIC index comparison of compound 3 and colistin in combination with antibiotics against P. aeruginosa PAO1 .
  • FIG. 7 Cytoplasmic membrane depolarization of P. aeruginosa PAO1 treated with tobramycin, colistin, compound 3, or 4 at 32 pg/rriL was measured using the membrane potential-sensitive dye diSC3-5. The fluorescent intensity was monitored at an excitation wavelength of 622 nm and an emission wavelength of 670 nm over a 1200 seconds period.
  • FIG. 9 (A) Hemolytic activity of compounds 1-4 was evaluated against pig red blood cells. 0.1 % Triton X-100 was employed as positive control to calculate the percentage of hemolysis. (B) Cytotoxicity of compound 3 was demonstrated against DU145 and JIMT-1 cell lines by MTS assay.
  • Figure 1 1 Scheme 1 . General synthetic scheme for the preparation of amphiphilic peptoid.
  • Figure 13 Time killing kinetics of minocycline, rifampicin, and 3 as monotherapy against P. aeruginosa PAO1 at 1 MIC, 2 ⁇ MIC, and 4 MIC, respectively.
  • Figure 14 Tolerability of Galleria mellonella treated with compound 3 and 4 at 100 and 200 mg/kg. The numbers of surviving larvae were scored daily for 4 days.
  • amphiphilic tobramycin-lysine conjugates preserve many of the known adjuvant properties of previously reported tobramycin-fluoroquinolone hybrids. From a medicinal chemistry point, linking a tobramycin C-12 vector to an amphiphilic lysine conjugate enhances the outer membrane destabilization effect of the amphiphilic lysine analog. As such our study suggests that a tobramycin-C12 tether at C-5 position in tobramycin serves as an effective vector to promote delivery of compounds through the outer membrane barrier of Gram-negative bacteria with an optimized effect on P. aeruginosa. However, the effect of the tobramycin-C12 tether appears to be not limited to the outer membrane but also involves the cytoplasmic membrane.
  • Ceftazidime/avibactam contains an older 3 rd generation cephalosporin ceftazidime, with avibactam a synthetic ⁇ - ⁇ -lactam, ⁇ -lactamase inhibitor that inhibits the activities of Ambler class A and C ⁇ -lactamases and some Ambler Class D enzymes. 34 Limited data suggest that the addition of avibactam does not improve the activity of ceftazidime versus Pseudomonas aeruginosa.
  • Ceftolozane is a novel cephalosporin with a chemical structure similar to that of ceftazidime, with the exception of a modified side-chain at the 3-position of the cephem nucleus, which confers potent antipseudomonal activity. 35
  • tazobactam extends the activity of ceftolozane to include most ESBL producers but not P. aeruginosa. Nevertheless, effective drug combinations often lead to inconclusive benefits of combination therapy over monotherapy during meta-analysis.
  • the tether is a tether that has a length corresponding to 2-20 carbons.
  • such a tether could be not only alkyl based but could be composed of anything that can approximate this length such as for example but by no means limited to aromatic tethers, polyethyleneglycol based or carbon chains with double or triple bonds.
  • amphiphilic tobramycin-lysine conjugate 4 possesses similar MICs against the eight strains (range 8-64 ⁇ g/mL) while tobramycin possesses a MIC range between 0.25 - 512 g/mL indicating that the conjugate is 32-fold less active against tobramycin susceptible strains while being 16-fold less active against tobramycin-resistant strains which supports a different mode of action.
  • tetracyclines such as minocycline
  • ⁇ transmembrane chemical gradient
  • the other component of PMF is electrical potential ( ⁇ ), which is known to drive aminoglycosides uptake.
  • ⁇ electrical potential
  • Bacteria control ⁇ and ⁇ reasonably to maintain a constant value of PMF, and disruption of either component is compensated for by a counteracting increase in the other.
  • 44 When a compound disrupts ⁇ , an antagonism effect will be observed in combination with aminoglycosides, while synergism will show in combination with tetracyclines due to the compensatory increase of ⁇ .
  • Tetracyclines and aminoglycosides have therefore been used as two relevant antibiotics in combination studies with other drugs to identify compounds that affect membrane PMF and specifically dissipate either component of PMF. 31
  • compound 3 displayed different synergistic effects with minocycline (synergism) and tobramycin (no interaction), an observation that is consistent with dissipation of ⁇ component of the PMF by 3.
  • the expected antagonistic effect of 3 with aminoglycosides was not observed, likely due to the membrane penetration induced by 3 that slightly affected aminoglycosides uptake into bacterial cells.
  • the effect of 3 on ⁇ was further corroborated by the increased diSC3-5 fluorescence ( Figure 7) and repression of swimming motility controlled by this parameter (Figure 8).
  • Compounds that collapse the PMF are known to inhibit ATP synthesis and flagellar motility, preventing or reducing swimming activity. 45
  • Membrane-associated efflux is another major mechanism that prevents bioaccumulation of drugs within the cytosol, thus preventing/reducing access of antibiotics to intracellular targets.
  • PMF periplasmic membrane fusion protein
  • aeruginosa efflux pump substrates were evaluated in PAO200 and PAO750, including chloramphenicol, erythromycin, trimethoprim, and moxifloxacin. 7 ' 48-50 The results showed weak synergy or additive effects of 3 in these combinations, corroborating the efflux pump inhibitory activity of 3. We posited that the dissipation of electrochemical gradient across the cytoplasmic membrane affected respiratory ATP production, thereby compromising efflux pump efficiency.
  • rifampicin which is not substrate of the five efflux pumps investigated in this study (MexAB-OprM, exCD-OprJ, MexEF-OprN, MexJK, and MexXY) was similarly strongly synergized by 3 against PAO200 and PAO750 (Table 5).
  • Rifampicin is known to kill bacteria by inhibiting RNA synthesis after binding to DNA- dependent RNA polymerase. 51 Both gram-positive and gram-negative bacteria are similarly sensitive to rifampicin, with the higher MICs reported in gram-negative bacteria due to its low penetration across the outer membrane.
  • minocycline In contrast to the bactericidal nature of rifampicin, minocycline is known to be only bacteriostatic, which was evident in the time-kill assay with constant bacterial cells number at all concentrations tested ( Figure 13), the observation that is consistent with previous studies. 54 ' 55 However, the increased killing efficiency of minocycline when used in combination with 3 is likely attributable to the effect of 3 on the membrane. Attempts to select for resistance with combination of 3 and minocycline during 25 serial passages resulted in a 4-fold increase in MIC, as opposed to minocycline and tobramycin alone that had 16- and 256-folds increase respectively (Figure 5). Indeed, it is more difficult for bacteria to develop resistance to simultaneously-acting drug combination, especially when one of the drugs acts on the membrane 56"58 .
  • Galleria mellonella injection model has been commonly used in accessing the in vivo efficacy of antimicrobials against P. aeruginosa because it shares a high degree of structural and functional homology to the immune systems of vertebrates with both cellular and humoral defenses. 60
  • single dose combination of 3 (75 mg/kg) plus minocycline (75 mg/kg) or 3 (75 mg/kg) plus rifampicin (75 mg/kg) effectively protected larvae from XDR P. aeruginosa P262 infection with more than 75 % survival after 24 h, indicating the therapeutic potential of amphiphilic tobramycin as an adjuvant to treat infection caused by XDR P. aeruginosa.
  • a compound comprising tobramycin connected by a tether to a lysine-based peptoid mimic.
  • the lysine-based peptoid mimic may be an L-lysine or a D-lysine
  • the lysine-based mimic comprises a positively-charged lysine, a hydrophobic aromatic core, and an alkyl chain.
  • the lysine may be L-lysine D-lysine.
  • the compound comprises a structure as set forth Formula (I):
  • n is an integer between 2-20, indicating that the tether is of a length of between 2-20 carbons.
  • the tether or linker may be for example but by no means limited to an alkyl-based tether, an aromatic-based tether, a polyethyleneglycol-based tether or carbon chains with for example double or triple bonds.
  • the tether may comprise a length corresponding to 2-20, 3-20, 4- 20, 5-20, 6-20, 2-19, 2-18, 2-17, 2-16, 2-15, 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 4-20, 4- 19, 4-18, 4-17, 4-16, 4-15, 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 6-20, 6-19, 6-18, 6-17, 6- 15, 6-15, 6-14, 6-13, 6-12, 6-1 1 , 7-1 1 or 8-1 1 carbon atoms.
  • any suitable tether or linker known in the art may be used to connect the lysine-based peptoid mimic to the tobramycin provided that the tether is sufficiently flexible and sufficiently long that the tobramycin and the lysine-based peptoid mimic are able to function as discussed herein.
  • the tether is a flexible tether, as shown in Figure 1 .
  • the tether may comprise 3-11 repeating alkyl units.
  • the compound may be one of compounds 1-3:
  • a method of treating a bacterial infection in an individual in need of such treatment comprising administering to said individual an effective amount of a compound comprising tobramycin connected by a tether to a lysine-based peptoid mimic.
  • a person in need of such treatment is a person who has or is suspected of having a bacterial infection, particularly a bacterial infection that has been difficult to treat with conventional or prior art antibiotics alone or is a bacterial infection that is suspected of or known to be caused by a bacterial strain that may be drug resistant.
  • the bacterial infection is caused by a pathogenic gram- negative bacteria.
  • the pathogenic gram-negative bacteria may be for example but by no means limited to E. coli, Acetinobacter, Salmonella, Shigella, Enterobacteriaceae, Pseudomonas, Moraxella, Heliobacter, Stenotrophomonas, Bdellovibrio, Legionella and the like.
  • the pathogenic gram-negative bacteria is P. aeruginosa, E. coli or Acetinobacter baumannii.
  • the P. aeruginosa strain is an extremely drug-resistant strain of P. aeruginosa.
  • an "effective amount” is an amount that is sufficient to accomplish at least one of the following: reduction in the severity of the symptoms associated with the bacterial infection; and reduction in the CFU per ml of bacteria in a sample taken from the individual. As discussed herein, this may be accomplished by increasing or enhancing the anti-bacterial activity of another compound. For example, while not wishing to be bound to a particular theory or hypothesis, the inventors believe that the compounds described herein enhance the permeability of the gram-negative outer membrane and/or inner membrane and thereby in effect act as a delivery system.
  • an "effective amount” is an amount that is sufficient to enhance the permeability of the outer membrane of the infective bacteria so that an antibacterial agent can enter the bacterial cell.
  • an antibacterial agent can enter the bacterial cell.
  • other anti-bacterial agents may be administered at a much lower effective amount, as discussed herein.
  • the compound may be co-administered with an antibacterial compound such as an antibiotic.
  • an antibacterial compound such as an antibiotic.
  • the compound and the antibiotic may be co-administered simultaneously although this is not necessary.
  • a compound comprising tobramycin connected by a tether to a lysine-based peptoid mimic as discussed above for enhancing the antibacterial activity of an agent.
  • the agent does not necessarily have antibacterial properties on its own and may need to be co-administered with the compound in order to enter the bacterial cell.
  • the agent may have antibacterial properties on its own but these antibacterial properties may be enhanced by increased entry of the agent into the bacterial cell by the membrane permeability enhancing activity of the agent, as discussed herein.
  • a method of preparing a medicament for treatment of a bacterial infection comprising admixing a compound comprising tobramycin connected by a tether to a lysine-based peptoid mimic and a suitable excipient such as for example but by no means limited to a pharmaceutically acceptable excipient, which are well-known in the art.
  • the compound may be formulated for co-administration with an agent having anti-bacterial properties either alone or when administered in combination with the compound, as discussed herein.
  • the compound and the antibiotic of choice may be formulated as a single unit dosage or the compound and the antibiotic of choice may be separate unit doses but may be otherwise packaged together.
  • the antibiotic of choice that is, the coadministered antibiotic may be a tetracycline.
  • the antibiotic may be an antibiotic against which bacterial resistance is mediated by efflux pump activity.
  • the antibiotic is selected from the group consisting of fluoroquinolones (such as for example ciprofloxacin, moxifloxacin, and levofloxacin), tetracyclines (such as for example minocycline, tetracycline, tigecycline, and oxycycline), fosfomycin, anthelminthic drugs (such as for example benzimidazoles (such as for example albendazole, thiabendazole), niclosamide, and oxyclozanide), rifampicin, macrolides (such as for example erythromycin and others), sulfadrugs (such as for example sulfamethoxazole), trimethoprim, vancomycin and combinations thereof.
  • fluoroquinolones such as for example ciprofloxacin, moxifloxacin, and levofloxacin
  • tetracyclines such as for example minocycline, tetracycline, tigecycline,
  • the antibiotic is selected from the group consisting of: moxifloxacin; novobiocin; minocycline; rifampicin; chloramphenicol; erythromycin; trimethoprim; and vancomycin.
  • the reference compound ultrashort peptoid mimic 4 was synthesized by reductive amination of aromatic aldehyde 5 with dodecylamine 6 generating secondary amine 7 which after coupling to di-Boc-protected lysine produced protected lysine- based peptoid 8. Deprotection of the Boc-protecting groups with TFA afforded ultrashort lysine peptoid 4 as previously described ( Figure 1 1 ). 23
  • the synthesis of tobramycin- lysine conjugates 1-3 was achieved by preparing amphiphilic tobramycin derivatives (5 steps), followed by a single-step reductive amination conjoining and a final deprotection (Figure 12).
  • tobramycin derivatives Preparation of the tobramycin derivatives commenced by protecting the amines on commercially available tobramycin with Boc anhydride, followed by silylation of the hydroxyl groups with a bulky protecting group such as TBDMSCI to give 9. This is to ensure the more hindered C-5 position of the Boc- and TBDMS-protected tobramycin intermediate, the desired point of alkylation, is unprotected. This is the preferred position because C-5-modified tobramycin derivatives retained antibacterial activity 24 and superior adjuvant properties against gram-negative bacteria like P. aeruginosa.
  • reference peptoid mimic compound 4
  • tobramycin hybrids 1-3 against a panel of gram-negative and gram-positive bacteria are presented as the minimum inhibitory concentration (MIC) in Table 1.
  • MIC minimum inhibitory concentration
  • Reference peptoid mimic 4 with a C12 hydrophobe displayed weaker antimicrobial activity compared to the reported C10 peptoid, 23 with MIC > 8 pg/mL against all the strains tested in this study.
  • tobramycin-lysine conjugates there was a positive correlation between antimicrobial activity and the length of the carbon chain tether.
  • Compound 3 with C12 tether was the most potent analog of all the hybrids, and displays moderate activity against gram- positive bacteria (MIC of 2-32 pg/mL) but a relatively weak activity against gram- negative bacteria (MIC > 16 pg/mL). Further, the anti-pseudomonal activities of all compounds were evaluated against wild-type and seven clinical isolates of P. aeruginosa, including MDR, XDR, and colistin-resistant strains (Table 2). A similar trend of longer carbon chain displaying better activity was observed for drug-resistant P. aeruginosa as well, suggesting C12 as the optimal tether length of all analogues tested.
  • aeruginosa strains when compared to the antipseudomonal agents ciprofloxacin and ceftazidime but also nonpseudomonal drugs such as moxifloxacin, minocycline, rifampicin, chloramphenicol, erythromycin, and trimethoprim (Table 2).
  • the absolute MICs (the MIC of antibiotics in the presence of adjuvants at 4 pg/mL) of minocycline or rifampicin in combination therapy with hybrids was dramatically lower than monotherapy, in particular with compound 3 where MICs of minocycline and rifampicin were reduced from 8 pg/rnL and 16 pg/mL in monotherapy to 0.25 pg/rnL (32-fold potentiation) and 0.0625 pg/rnL (256- fold potentiation), respectively.
  • combinations of 3 with minocycline and rifampicin were selected for further synergy studies against a panel of P. aeruginosa clinical isolates (Table 4).
  • the susceptible breakpoints of minocycline (MIC ⁇ 4 pg/rnL) against Acinetobacter spp. were reached for all minocycline-resistant, MDR, or XDR P. aeruginosa isolates at 4 g/mL of 3.
  • PAO200 is a MexAB-OprM deletion strain while PAO750 is an efflux-sensitive strain that lacks five different clinically-relevant RND pumps (MexAB-OprM, MexCD-OprJ, MexEF-OprN, MexJK, and MexXY) and the outer membrane protein OpmH. Some of these pumps are homologues of broad substrate specificities that expel different classes of antimicrobial agents and confer resistance on P. aeruginosa.
  • the tripartite protein system MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY-OprM allow the translocation of a wide variety of substrates such as quinolones, chloramphenicol, trimethoprim, imipenem, and tetracyclines out of the cell.
  • substrates such as quinolones, chloramphenicol, trimethoprim, imipenem, and tetracyclines out of the cell.
  • the synergistic MIC of rifampicin in combination with 4 pg/mL of 3 against rifampicin-resistant E. cloacae 1 17029 was 0.063 pg/mL, which is 16-fold lower than rifampicin-susceptible breakpoints of ⁇ 1 pg/mL
  • Minocycline, rifampicin, or 3 at sub-inhibitory concentrations were unable to suppress bacteria growth in monotherapy, even after 6 h exposure (Figure 4).
  • sub-MIC (1 /8 to 1 /2 MIC) of 3
  • in vitro bactericidal activities of minocycline and rifampicin were both enhanced, yielding synergistic killing at sub-MIC concentration (1 /8 ⁇ MIC) after 90 mins of incubation (Figure 4).
  • a potential problem usually associated with membrane-active agents is their toxicity towards eukaryotic cells.
  • the hemolytic properties of the hybrid molecules were first examined using freshly collected pig erythrocytes. All hybrids demonstrated lower hemolytic activities ( ⁇ 20 %) relative to 4, which is highly toxic with 87 % hemolysis at the highest measured concentration of 512 g mL ( Figure 9A).
  • SAR structure- activity relationships
  • NMR spectra ( 1 H, 13 C, DEPT, COSY, HSQC and HMBC) were recorded on a Bruker Avance 500 spectrometer (500 MHz for 1 H NMR, 126 MHz for 13 C). All reactions were monitored by analytical thin-layer chromatography (TLC) on pre-coated silica gel plates 60 F254 (0.25 mm, Merck, Ontario, Canada), and the spots were visualized by ultraviolet light and/or by staining with ninhydrin solution in n-butanol.
  • TLC analytical thin-layer chromatography
  • Mass spectrometry was carried by ESI analyses on a Varian 500 MS Ion Trap Mass Spectrometer, and MALDI-TOF on a Bruker Daltonics Ultraflex MALDI TOF/TOF Mass Spectrometer. Chromatographic separations were performed on a silica gel column by flash chromatography (Kiesel gel 40, 0.040-0.063 mm; Merck, Ontario, Canada). Yields were calculated after purification. When reactions were carried out under anhydrous conditions, the mixtures were maintained under nitrogen atmosphere. Analytical HPLC was performed on Hitachi LC system equipped with autosampler, using Superspher 100 RP-18 column and a detection wavelength of 260 nm. The purity of final compounds determined by HPLC analysis were > 95%.
  • reaction mixture was subsequently concentrated, re-dispersed in diethyl ether, and treated with 2N NaOH (20.0 mL) at room temperature for additional 15 mins.
  • the organic layer was separated from the aqueous phase, washed with water and brine, dried over anhydrous Na2SO4, concentrated, and purified by flash column chromatography (eluted with CH2Cl2/MeOH from 300:0 to 30:1 , v/v) to afford the desired compound as solid (Yield: 70 %).
  • Boc-Lys(Boc)-A/-dodecyl-10-Aminomethyl-9-chloroanthracene Boc-Lys(Boc)-OH (0.520 g, 1 .5 mmol) dissolved in DMF:CHCI 3 (5:2, v/v) (7.0 ml_) was activated with DIPEA (0.388 g, 3.0 mmol) and HBTU (0.569 g, 1 .5 mmol) at 0 °C for 15 mins, and treated with 7 (0.410 g, 1 .0 mmol).
  • Boc-Lys(Boc)-N-alkyl-aromatic compound 8 (0.222 g, 0.3 mmol) was dissolved in CH2Cl2:TFA (2:1 , v/v) (20.0 ml_) and stirred at room temperature for 1 h. The reaction was monitored by TLC (ChbCla/NHUOH/MeOH, 5:1 :1 ). At the end of the reaction, the mixture was evaporated to dryness, and purified by C-18 reverse-phase flash column chromatography (eluted with deionized water) to get analytically pure compound 4 (Yield: 62 %).
  • Boc-Lys(Boc)-OH (0.520 g, 1 .5 mmol) dissolved in DMF (30.0 mL) was activated with DIPEA (0.388 g, 3.0 mmol) and HBTU (0.569 g, 1 .5 mmol) at 0°C for 15 mins and subsequently treated with 13a-c (1.0 mmol). The mixture was stirred at 0°C to room temperature overnight. The reaction progress was monitored by TLC (CH 2 CI 2 /MeOH, 35:1 ), and at the end, the mixture was diluted with water and extracted with EtOAc. The organic layer was washed with water and brine, dried over anhydrous Na2S04, and concentrated.
  • Lys 1 .64 - 1 .57 (m, 2H, 6-CH 2 of Lys), 1 .44 - 1.35 (m, 3H, ⁇ - CH of Lys, CH 2 of linker), 1 .32 - 1 .27 (m, 1 H, CH 2 of linker), 1 .1 1 - 1 .04 (m, 1 H, CH 2 of linker), 1 .02 - 0.91 (m, 2H, CH 2 of linker), 0.83 - 0.63 (m, 4H, Ctf 2 of linker), 0.62 - 0.53 (m, 3H, CH 2 of linker).
  • Bacterial isolates were obtained as part of the Canadian National Intensive Care Unit (CAN-ICU) study 61 and Canadian Ward Surveillance (CANWARD) studies 62 ' 63 .
  • the CAN-ICU study included 19 medical centres across Canada with active ICUs. From September 2005 to June 2006, 4180 isolates represented in 2580 ICU patients were recovered from clinical specimens including blood, urine, wound/tissue, and respiratory specimens (one pathogen per cultured site per patient). Only "clinically significant" specimens from patients with a presumed infectious disease were collected. The isolates obtained were shipped to the reference laboratory (Health Sciences Centre, Winnipeg, Canada) on Amies charcoal swabs.
  • isolates were sub-cultured onto appropriate medium and stocked in skim milk at -80 °C until subsequent MIC testing was carried out.
  • the quality control strains including Staphylococcus aureus ATCC 29213, methicillin-resistant S. aureus (MRSA) ATCC 33592, Enterococcus faecalis ATCC 29212, Enterococcus faecium ATCC 27270, Streptococcus pneumoniae ATCC 49619, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Klebsiella pneumoniae ATCC 13883 were acquired from the American Type Culture Collection (ATCC).
  • MRSA methicillin-resistant S. aureus
  • CANWARD-2008 81388 was obtained from the 2008 CANWARD study, while gentamicin-resistant tobramycin-resistant ciprofloxacin-resistant [aminoglycoside modifying enzyme aac(3)-lla present] E. coli CANWARD-2011 97615, and gentamicin-resistant tobramycin-resistant P. aeruginosa CANWARD-201 1 96846 were obtained from the 201 1 CANWARD study.
  • P. aeruginosa PA01 P. aeruginosa P259-96918
  • aeruginosa P264-104354 colistin-resistant P. aeruginosa 91433, colistin-resistant P. aeruginosa 101243, A. baumannii AB027, A. baumannii AB030, A. baumannii AB031 , A. baumannii 1 10193, Enterobacter cloacae 1 17029, and Klebsiella pneumonia 1 16381 were kindly provided by Dr. George G. Zhanel.
  • the efflux pump-mutated strains, P. aeruginosa PAO200 and P. aeruginosa PAO750 were provided by Dr. Ayush Kumar from University of Manitoba in Canada.
  • Multi-drug resistance in P. aeruginosa was defined as concomitant resistance to 3 or more chemically unrelated antimicrobial classes, while extremely drug resistant was defined as concomitant resistance to 5 or more chemically unrelated antimicrobial classes.
  • the antimicrobial activity of the compounds against a panel of bacteria was evaluated by microliter dilution method in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines.
  • CLSI Clinical and Laboratory Standards Institute
  • the overnight bacterial culture was diluted in saline to 0.5 McFarland turbidity, and then 1 :50 diluted in Mueller-Hinton broth (MHB) for inoculation.
  • the antimicrobial agents were 2-fold serially diluted in MHB in 96-well plate and incubated with equal volumes of inoculum for 18 h at 37 °C. The lowest concentration that prevented visible bacterial growth was taken as the MIC for each antimicrobial agent.
  • the broth with or without bacterial cells was employed as positive or negative controls, respectively.
  • FIC index was determined by setting up standard checkerboard assay in 96-well plate as previously described. 64 Each antibiotic to be tested was serially diluted along the abscissa in MHB, while adjuvant was diluted along the ordinate to create a 10 ⁇ 7 matrix.
  • the bacterial culture was prepared in MHB by 1 :50 dilution from the 0.5 McFarland turbidity culture in saline. The inoculum was added to each well of the plate and incubated for 18 h at 37 °C. After the incubation, plates were read on EMax ® Plus microplate reader (Molecular Devices, Sunnyvale, CA, USA). MIC was recorded as wells with the lowest concentration of drugs with no bacterial growth.
  • the FIC for each antibiotic was calculated as the concentration of the antibiotic for a well showing no growth in the presence of adjuvant divided by the MIC for that antibiotic alone.
  • the FIC for each adjuvant was calculated as the concentration of the adjuvant for a well showing no growth in the presence of antibiotic divided by the MIC for that adjuvant alone.
  • the FIC index is the sum of the two FICs. Chemical-chemical interactions with an FIC index ⁇ 0.5 were deemed synergistic; 0.5 - 4, no interaction; and > 4, antagonism.
  • the kinetics of bacterial killing was measured using P. aeruginosa PA01 , as previously described. 59 Overnight bacterial culture was diluted in saline to 0.5 McFarland turbidity and then 1 :50 diluted in Luria-Bertani (LB) broth. The cell suspension was incubated with minocycline, rifampicin, or hybrid 3 diluted in PBS (pH 7.2) alone at desired concentrations (1/2 ⁇ , 1 ⁇ , 2 ⁇ , 4 ⁇ MIC).
  • Wild-type P. aeruginosa PAO1 was used to study resistance development against antibiotics by sequential passaging method as previously described. 65 Briefly, MIC testing was first conducted for all drugs or drug combinations to be tested, as described above. After 18 h incubation, the bacterial cells growing in the well of half- MIC concentration were harvested and diluted to 0.5 McFarland in saline followed by 1 :50 dilution in fresh MHB broth. The inoculum was subjected to next passage MIC testing, and the process repeated for 25 passages. The fold change in MIC was plotted against the number of passages.
  • the CFDASE dye was used to determine the outer membrane permeability of drugs against P. aeruginosa PAO1 , following established protocols. 28 Logarithmic phase P. aeruginosa was harvested by centrifugation and washed twice with PBS. The bacterial cells were resuspended in the same buffer to OD600 of 0.5, followed by staining with CFDASE at 100 ⁇ for 30 mins at 37°C. The unbound dye was then removed by washing the cells with excess buffer, and the cells were again resuspended to the initial volume.
  • the bacterial suspension was treated with drugs at 37 °C for 30 mins at desired concentration and the supernatant obtained by centrifugation was transferred to 96-well black plate for measuring the fluorescence at an excitation wavelength of 488 nm and an emission wavelength of 520 nm using a microplate reader FlexStation 3 (Molecular Devices, Sunnyvale, CA, USA).
  • the membrane-potential-sensitive fluorescent dye was utilized to determine the membrane depolarization of P. aeruginosa PAO1 as previously described. 19 Overnight growth P. aeruginosa PAO1 was diluted in fresh LB broth and cultured to the mid-log phase. The bacterial cells were harvested and washed three times with 5 mM sodium HEPES buffer, pH 7.4, containing 20 mM glucose, and resuspended to ⁇ of 0.05 in the same buffer. The cell suspension was incubated with 0.2 mM EDTA and 0.4 ⁇ diSC3-5 in the dark for 2 h at 37 °C under constant shaking (150 rpm).
  • Cell motility assay was performed on 0.3 % (w/v) agar media supplemented with tryptone (5 g/L) and NaCI (2.5 g/L).
  • 66 Antimicrobial agents were added to 25 ml_ medium to the desired concentration and poured on 100 ⁇ 15 mm petri dishes followed by 2 h drying.
  • Overnight P. aeruginosa PAO1 culture was diluted in 0.85 % saline to 1 .0 McFarland and point inoculated into the center of the motility agar plates. Plates were incubated at 37 °C for 20 h. The images presented were taken using a FluroChem®Q (Cell biosciences).
  • the hemolytic activity of the compounds was determined as the amount of hemoglobin released by lysing pig erythrocytes.
  • Fresh pig blood (provided by Dr. Charles M. Nyachoti from University of Manitoba) drawn from pig antecubital were centrifuged at 1 ,000 ⁇ g for 5 mins at 4 °C, washed with PBS thrice, and resuspended in the same buffer.
  • Compounds were 2-fold serially diluted in PBS in 96-well plate and mixed with equal volumes of erythrocyte solution.
  • DU145 ATCC, Manassas, VA, USA
  • JIMT-1 DSMZ, Braunschweig, Germany
  • Dulbecco's modified Eagle's References medium supplemented with 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 10 % (v/v) fetal bovine serum (FBS) at 37 °C under a humidified atmosphere of 5 % CO2 and 95 % air.
  • FBS fetal bovine serum
  • the methanethiosulfonate (MTS) cell viability assay was employed to measure the cytotoxicity of compound 3 as previously described.
  • the cells were seeded in 96-well plate with a final concentration of 7500-9000 cells per well and incubated for 24 h. Then the cells were treated with test compound at final concentrations of 2.5 to 30 ⁇ and incubated for an additional 48 h at the same condition. MTS reagent (20 %, v/v) was further added to each well and the plates were incubated for 4 h on a Nutating mixer in the incubator. The optical density was measured using a SpectraMax M2 plate reader (Molecular Devices, Sunnyvale, CA, USA) at 490 nm. Only medium without cells were served as blank and the blank values were subtracted from each sample value. The cell viability relative to the control with vehicle was calculated.
  • Galleria mellonella waxworms were obtained from The Worm Lady ® Live Feeder Insects. Worms (average weight at 250 mg) were used within 7 days of delivery to determine the survival rate after bacteria or antimicrobials injection using previously described methods. 68 The tolerability study was performed by only injecting antimicrobial agent into the worms at 100 and 200 mg/kg without bacteria. The worms (ten larvae in each group) were incubated at 37 °C and monitored for 96 h for survival. For therapeutic study, overnight XDR P. aeruginosa P262 culture was diluted in PBS to a final concentration of 1 .0 ⁇ 10 3 CFU/mL. 15 larvae per group were infected with 10 pL bacterial suspensions.
  • worms in monotherapy experimental groups received a 10 pL injection of minocycline, rifampicin, or compound 3 individually at 75 mg/kg.
  • 3 plus minocycline and 3 plus rifampicin were injected to give final dosages of 12.5 + 12.5, 25 + 25, 37.5 + 37.5, and 75 + 75 mg/kg respectively.
  • vehicle (PBS) without antimicrobials was injected as control group. The larvae were monitored for 24 h at 37 °C in petri dishes lined with filter paper and scored for survivability. Larvae considered dead if they do not respond to touch.
  • E.coli CAN-ICU 63074 (AMK 32) 8 b 64 64 128 16
  • MIC Minimum inhibitory concentration
  • MRSA Methicillin-resistant S. aureus
  • MSSE Methicillin-susceptible S. epidermidis
  • MRSE Methicillin-resistant S. epidermidis
  • CANWARD Canadian Ward surveillance
  • CAN-ICU Canadian National Intensive Care Unit surveillance
  • CAZ Ceftazidime
  • GEN-R Gentamicin-resistant
  • AMK Amikacin
  • TOB-R Tobramycin-resistant
  • CIP-R Ciprofloxacin-resistant.
  • Minocycline 8 0.5 0.063 3 32 1 0.031 0.094
  • Colistin 1 1 1 3 32 1 0.031 1.031
  • Vancomycin >1024 128 ⁇ 0. 125 3 32 4 0.125 0.125 ⁇ 0.25
  • MIC a FIC Hybrid (MIC) FIC FIC index Absolute MIC b Potentiation 0 strain
  • PAO750 Rifampicin (8) 3 (8) 0. 156
  • PAOl Minocycline 8 1 Tobramycin 0.25 0.016 0.064 1 .064
  • PAOl Minocycline 8 8 1 4 256 1 0.004 1.004
  • PAOl Minocycline 8 4 0.5 1 >256 1 ⁇ 0.004 0.5 ⁇ ; ⁇ 0.504
  • PAOl Minocycline 8 0.5 0.063 2 128 1 0.008 0.071
  • PAO l Minocycline 8 0.5 0.063 3 32 0.031 0.094
  • PAOl Rifampicin 16 8 0.5 Tobramycin 0.25 0.125 0.5 1

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Abstract

L'invention concerne des conjugués amphiphiles de tobramycine liés à un mimétique peptoïde à base de lysine par l'intermédiaire d'une attache. Lesdits mimétiques peptoïdes à base de lysine comprennent une L-lysine chargée positivement, un noyau aromatique hydrophobe et une attache alkylène assemblée par l'intermédiaire d'une liaison amide tertiaire. L'optimisation du conjugué obtenu est fournie au moyen d'une attache alkylène en C12. Ces conjugués sont utiles en tant qu'agents antibactériens, notamment lorsqu'ils sont utilisés conjointement avec un autre agent antibactérien (tel que la rifampicine ou la minocycline), cette combinaison résultant en une activité synergique contre les bactéries résistant aux médicaments (en particulier P. aeruginosa multirésistant). Par conséquent, ces conjugués constituent des adjuvants efficaces aux antibiotiques qui contribuent à vaincre la résistance aux antibiotiques de bactéries à Gram négatif.
PCT/CA2018/050439 2017-04-13 2018-04-10 Conjugués amphiphiles de tobramycine liés à un mimétique peptoïde à base de lysine par l'intermédiaire d'une attache Ceased WO2018187867A1 (fr)

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