In vitro studies
Thirty clinical isolates of MRSA were studied. The minimum inhibitory concentration (MIC) of oxacillin (Sigma-Aldrich, St. Louis, MO), vancomycin HCl (Sigma-Aldrich), and BT2-peg2-vancomycin diacetate containing 82.4% vancomycin (Pradama Inc., Louisville, KY) were determined using broth microdilution according to Clinical and Laboratory Standards Institute (CLSI) guidelines (Clinical and Laboratory Standards Institute 2012; Clinical and Laboratory Standards Institute 2013). The minimum bactericidal concentration (MBC) of vancomycin HCl and BT2-peg2-vancomycin diacetate were also determined according to CLSI guidelines (Clinical and Laboratory Standards Institute 1999).
Hydroxyapatite (HA) binding assay
10-5 M solutions of tetracycline, vancomycin HCl or BT2-peg2-vancomycin diacetate in 50 mM Tris–HCl buffer, pH 7.4, 1% DMSO were prepared. Tetracycline was used as a reference analyte; at 10-5 M, approximately 50% was bound to HA. An HA slurry of 0.5 g HA/100 ml 50 mM Tris–HCl buffer, 1% DMSO was prepared. For each analyte, two samples were tested in triplicate. For one sample, 1 ml of 10-5 M analyte and 100 μl 50 mM Tris–HCl buffer, 1% DMSO was pipetted into a microcentrifuge tube. For the second sample, 1 ml of 10-5 M analyte and 100 μl of the HA slurry was pipetted into a microcentrifuge tube. The samples were mixed gently by inversion for five minutes and then centrifuged at 12,000 g for three minutes to sediment the HA. The supernatant was transferred to another microcentrifuge tube. An electronic spectral scan (ultraviolet–visible) from 220–520 nm was obtained for each analyte using a Varian Cary 300 Bio Scan (Agilent Technologies, Santa Clara, CA). The blank was 50 mM Tris–HCl buffer, 1% DMSO. The wavelength of maximum absorbance (λmax) was determined, and the extinction coefficient (ϵ) calculated using the Beer-Lambert law. Absorbance of the samples incubated with HA was measured at λmax, and the molar concentration of the analyte determined using the Beer-Lambert law and the previously calculated extinction coefficient. The fraction adsorbed to HA was calculated for each sample. The HA binding capacity of each analyte was normalized to tetracycline by calculating the HA binding index, defined as percentage of analyte bound to HA/percentage of tetracycline bound to HA × 100 (Neale et al. 2009).
In vivo studies
The experimental model described was performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Mayo Clinic. Experimental osteomyelitis was established in 90 male Wistar rats using a modification of Zak’s model of experimental osteomyelitis (O'Reilly & Mader 1999). In earlier studies by our laboratory, we found that this model was highly reproducible with histological changes similar to those observed in humans with chronic osteomyelitis (Patel et al. 2000; Rouse et al. 2006). Animals were anesthetized with ketamine and xylazine and the proximal third of the medial left tibia surgically exposed. A 0.5 mm hole was drilled into the medullary cavity. Fifty microliters of morrhuate sodium (a sclerosing agent) was injected into the cavity, followed by 50 μl sterile saline containing 107 colony forming units (cfu) of MRSA IDRL-4293. The muscle was reattached with coated vicryl sutures (Ethicon, Somerville, NJ). The skin was closed with tissue glue (Tissuemend, Butler Animal Health Supply, Dublin, OH) and wound clips (Harvard Apparatus, Holliston, MA). The wound was sprayed with a wound adhesive (Aluspray, Butler Animal Health Supply, Dublin, OH), along with a chewing repellent (Chewguard, Summit Hill Laboratories, Tinton Falls, NJ).
Four weeks after establishing infection, five rats were sacrificed, the left tibia aseptically removed and quantitative cultures performed. The remaining animals underwent surgical débridement of the infection site with sterile saline and were arbitrarily assigned to one of five study arms for a duration of 21 days: No treatment (n=17), vancomycin HCl every 12 hours (n=17, 42 doses), BT2-peg2-vancomycin diformate every 12 hours (n=17, 42 doses), BT2-peg2-vancomycin diformate every 12 hours for 3.5 days and then every fourth day thereafter (n=16, 11 doses), and BT2-peg2-vancomycin diformate once per week (n=15, 3 doses). Vancomycin HCl was administered at 50 mg/kg and BT2-peg2-vancomycin diformate at 63.85 mg/kg (equivalent to 50 mg/kg active vancomycin); both were administered intraperitoneally. Twelve hours after completion of therapy (day 50), blood was collected via cardiac puncture for hematology and chemistry studies [performed by Charles River (Wilmington, MA)], and drug concentration studies. Rats were sacrificed with CO2. The left (infected) tibia from one animal in each group was collected for histopathology with bones placed in 10% neutralized buffered formalin (NBF), decalcified, embedded in parafilm, prepared as longitudinal cuts and stained with hematoxylin and eosin. The left (infected) tibias from the remaining animals were weighed, cryopulverized and processed for quantitative and qualitative bacterial culture. Culture results were expressed as log10 cfu/g of bone tissue.
Ten MRSA colonies per animal recovered from quantitative bacterial cultures were tested for susceptibility to BT2-peg2-vancomycin diformate and/or vancomycin HCl.
Kidneys from all animals were weighed, observed for abnormalities in size or color, and placed in NBF. Histological analysis was performed by Seventh Wave (Chesterfield, MO). Microscopic lesions were graded on a scale of one to four, with four being the most severe. Tubulointerstitial nephritis was characterized by multifocal non-suppurative interstitial inflammation, interstitial fibrosis, tubular epithelial degeneration, necrosis, and regeneration with frequent intra-tubular neutrophilic inflammation and cellular debris.
Three right tibias from each group were collected, weighed, placed into NBF, and sent to Histion LLC (Everett, WA). They underwent decalcification and were embedded in paraffin, cut into three frontal sections, and stained with either Masson’s trichrome, hematoxylin and eosin, or toluidine blue. Sections were sent to Think Bone Consulting (Seattle, WA) for histomorphometry performed using an OsteoMeasureXP system (Osteometrics, Inc., Atlanta, GA). Bone mass and possible toxic effects were assessed by measuring tissue area (bone/marrow), bone area and perimeter, and perimeter of bone lined by osteoblasts.
Plasma concentrations of BT2-peg2-vancomycin in infected animals were measured by liquid chromatography/mass spectrometry (LC/MS). Plasma samples (50 μl) were mixed with 200 μl of 25 μg/ml teicoplanin (Sigma, St. Louis, MO) in 1% formic acid. Proteins were precipitated with 750 μl of acetonitrile (ACN) and centrifuged at 13,000 RPM for two minutes. The supernatant was collected and concentrated to 50 μl by speedvac, diluted with 100 μl 5% ACN in 0.1% formic acid, loaded onto a prewashed C18 spin column (Nest Group, Inc, Southborough, MA), washed three times with 100 μl 5% ACN in 0.1% formic acid, and eluted twice with 100 μl 5% ACN in 0.1% formic acid. The elutes (50 μl) were diluted with 450 μl 5% ACN in 0.1% formic acid and analyzed by Accela LC System (Thermo Scientific, San Jose, CA) coupled with a LTQ-Orbitrap XL mass spectrometer (Thermo Scientific, San Jose, CA). Samples were loaded onto a 50 × 2.1 mm × 1.9 μm Hypersil GOLD column (Thermo Scientific, San Jose, CA) and eluted with a 17 minute binary solvent gradient (solvent A: 5% ACN/0.1% formic acid and solvent B: 95% ACN/0.1% formic acid) at 100 μl/minute. The gradient started from 5% solvent B, increased linearly to 80% solvent B in 12 minutes, and then remained at 80% B for 5 minutes. Elutes from the LC column were ionized by electrospray ionization and BT2-peg2-vancomycin and teicoplanin were detected by multiple reaction monitoring. Full scan fragment spectra from precursor ions (double charged m/z 880.3 from 5.5 to 7 minutes for BT2-peg2-vancomycin and double charged m/z 940.8 from 7 to 10 minutes for teicoplanin) were acquired by orbitrap. Compound confirmation was obtained from fragment spectra and concentration was calculated from peak areas of fragment ion chromatograms (m/z 1614.47 at 6.25 minutes for BT2-peg2-vancomycin diformate and m/z 1563.36 at 7.95 minutes for teicoplanin) and a calibration curve from control rat plasma (Biochemed Services Winchester, VA) spiked with authentic BT2-peg2-vancomycin diformate (0 to 200 μg/ml).
Plasma concentrations of vancomycin were measured by LC/MS. Protein precipitation and C18 spin column cleanup were performed as described for BT2-peg2-vancomycin diformate except that the teicoplanin concentration was 2.5 μg/ml and 0.1% formic acid was used as washing solvent. Elutes from the C18 column were concentrated to approximately 10 μl by speedvac, diluted to 100 μl with 0.1% formic acid, and analyzed by LC/MS with same column, gradient and instruments. Full scan fragment spectra from precursor ions (double charged m/z 725.7 from 0 to 5.5 minutes for vancomycin and double charged m/z 940.8 from 7 to 10 minutes for teicoplanin) were acquired by orbitrap. Compound confirmation was obtained from fragment spectra and concentration was calculated from peak areas of fragment ion chromatograms (m/z 1305.34 at 5.0 minutes for vancomycin and m/z 1563.36 at 7.95 minutes for teicoplanin) and a calibration curve from control rat plasma spiked with vancomycin standard (0 to 5 μg/ml).
Descriptive summaries for bacterial culture, hematology and chemistry studies are reported as medians and ranges. Comparisons between groups were performed using the Kruskall Wallis test. If the overall test among the groups was significant, further pairwise comparisons were made using the Wilcoxon rank-sum test. Given the small sample sizes, no adjustments for multiple comparisons were made. All tests were two sided and p-values less than 0.05 were considered statistically significant. Statistical analysis was performed using SAS version 9.2 (SAS Inc., Cary, NC).