A potent antibiotic-loaded bone-cement implant against staphylococcal bone infections

Abstract

New antibiotics should ideally exhibit activity against drug-resistant bacteria, delay the development of bacterial resistance to them and be suitable for local delivery at desired sites of infection. Here, we report the rational design, via molecular-docking simulations, of a library of 17 candidate antibiotics against bone infection by wild-type and mutated bacterial targets. We screened this library for activity against multidrug-resistant clinical isolates and identified an antibiotic that exhibits potent activity against resistant strains and the formation of biofilms, decreases the chances of bacterial resistance and is compatible with local delivery via a bone-cement matrix. The antibiotic-loaded bone cement exhibited greater efficacy than currently used antibiotic-loaded bone cements against staphylococcal bone infections in rats. Potent and locally delivered antibiotic-eluting polymers may help address antimicrobial resistance.

Extended Data Fig. 1.. Physico-chemical characterization of VCD-077 impregnated PMMA beads

(a-c) FT-IR spectrum of different groups (a) VCD-077, (b) PMMA, (c) VCD-077 impregnated PMMA at (40:1) with VCD-077 peaks 1 (3352.75 cm⁻¹), 2 (3114.27 cm⁻¹), 3 (1643.82 cm⁻¹), 4 (1615.48 cm⁻¹), 5 (1595.79 cm⁻¹), 6 (1550.68 cm⁻¹), 7 (1352.31 cm⁻¹), 8 (1313.02 cm⁻¹). (d) Release of VCD-077 from Smartset HV^® (PMMA) bead at different drug:polymer ratio (1:40, 2:40 and 3:40) in pH 7.4 buffer. Data is represented as mean ± SD (n=3). (e) Release of VCD-077 from Smartset HV^® (PMMA) bead at different particle size from drug:polymer ratio (1:40) or (f) at different temperatures, in pH 7.4 buffer. Data is represented as mean ± SD (n=3).

Fig. 1.. Design and in silico screening a library of next-generation antibiotics.

(a) Schematic showing the design of a library of next-generation antibiotics starting with a fluoroquinolone (FQ) backbone and functionalized bio-active heterocyclic pharmacophores with general formula I, II, III and IV. IA and IIA belong to the sub-family of I and II respectively. Distance between piperazine nitrogen to C5 in case of five membered heterocyclic motif (Formula I to III) and C6 for 9-memebered fused heterocyclic unit (Formula IV) was maintained at 6.1 to 6.8 A in all the energy minimized docked poses of ligands. (b) Schematic showing the screening of lead molecule from a library of 17 FQ derivatives. (c-d) Surface diagram of QBP of gyrase-DNA complex in (c), wild type (PDB ID: 5CDQ) and (d), mutant variant of the protein. In the mutant variant, 2 critical residues Ser84 and Glu88 were changed to Leu84 and Lys88 to reflect the mutations in nature. The surface area of QBP has been altered to 4181.9 sq. Å compared to 4383.6 sq. Å in wild type. (e-h) Docking poses of selected VCD molecules to S. aureus DNA gyrase in complex with DNA. Binding pose of (e), VCD-068 and (f) VCD-077 to wild type S. aureus DNA gyrase-DNA complex. The crystal structure has been taken from PDB ID: 5CDQ. VCD-068 and VCD-077 both maintained stable water-Mg²⁺ bridge interaction while interacting with at GyrA amino acid residues and additional interactions with GyrB residues. Binding pose of (g) VCD-068 and (h) VCD-077 to mutated S. aureus DNA gyrase-DNA complex with mutations Ser84Leu and Glu88Lys. Mutation generally causes perturbation in water-Mg²⁺ bridge interaction and steric clashes between bulky Leu residues and VCDs. However, in both cases, docking pose is marginally affected and interactions with GyrB are maintained. The residue numbers mentioned in the structures are based on Staphylococcus aureus DNA numbering of the PDB file. Dotted lines denote the H-bond interactions between ligand (cyan) and amino acid residues (green). The mutated residues are labeled in red and drawn in yellow. The DNA residues (orange) are coded as DA: deoxyadenosine; DG: deoxyguanosine; DT: deoxythiamine and DC: deoxycytosine. Figures were drawn using PyMol. (i) The docking score (kcal/mol) of seventeen VCD compounds against mutated S. aureus gyrase protein compared to moxifloxacin (number 18; closed triangle). Based on interactions and docking scores, five VCDs, VCD-068, VCD-077, VCD-176, VCD-367 and VCD-380 were found to maintain favourable docking pose with mutated gyrase protein as compared with moxifloxacin, a clinically-approved fluoroquinolone, which was used as control.

Fig. 2. In vitro efficacy against clinical isolates of sensitive and resistant bacterial strains.

MIC distribution of VCD-077 compared to moxifloxacin when screened against 50 clinical isolates of Staphylococcus aureus collected from US medical centers within the SENTRY Antimicrobial Surveillance Program. VCD-077 exhibited an MIC value of less than 1μg/ml in most strains.

Fig. 3.. VCD-077 retards resistance development.

Generation of resistant mutants by VCD-077 using serial passage assay showing changes in MIC through 28 passages against (a) S. aureus (ATCC 25923) and (b) MRSA (ATCC 43300). VCD-077 has low propensity to develop resistance even after prolonged exposure (c) Effect of VCD-077 on DNA supercoiling using S. aureus DNA gyrase. The data is represented as percentage of DNA supercoiling (mean ± S.D., n=3). The percentage of supercoiling of the relaxed plasmid DNA in presence of S. aureus DNA gyrase was measured by estimating the intensity of the supercoiled plasmid DNA band. Ciprofloxacin was used as a positive control. The intensity of the supercoiled DNA band in absence of any drug was taken as 100%. Statistical analysis was performed using unpaired t-test with Welch correction. VCD-077 inhibited S. aureus DNA gyrase-mediated DNA supercoiling in a dose-dependent manner and to a greater degree than equimolar concentration of ciprofloxacin. (d) Cell viability of E. histolytica (which is responsive to the effect of nitro-heterocyclic but not quinolone motifs) upon the treatment of VCD-077. Moxifloxacin was used as a negative control and nitazoxanide, a nitroheterocyclic compound, was used as a positive control. Data is represented as percentage of cell viability (mean ± S.D., n=6). Statistical analysis was performed using two-tailed t-tests (*p<0.0001 vs vehicle control).

Fig. 4.. Antibiofilm activity of VCD-077.

Confocal images showing effect of drug (16 μg/ml) on SYTO^® 9 stained biofilms of S. aureus ATCC 6538P grown for 72 h. (b) SEM images showing effect of drug (16 μg/ml) on matured biofilms (72 h) of S. aureus ATCC 6538P. The magnification of the image was 5000×. VCD-077 has potent anti-biofilm activity against S. aureus unlike vancomycin or gentamicin.

Figure 5:. Physico-chemical characterization of PMMA loaded beads and cylinders.

(a) Pictorial representation of VCD-077 loaded beads with 4.6 mm height and 4.2 mm diameter and cylinders of 12 mm height and 6 mm diameter. (b) Drug release kinetics from 3 different bone cements. Data is represented as mean ± SD (n=3). The best fit curves for the release kinetics of VCD-077 from Palacos (R² = 0.9971), Simplex (R² = 0.9909) and Smartset HV^® (R² = 0.9849) bone cements. A quick onset and sustained release were observed from Smartset HV^® versus the other two variants. (c) Comparative drug release rate from different bone cement beads at 40:3 polymer drug ratio calculated from the release kinetics. The data is represented as mean ± S.D. (n=3). Statistical analysis was performed using two-tailed t-tests (*p<0.005 compared to other two cements). Smartset HV^® shows higher drug release rates (3.6682 μg/ml/√hr) compared to Palacos R^® (0.8309 μg/ml/√hr) and Simplex P^® (0.7301 μg/mL/√hr) PMMA bone cements. (d) Comparative drug release rate from VCD-077 impregnated Smartset HV^® bone cement beads at different polymer:Drug ratio. Data is represented as mean ± S.D. (n=2). Statistical analysis was performed using two-tailed t-tests (*p<0.005 compared to other two cement:drug combinations). Higher drug loading enhances drug release rates (3.277 μg/ml/√hr) by 10X compared to drug release rate (0.315 μg/ml/√hr) at low drug : polymer ratios (1 : 40) in Smartset HV^® PMMA bone cement. (e) Surface morphology (SEM images) of Smartset HV^® beads with or without drug (at 40:1 polymer-drug ratio) at day 0 and at 14^th day of drug release stage. Solvent channels observed after drug release on day 14 are denoted with yellow arrows. The conspicuous micro channels in the cement mantle can lead to widening of the open spaces in the drug polymer network, compared to one without drug. (f) Compression strength as per ASTM F451 of Smartset HV^® (PMMA) cylinders alone, VCD-077 loaded PMMA cylinders with drug:polymer ratio 3:40 on Days 0, 7 and 14 during drug release. Data is represented as mean ± SD (n=5). Statistical analysis was performed using two-tailed t-tests (ns: nonsignificant; p>0.5 w.r.t Smartset HV^® without drug).

Figure 6:. VCD-077-embedded bone cement beads are effective against bone infections.

(a) Procedure and experimental design for in vivo bone infection model to test the prophylactic activity of antibiotics-loaded bone cement. (b) Graph shows bacterial load (log₁₀CFU/ml) in bone tissue of the S. epidermidis-infected tibia, determined at designated time points during the efficacy experiment. Drug-loaded PMMA beads were implanted at the site of infection on day 0 prophylactically (except in infection control group). Smartset PMMA bone cement served as the negative control. The data is represented as mean ± SD (n = 4) (*p=0.004; **p<0.0001). (c) H&E-stained images of bone sections at Day 28 from animal efficacy model. (d) Schematic shows experimental design for testing VCD-077-impregnated PMMA bone cement in an experimental model of established osteomyelitis. S. epidermidis (ATCC 35984, 25 μl of bacterial innoculum at 2 ×10⁷ CFU/ml) was injected through a hole drilled into the medullary cavity of the tibia, and the wound was closed. Twenty-one days post-infection, the animals underwent limited surgical debridement of the infection site, and a SmartSet HV PMMA loaded with VCD-077 (3 mm bead) placed by boring a ~3.2 mm hole through the cortical bone. SmartSet HV PMMA (Veh) served as negative control while SmartSet HV PMMA loaded with either gentamicin or rifampicin served as positive controls. Graph shows the efficacy of different bone cements in reducing bacterial load at day 10 post-application. Data shown are mean ± SEM (n=3–4, ANOVA followed by Tukey’s multiple comparison test).