Design, Synthesis, and Antimicrobial Evaluation of a Novel Bone-Targeting Bisphosphonate-Ciprofloxacin Conjugate for the Treatment of Osteomyelitis Biofilms

Article abstract of DOI:10.1021/acs.jmedchem.6b01615

Osteomyelitis is a major problem worldwide and is devastating due to the potential for limb-threatening sequelae and mortality. Osteomyelitis pathogens are bone-attached biofilms, making antibiotic delivery challenging. Here we describe a novel osteoadsorptive bisphosphonate-ciprofloxacin conjugate (BV600022), utilizing a “target and release” chemical strategy, which demonstrated a significantly enhanced therapeutic index versus ciprofloxacin for the treatment of osteomyelitis in vivo. In vitro antimicrobial susceptibility testing of the conjugate against common osteomyelitis pathogens revealed an effective bactericidal profile and sustained release of the parent antibiotic over time. Efficacy and safety were demonstrated in an animal model of periprosthetic osteomyelitis, where a single dose of 10 mg/kg (15.6 μmol/kg) conjugate reduced the bacterial load by 99% and demonstrated nearly an order of magnitude greater activity than the parent antibiotic ciprofloxacin (30 mg/kg, 90.6 μmol/kg) given in multiple doses. Conjugates incorporating a bisphosphonate and an antibiotic for bone-targeted delivery to treat osteomyelitis biofilm pathogens constitute a promising approach to providing high bone-antimicrobial potency while minimizing systemic exposure.

Full text of DOI:10.1021/acs.jmedchem.6b01615

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Article
Design, synthesis, and antimicrobial evaluation of a
novel bone-targeting bisphosphonate-ciprofloxacin
conjugate for the treatment of osteomyelitis biofilms
Parish P. Sedghizadeh, Shuting Sun, Adam F. Junka, Eric Richard, Keivan Sadrerafi, Susan Mahabady,
Neema Bakhshalian, Natalia Tjokro, Marzenna Bartoszewicz, Monika Oleksy, Patrycja Szymczyk, Mark
W Lundy, Jeffrey D. Neighbors, R. Graham G. Russell, Charles E. McKenna, and Frank H. Ebetino
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01615 • Publication Date (Web): 25 Jan 2017
Downloaded from http://pubs.acs.org on January 27, 2017
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Design, synthesis, and antimicrobial evaluation of a novel boneꢀtargeting bisphosphonateꢀ
ciprofloxacin conjugate for the treatment of osteomyelitis biofilms
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Parish P. Sedghizadeh*1, Shuting Sun2, Adam F. Junka3, Eric Richard2,4, Keivan Sadrerafi2,
Susan Mahabady1, Neema Bakhshalian1, Natalia Tjokro1, Marzenna Bartoszewicz3, Monika
Oleksy3, Patrycja Szymczyk4, Mark W. Lundy2, Jeffrey D. Neighbors2,6, R. Graham G. Russell7,
Charles E. McKenna4, Frank H. Ebetino*2
1. Center for Biofilms, Ostrow School of Dentistry, University of Southern California, Los
Angeles, California, United States of America
2. BioVinc LLC, Pasadena, California, United States of America
3. Department of Pharmaceutical Microbiology and Parasitology, Wroclaw Medical University,
Wroclaw, Poland
4. Department of Chemistry, University of Southern California, Los Angeles, California, United
States of America
5. Centre for Advanced Manufacturing Technologies, Wrocław University of Technology,
Wrocław, Poland
6. Department of Pharmacology and Medicine, Pennsylvania State University College of
Medicine, Hershey, Pennsylvania, United States of America
7. The Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology &
Musculoskeletal Sciences, University of Oxford, and The Mellanby Centre for Bone Research,
Department of Oncology and Metabolism, University of Sheffield, United Kingdom
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ABSTRACT
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Osteomyelitis is a major problem worldwide, and is devastating due to the potential for limbꢀ
threatening sequelae and mortality. Osteomyelitis pathogens are boneꢀattached biofilms, making
antibiotic delivery challenging. Here we describe a novel osteoadsorptive bisphosphonateꢀ
ciprofloxacin conjugate (BV600022), utilizing a ‘target and release’ chemical strategy, which
demonstrated a significantly enhanced therapeutic index versus ciprofloxacin for the treatment of
osteomyelitis in vivo. In vitro antimicrobial susceptibility testing of the conjugate against
common osteomyelitis pathogens revealed an effective bactericidal profile and sustained release
of the parent antibiotic over time. Efficacy and safety were demonstrated in an animal model of
periprosthetic osteomyelitis, where a single dose of 10 mg/kg (15.6 µmol/kg) conjugate reduced
the bacterial load by 99% and demonstrated nearly an order of magnitude greater activity than
the parent antibiotic ciprofloxacin (30 mg/kg, 90.6 µmol/kg) given in multiple doses. Conjugates
incorporating a bisphosphonate and an antibiotic for boneꢀtargeted delivery to treat osteomyelitis
biofilm pathogens constitute a promising approach to providing high boneꢀantimicrobial potency
while minimizing systemic exposure.
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Keywords: osteomyelitis, biofilm, antimicrobial, antibiotic, conjugate, bisphosphonate,
fluoroquinolone, ciprofloxacin, staphylococcus, pseudomonas, aggregatibacter
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INTRODUCTION
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Infectious bone disease, or osteomyelitis, is a major problem worldwide in human1 and
veterinary2 medicine and can be devastating due to the potential for limbꢀthreatening sequelae3
and mortality.4 The current approach to treat osteomyelitis is mainly antimicrobial, and often
intravenous and longꢀterm, with surgical intervention in many cases to control infection. The
causative pathogens in the majority of long bone osteomyelitis cases are biofilms of
Staphylococcus aureus; these microbes are bound to bone (Figure 1) in contrast to their
planktonic (freeꢀfloating) counterparts.5
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Figure 1. Scanning electron micrograph of a surgical bone specimen from a patient with chronic
osteomyelitis (100x magnification). Characteristic multiꢀlayered and matrixꢀenclosed biofilms
colonizing bone surfaces internally and externally can be visualized; inset top right shows highꢀ
power view (5000x magnification) of the causative staphylococcal biofilm pathogens. [The
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sample was processed for SEM, sputter coated with platinum and imaged with an XL 30S SEM
(FEG, FEI Co., Hillsboro, OR) operating at 5 kV in the secondary electron mode]
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The biofilmꢀmediated nature of osteomyelitis is important in clinical and experimental
settings because many biofilm pathogens are uncultivable and exhibit an altered phenotype with
respect to growth rate and antimicrobial resistance.5, 6 The difficulty in eradicating biofilms with
conventional antibiotics partly explains why the high success rates of antimicrobial therapy in
general have not yet been realized for orthopedic infections, along with the development of
resistant biofilm pathogens, poor penetration of antimicrobial agents into bone, and adverse
events related to systemic toxicity.3
To overcome the many challenges associated with osteomyelitis treatment,7 there is
increasing interest in drug delivery approaches using boneꢀtargeting conjugates to achieve higher
or more sustained local therapeutic concentrations of antibiotic in bone while minimizing
systemic exposure.8 Conjugation of fluoroquinolone antibiotics to osteoadsorptive
bisphosphonates (BPs) (Figure 2) represents a promising approach because of the long clinical
trackꢀrecord of safety of each constituent, and their advantageous biochemical properties.9, 10
Ciprofloxacin (Figure 2) has several advantages for repurposing in this context: 1) it can be
administered orally or intravenously with relative bioequivalence, 2) it is already FDA approved
and indicated for bone and joint infections caused by Pseudomonas aeruginosa and several other
pathogens, 3) it has broad spectrum antimicrobial activity that includes the most commonly
encountered osteomyelitis pathogens like Staphylococcus aureus (methicillinꢀsusceptible),
Pseudomonas aeruginosa for long bone osteomyelitis,11 and Aggregatibacter
actinomycetemcomitans for jawbone osteomyelitis,12 4) it demonstrates bactericidal activity in
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clinically achievable doses,13 and 5) it is the least expensive drug in the fluoroquinolone family.
However, like most antibiotics, fluoroquinolones suffer from reduced activity against biofilms as
compared to the same bacteria in planktonic forms; this has been shown specifically for
ciprofloxacin against S. aureus in addition to many other bacterial strains and antibiotic
classes.14ꢀ17 Such studies have demonstrated that biofilms can be one to several orders of
magnitude more resistant to the same antimicrobial agents as compared to their planktonic
counterparts. This highlights the importance of a boneꢀtargeted approach for treating
osteomyelitis, in order to achieve higher local concentrations of antibiotic against causative
biofilms and overcome potential resistance.
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The specific boneꢀtargeting properties of the BP family make these drugs ideal carriers
for targeting antibiotics to bone in osteomyelitis pharmacotherapy.18ꢀ20 BPs form strong bidentate
or tridentate bonds with calcium phosphate mineral, and as a result concentrate in hydroxyapatite
(HA), particularly at skeletal sites of active metabolism including sites of infection and
inflammation.21 BPs also exhibit exceptional stability against both chemical and biological
degradation.22 BPꢀfluoroquinolone antimicrobial activity is complex and is related to the specific
strain of pathogen tested, the choice of antibiotic and covalently bound BP moiety, the tether
length between the two constituents, the bone binding affinity of the BP, the adsorptionꢀ
desorption equilibria of the BP, and the stability/lability and kinetics of the linkage moiety used
for conjugation.18ꢀ20 Therefore, accumulating evidence suggests that a ‘target and release’ linker
strategy (Figure 2) where a conjugate is stable in circulation, but labile at the bone surface, may
offer more opportunities for optimization and success in this context. We thus hypothesized that
conjugation of ciprofloxacin to a phenyl BP moiety, through metabolically hydrolyzable
carbamate linkers, should mitigate the problems seen with antibiotic dosing in osteomyelitis
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pharmacotherapy. The cleavable carbamate linkage is a key functionality in many drugs
designed for target and release in specific tissues,23, 24 and confers pharmacokinetic advantages
such as stability in serum and lability at infected bone surfaces in the presence of an acidic and
enzymatic environment (e.g. inflammation or infection).25
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Figure 2. Design of bisphosphonateꢀciprofloxacin conjugate via a “target and release” strategy.
A recent apparent success utilizing a boneꢀtargeting and release strategy is provided by
Morioka et al26 who designed an estradiol analog conjugate using a cleavable variant
(carbamate) of the more stable amide peptide bond. Several versions of this linkage were
attempted before the identification of a pharmacologically active variant (aryl carbamate).
Importantly, they demonstrated that a single dose of a similarly linked BPꢀestradiol conjugate (at
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a dose nearly 5,600 times lower than the total dose of estradiol alone) produced a similar effect
on bone to that of the estradiol dosed alone.26 The conjugate also provided an even greater
therapeutic index, as there were minimal effects systemically and in uterine tissues compared to
the estradiol alone. Pharmacokinetic studies completed by Arns et al27 are in agreement with this
dramatic enhancement of potency in studies based on a BPꢀprostaglandin with a more labile
linker. Other synthetic examples of this approach in the antimicrobial field are reported for the
macrolide class;28 however, only alkyl carbamates were explored and the lack of further success
suggests that target and release strategies are likely chemical classꢀdependent (taking into
consideration compatibilities of the functional groups of each component) as well as biochemical
targetꢀdependent, and the design for any particular chemical class must be customized for its use.
Here, we describe a novel aryl carbamate BPꢀciprofloxacin conjugate 6 (BV600022),
evaluate its antimicrobial activity in vitro against common osteomyelitis pathogens, and assess
its in vivo safety and efficacy in an animal model of periꢀprosthetic osteomyelitis. Importantly,
the studies presented herein utilize biofilm models and methodology, in addition to planktonic
cultures, to provide greater clinical or translational relevance.
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RESULTS
Chemistry
The overall synthetic route to 6 is shown in Scheme 1, starting from the relatively
pharmacologically inert 4ꢀhydroxyphenylethylidene BP (3). The rationale for this BP design was
to retain the boneꢀseeking ability of the BP moiety while suppressing its unneeded antiresorptive
activity, minimizing confounding factors to focus on evaluating the antimicrobial effect due to
the parent ciprofloxacin compound. BP ligands can also be designed to have antiresorptive
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functionality (of varying potency) if needed to provide a dualꢀaction effect of bone tissue
protection in addition to antimicrobial effects at the anatomic site of infection. We also chose this
phenyl BP with consideration of bone binding affinity and tether length, as previous studies have
demonstrated that weak binding affinity decreases targeting efficiency.13, 14 Importantly, we
postulated that the use of an aryl carbamate as a linker might offer optimized stability in plasma
and adequate release on bone for this biochemical target as compared to previously derived BPꢀ
fluoroquinolone conjugates.
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Scheme 1.ᵅ Overall synthetic scheme of aryl carbamate BPꢀciprofloxacin conjugate 6
ᵅ Reagents and conditions: (a) BTMS (2 equiv), Et2O, 0 °C ꢀ rt, 17 h, yield 95%. (b) i)
tetraisopropyl methylene bisphosphonate (1 equiv), NaH (1 equiv), THF, rt, 10 min; ii) 1 (1
equiv), rt, 2 h, yield 52%. (c) Pd/C (Catalyst) (0.07 equiv), H2, MeOH, rt, overnight, yield 88%.
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(d) 4ꢀnitrophenyl chloroformate (1.1 equiv), Et3N (3 equiv), DCM, rt, 2.5 h, yield 44%. (e)
Ciprofloxacin (1.2 equiv), water (pH 8.5), THF, 0 °C ꢀ rt, overnight, yield 52%. (f) i) BTMS
(excess), DCM, 35 °C, 24 h, ii) MeOH, rt, overnight, yield 86%.
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Additionally, we synthesized a similar BPꢀciprofloxacin conjugate having an amide
linkage as opposed to a carbamate linkage as outlined in Scheme 2 as a control conjugate 11
(BV600026). Previous investigations have indicated that amide conjugates are not able to release
the parent antibiotic and are thus less effective in vitro and in vivo,11 which we sought to verify
in this instance.
Scheme 2.b Overall synthetic scheme of amide BPꢀciprofloxacin conjugate 11
b
Reagents and conditions: (a) i) NaH (1.4 equiv), THF, 0 °C ꢀ rt, 1 h; ii) methyl 4ꢀ
(bromomethyl)benzoate (0.7 equiv), THF, 0 °C ꢀ rt, overnight, yield 37%. (b) LiOH•H2O (5
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equiv), MeOH, rt, overnight, yield 91%. (c) SOCl2 (2 equiv), DMF (0.05 equiv), DCM, rt, 2 h,
yield quantitative. (d) Ciprofloxacin (1 equiv), DIPEA (6 equiv), CHCl3, rt, overnight, yield
65%. (e) i) BTMS (excess), DCM, 35 °C, overnight, ii) MeOH, rt, 30 min, yield 82%.
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Antibacterial properties of BPꢀciprofloxacin conjugates
Minimal inhibitory concentration (MIC) assays: The antimicrobial activity of both conjugates
(6 and 11) and the parent antibiotic ciprofloxacin in standard laboratory planktonic culture
systems was evaluated against a panel of S. aureus clinical strains associated with bone
infections, including methicillinꢀsensitive S. aureus (MSSA) and methicillinꢀresistant S. aureus
(MRSA). Following EUCAST (European Committee on Antimicrobial Susceptibility Testing)
guidelines,29 results from disc diffusion inhibition zone assays revealed diameters ranging from
25ꢀ40 mm (mean 31.5, SD±5), and every strain demonstrated antimicrobial susceptibility to the
parent antibiotic ciprofloxacin according to EUCAST clinical breakpoints. Minimal inhibitory
concentration (MIC) results for 6 and 11 against eight S. aureus strains using microdilution
methodology are shown in Table 1. MICs for the parent compound ciprofloxacin were
determined concurrently for reference (see Table 1) and were found to be consistent with
established clinical breakpoints.29
Table 1. Antimicrobial susceptibility testing results for ciprofloxacin, 6 and 11 tested
against a panel of clinically relevant S. aureus osteomyelitis pathogens. (MSSA=methicillinꢀ
susceptible S. aureus; MRSA=methicillinꢀresistant S. aureus)
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Antibiotic
Sensitivity (S)
or Resistance
MIC for
ciprofloxacin
[µg/mL]
MIC for 6 MIC for 11
S. aureus strain
[µg/mL]
[µg/mL]
(R)
S
S
S
S
S
S
S
MSSA1
MSSA2
MSSA3
MSSA4
MSSA5
MSSA6
1
0.25
0.25
1
3.9
0.49
0.49
3.9
31.2
31.2
15.6
62.5
0.5
1
1.9
7.8
7.8
15.6
MSSA: ATCCꢀ6538
MRSA: MR4ꢀCIPS
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31.2
15.6
62.5
Not tested
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S
Hydroxyapatite (HA) binding assay: Having established the antimicrobial efficacy of 6, we
next sought to evaluate HA binding ability. When we added HA spherules to our microbiological
media and then introduced 6 at various concentrations similar to those used in the antimicrobial
testing. Quantitative spectroscopic analysis of supernatant (without HA spherules) confirmed
significant adsorption and retention of the conjugate by HA (Figure 3).
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Figure 3. Spectroscopic analysis of 6 in TSB microbiological media with the addition of HA
spherules. The significant decreases from 0 hr to 24 hrs confirms conjugate adsorption to HA
since the measurement is only performed in the supernatant where the conjugate adsorbed to the
HA spherules is absent. [Results for 1.95ꢀ250 µg/mL are all statistically significant: p<0.05,
ANOVA; triplicate; *results for 0.12ꢀ0.48 µg/mL (with a horizontal bar above) are inconclusive
relative to control medium].
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pH effect in antimicrobial susceptibility testing (AST) on planktonic S. aureus strain
ATCCꢀ6538: We selected S. aureus strain ATCCꢀ6538 for further investigation because it
demonstrated the lowest MIC profile for both ciprofloxacin and 6 (see Table 1) compared to the
other strains tested. This ATCC strain is also a wellꢀknown and robust biofilmꢀforming
pathogen. Consequently, we could test our conjugates against the most challenging pathogen to
limit bias and overestimated results, while also facilitating assessment of antimicrobial activity in
biofilmꢀbased and clinically relevant models. Antimicrobial susceptibility testing (AST) on
planktonic S. aureus strain ATCCꢀ6538 with 6 under both acidic and physiological pH was
performed to assess the effect of pH on conjugate activity. Quantitative results from standard
microdilution methodology indicated that under acidic conditions (pH 5) the antimicrobial
activity of 6 was improved overall as the MIC50 was reached at half the conjugate concentration
required to reach MIC50 under physiological conditions (Figure 4). It is important to note that in
these results and in results presented later in this paper, the minimum inhibitory concentration
terms MIC50 or MIC90 refer to a reduction of 50% or 90% of bacterial load, respectively; and the
biofilmꢀrelated terms of minimum biofilm inhibitory concentrations (MBIC50 or MBIC90) refer to
similar reductions (50% or 90%) but in biofilm bacterial load.
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Figure 4. Antimicrobial susceptibility testing of 6 against planktonic cultures of S. aureus strain
ATCCꢀ6538 in acidic (top graph) and physiological (bottom graph) pH. Results show an
improved bactericidal profile in acidic versus physiological pH as half the concentration of
conjugate is required to reach the MIC50 in acidic conditions as compared to physiological
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conditions. Note that the MIC90 of 31.25 µg/mL in this assay in acidic and physiological
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conditions is consistent with the MIC results shown in Table 1 for 6 against the same strain.
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Timeꢀkill assays of 6: Next, kinetic assays were performed with 6 according to CLSI (Clinical
Laboratory Standards Institute) methods.30 Results indicated that this conjugate was bactericidal
at the previously established MIC for methicillinꢀsusceptible (ATCCꢀ6538) and methicillinꢀ
resistant (MR4ꢀCIPS) isolates of planktonic S. aureus within 1 hr and up to 24 hrs, preventing
100% of bacterial growth; these kinetic studies also revealed that at half the MIC value,
prevention of bacterial growth became evident after 2 hrs and inhibition was at 50% of control
after 24 hrs (Figure 5).
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Figure 5. Timeꢀkill results for 6 against S. aureus strain ATCCꢀ6538 (top graph) and MRSA
strain MR4ꢀCIPS (bottom graph) at their established MIC values (see Table 1) and at half that
value. Kinetic results reveal that at half the MIC value, prevention of bacterial growth became
evident after 2 hrs and inhibition was at 50% of control after 24 hrs.
Evaluation of antimicrobial efficacy of 6 against biofilms: We then tested 6 against preꢀ
formed bacterial biofilms on two different substrates (polystyrene and HA discs) to evaluate
antimicrobial efficacy against biofilms, and to also determine if substrate bindingꢀspecificity
plays any role in the observed antimicrobial efficacy. Biofilms of S. aureus (ATCCꢀ6538), and
additionally biofilms of P. aeruginosa (ATCCꢀ15442), were grown on polystyrene or HA as
substrates and were subjected to varying concentrations of 6 for assessment of antimicrobial
activity. We tested P. aeruginosa here because it is a Gram negative pathogen and the second
most common clinical pathogen in osteomyelitis, though less frequent in prevalence than Gram
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positive S. aureus. Figure 6 shows results for polystyrene as the substrate for biofilm growth,
and the minimal biofilm inhibitory concentration (MBIC50) of 6 was 15.6ꢀ31.2 µg/mL for S.
aureus ATCCꢀ6538, which was comparable to the MIC for this strain in planktonic cultures. No
MBIC50 was observed for P. aeruginosa ATCCꢀ15442 in the tested range of concentrations and
no MBIC90 was observed for either pathogen.
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Figure 6. Antimicrobial susceptibility testing of 6 against biofilms of S. aureus strain ATCCꢀ
6538 (top graph) and P. aeruginosa strain ATCCꢀ15442 (bottom graph) formed on polystyrene
as a substrate. The conjugate demonstrates minimal activity against biofilms on polystyrene, and
no MBIC90 is observed for either pathogen.
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However, when HA discs were used as the biofilm substrate, marked bactericidal activity
was observed with 6. As shown in Figure 7, all tested concentrations of this conjugate resulted
in statistically significant (p<0.05, KruskalꢀWallis test) bactericidal activity and reduction of
colony forming units (CFUs). The MBIC50 of 6 was 16 µg/mL and the MBIC90 was 100 µg/mL
against S. aureus strain ATCCꢀ6538; the MBIC90 for the parent drug ciprofloxacin was 8 µg/mL
against this pathogen. However, against P. aeruginosa strain ATCCꢀ15442 ciprofloxacin had no
inhibitory or bactericidal activity in this setting while the conjugate was bactericidal in acidic and
physiological conditions at 50 µg/mL, and showed improved bactericidal activity in
physiological conditions as compared to S. aureus where improved antimicrobial activity was
observed in acidic conditions.
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Figure 7. Antimicrobial susceptibility testing of 6 against biofilms of S. aureus strain ATCCꢀ
6538 (top graph) and P. aeruginosa strain ATCCꢀ15442 (bottom graph) formed on HA discs as
the substrate. All tested concentrations of 6 (dotted bars top graph) and the parent antibiotic
ciprofloxacin resulted in statistically significant bactericidal activity against S. aureus; c=
negative control comparator. Against P. aeruginosa, 6 was most effective at physiological pH at
8 µg/mL, and also effective at acidic pH at this concentration, but ciprofloxacin was inactive
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under either acidic or physiological conditions compared to the controls [*p<0.05, Kruskalꢀ
Wallis test; triplicate].
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Preventative antimicrobial assays: Next, we performed antimicrobial tests with 6 in a
preventative type of experimental setting with planktonic and biofilm cultures, which could also
have clinical relevance in antibiotic prophylactic scenarios for osteomyelitis pharmacotherapy.
Here HA spherules were introduced to varying concentrations of 6 and then inoculated with S.
aureus for 24 hrs, and quantitative assessments indicated no bacterial growth at concentrations as
low as 15.6 µg/mL and up to 250 µg/mL of 6, and minimal bacterial growth with strong
inhibition at conjugate concentrations ranging from 0.24 to 7.8 µg/mL as shown in Figure 8.
Figure 8. Results from preventative experiments where HA spherules were pretreated with 6 and
then inoculated with S. aureus. Controls (black bars) represent cultured bacteria without HA and
without conjugate treatment. After 24 hrs, significant increase in planktonic growth is observed
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when the supernatant is measured. Control + HA (C+HA bar) represents cultured bacteria with
HA, but still without treatment: after 24 hrs some bacterial growth is observed but not as much as
in the HA negative control (red bar), because bacteria bind to HA and form biofilms which are
not measured in the HAꢀfree supernatant. Comparing controls to treatments, at 15.6 to 250
µg/mL of the conjugate, there is complete bacterial inhibition after 24 hrs (right graph); at lower
concentrations ranging from 0.24 to 7.8 µg/mL, bacteria grew slightly but were still strongly
inhibited.
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Next, we tested the amide conjugate (11) for ability to treat S. aureus strain ATCCꢀ6538
biofilms in experimental conditions similar to those used to test the carbamate conjugate 6.
When evaluating the activity of 11 against established S. aureus biofilms grown on HA, and HA
pretreated with 11 prior to biofilm growth in a preventative experimental setting, antimicrobial
activity of 11 even at higher doses than those used to test 6, was insignificant in both cases as
shown in Figure 9.
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Figure 9. Antimicrobial susceptibility testing (top graph) of 11 at increasing concentrations
against biofilms of S. aureus strain ATCCꢀ6538 formed on HA as the substrate. No significant
activity is observed at any concentration as compared to the control C+ [p>0.05, KruskalꢀWallis
test; triplicate]. The bottom graph shows results from preventative experiments where HA is
pretreated with 11 or the parent antibiotic ciprofloxacin and then inoculated with S. aureus, and
again no antimicrobial activity is observed for 11; the only significant reduction is seen with the
parent drug at a relatively high dose of 400 µg/mL [*p<0.05, KruskalꢀWallis test; triplicate].
When 6 was tested for ability to prevent S. aureus ATCCꢀ6538 biofilms from forming on
pretreated HA, the conjugate showed superior antimicrobial activity as compared the parent
antibiotic and in contrast to 11 which showed no significant antimicrobial activity. Table 2
shows results of quantitative biofilm cultures and CFU counts after 24 hrs of growth, and at 100
µg/mL the parent drug ciprofloxacin inhibited all biofilm growth whereas at 10 µg/mL, 6
inhibited all growth. Since the molecular mass of ciprofloxacin is approximately half that of 6, 6
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was 20 times more active in achieving complete bactericidal action as compared to ciprofloxacin
alone.
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Table 2. Survival of S. aureus biofilms and CFU counts after 24 hrs of incubation on HA
discs pretreated with 6 or ciprofloxacin
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Concentration [µg/mL]
0
0
Compound
(Control
discs in
DMSO)
(Control
discs in
PBS)
1
5
10
25
50
100
200
400
No
growth
No
growth
No
No
No
No
growth
Not
applicable
7.1x1010
5.5x1010
3.3x109
4.5x1010
6
growth
growth
growth
No
No
No
No
growth
Not
applicable
9.2x1010 3.7x109 8.9x107
8.1x1010
Ciprofloxacin
growth
growth
growth
In vivo safety and efficacy: Since 6 demonstrated promising activity in vitro, we sought to
assess drug safety and efficacy in vivo in an animal model of periprosthetic osteomyelitis. This
model is a unique inꢀhouse jawbone periꢀimplant osteomyelitis model that was developed
specifically for translational value to study biofilmꢀmediated disease and host response in vivo.31
Because a systemic treatment regimen is utilized, this assay also serves to model any infected
bone surface, since the resulting osteolysis involved is key to attracting (targeting) high
concentrations of a BPꢀconjugate, like any high turnover site on bone, and to subsequently
release the active ciprofloxacin component of the conjugate at this diseased bone surface.
Briefly,
biofilms
of
the
jawbone
osteomyelitis
pathogen
Aggregatibacter
actinomycetemcomitans (Aa; wildꢀtype rough strain D7Sꢀ1; serotype a), which is not indigenous
to rat normal flora and specific to jawbone infections, were preꢀinoculated on miniature titanium
implants at 109 CFU. To confirm Aa sensitivity to the parent drug ciprofloxacin prior to our
animal studies, we performed AST and MIC assays as performed for the long bone osteomyelitis
pathogens described previously. Disc diffusion inhibition zone assays revealed diameters >40
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mm, and the MIC90 was 2 µg/mL, indicating strong susceptibility of this microbe to the parent
drug ciprofloxacin. Aa has also been tested previously for susceptibility to a pHꢀsensitive
biotinylated ciprofloxacin prodrug and was found to be sensitive to the parent antibiotic.32 As
with previous pathogens in this study, we also tested Aa biofilm pathogens grown on HA for
sensitivity to 6 and found our conjugate displayed effective antimicrobial activity as shown in
Figure 10.
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Figure 10. Antimicrobial susceptibility of 6 against biofilms of Aggregatibacter
actinomycetemcomitans strain D7Sꢀ5 grown on HA shows an effective antimicrobial profile for
conjugate 6 at > 15 µg/mL.
After Aa biofilms are established on implants in vitro, they are surgically transferred to
the jawbone of each rat. Animals are anesthetized, the cheeks are retracted and a transmucosal
osteotomy is performed so implants can be manually inserted into the osteotomy and secured.
Two biofilmꢀinoculated implants are placed in each rat (n=12 rats, 24 implants total) in the
palatal bone bilaterally. This model allows standardized and reproducible quantities of viable
bacteria to be formed as wellꢀestablished biofilms on each implant, which we have previously
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demonstrated persists in vivo for several weeks after placement and causes infection,
inflammation, and bone destruction locally.31
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Once periꢀimplant infection was established 1 week postꢀoperatively, the animals were
dosed with 6, ciprofloxacin alone as a positive control, and sterile endotoxinꢀfree saline as a
negative control at the dosing regimens specified in the experimental section. To determine
appropriate dosing concentrations, we calculated approximate initial doses for the conjugate
based on previous studies and pharmacokinetic data using similar target and release strategies
also in rodents.13, 26 We expected that increasing doses of 0.1, 1 and 10 mg/kg molar equivalents
of 6 will allow us to determine antimicrobial activity in 2 test animals per group based on sample
size estimations and previous experience with the animal model.32 Animals were dosed via
intraperitoneal injection under general anesthesia, and all compounds were constituted in sterile
physiological injectable saline at appropriate pH. Intraperitoneal injection was used because of
the ease of administration in small rodents as compared with other parenteral methods like tail
vein injection, and because the pharmacokinetics of ciprofloxacin following gastrointestinal
administration shows excellent bioavailability; serum drug levels achieved after such
administration are slightly less but comparable to those with intravenous dosing with no
substantial loss after first pass metabolism.33 One week after pharmacotherapy, all animals were
sacrificed and en bloc resection of periꢀimplant hard and soft tissues was performed and
homogenized for quantitative assessment of microbial load.
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All animals tolerated the pharmacotherapy well with no cutaneous injectionꢀsite reactions
or inflammation. There were no signs of gross tolerability issues during therapy. Treatment
efficacy was quantitatively measured in terms of the logarithmic reduction of the amount of
viable bacteria (mean log10 CFU/gram of tissue) as shown in Figure 11.
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Figure 11. Antimicrobial results from in vivo animal testing. Data show efficacy of tested
compounds for reducing bacterial load. The greatest efficacy was observed at a single high dose
(10 mg/kg) of 6 where a 2 log reduction (99% bactericidal activity) was seen as compared to the
negative control.
In vivo, the single dose of 6 at 10 mg/kg showed the highest efficacy with a 2 log
reduction in bacterial count (99% bacterial killing) and nearly an order of magnitude greater
activity than ciprofloxacin alone given at the same per dose concentration (mg/kg) but in
multiple doses (30 mg/kg total dose). Thus, given the greater molecular weight of 6 (~2x of
ciprofloxacin), the administered single dose of 6 at 10 mg/kg could deliver roughly 5 mg/kg of
effective ciprofloxacin assuming full release, which is 1/6th of the ciprofloxacin molar dose of
the control ciprofloxacin arm (30 mg/kg total). Ciprofloxacin alone in a multiple dosing regimen
resulted in a 1 log reduction in bacterial counts (90% bacterial killing). Concentrations of 6 at 0.1
and 1 mg/kg had little effect, suggesting that a minimum dose is necessary for clinical effect and
that further chemistry optimization may be possible in this context.
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To validate our animal study findings, and to provide for greater power and larger sample
size for statistical analysis, we conducted a second animal experiment nearly identical to the first
except for allocation of dosing regimens. Based on dosing data and antimicrobial results from
our first animal study described above, we focused this second animal study on three treatment
groups: negative control (n=5 rats), 6 at a single high dose of 10 mg/kg (n=5 rats), and 6 at a
multiple low dose regimen of 0.3 mg/kg 3x/week (n=2 rats). Dosing groups of 0.1 and 1 mg/kg
were excluded as they showed no efficacy previously, and the parent antibiotic alone was also
excluded since robust historical data exists for ciprofloxacin efficacy which we also confirmed in
our initial animal study. The multiple dosing regimen was utilized again to ascertain whether the
lack of recoverable bacteria could be attributed to treatment effect or experimental and sampling
error. All other experimental parameters were identical to the first animal experiment, and each
animal had two implants placed as before allowing for two results per animal and providing
sufficient power for statistical analyses as determined by sample size estimations.
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All animals again tolerated treatment and pharmacotherapy well and there were no signs
of gross tolerability issues during therapy. Clinically during euthanasia and surgical resection, we
observed that the majority of the animals in the control group demonstrated evidence of localized
periꢀprosthetic inflammation as compared to the majority of the animals in the treatment groups
which had nonꢀinflamed periꢀimplant tissues, and implant retention was 23/24 implants (96%)
which is a high retention rate and provided robust power for subsequent analyses. Quantitative
antimicrobial results from this second animal experiment are shown in Figure 12. Single factor
ANOVA testing (α=0.05) comparing CFUs between treatment groups resulted in a pꢀ
value=0.006 for significance between groups, and postꢀhoc testing utilizing an unpaired tꢀtest
(p=0.0005; df=20) and Dunnett’s multiple comparisons test (p<0.05) revealed significance for
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the single high dose of 6 treatment as compared to the control, but not for the multiple low dose
5
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group (p>0.05) when compared to the control or to the single high dose treatment group.
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Figure 12. Antimicrobial results from the second animal experiment. Data shows efficacy of 6
for reducing bacterial load or mean CFU/gram of tissue (yꢀaxis). The greatest efficacy was
observed at a single high dose (10 mg/kg) of the conjugate compared to the control and the
multiple low dose group (0.3 mg/kg X 3) [*p=0.0005; unpaired tꢀtest, errors bars represent
Standard Error].
DISCUSSION
Targeting antibiotics to bone by conjugation to a BP moiety (via a releasable carbamate
linker) is a promising approach for the treatment of osteomyelitis biofilms. Results of AST
testing and MIC data presented herein indicate that against planktonic S. aureus, ciprofloxacin
and 6 have effective bactericidal activity, and that the conjugation linkage impacts antimicrobial
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activity of the parent drug as evidenced by the weaker activity of 11 (Table 1). Higher
concentrations of 6 were required to reach MIC, which is anticipated since conjugation is a
chemical modification that can alter the biochemical interactions of the antibiotic prior to release
from the conjugate. As a result, properties of the parent drug, including its pharmacodynamic
effect, can be altered by such modification. MIC results for 6 were consistent with previous
literature indicating that conjugates in this class can retain the antibacterial activity of the parent
compound, although at slightly lower levels.9, 10
10
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Of interest was the wide distribution of MIC values for both conjugates against tested S.
aureus strains, as compared to ciprofloxacin alone which demonstrated little variance in
antimicrobial efficacy against the same strains (Table 1). There are several possible explanations
for these results. Different strains of bacteria within the same species are known to show
significant variance in terms of virulence and antimicrobial susceptibility/resistance to an
antibiotic. It is wellꢀestablished that strainꢀspecific variances exist in antibiotic transport and
efflux mechanisms, bacterial cell wall density, enzymatic activity levels, resistance mechanisms,
and ability to alter pH of the environment.34 Ciprofloxacin bactericidal activity results from
intracellular inhibition of enzymes required for DNA replication – topoisomerase II and IV.35
The mechanisms underlying the conjugate antimicrobial activity have not yet been elucidated. It
is unclear whether these conjugates are completely cleaved outside the bacterial cell and the
antibiotic is then transported inside, or whether intact conjugate can enter the cell wall and is
then cleaved to active drug, or if both scenarios occur significantly; in all cases, difference
between species or strains may occur. Specific microbial biochemistries are likely to play an
important role in determining conjugate activities.
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It has been established that intact conjugates in this class generally lack significant
intrinsic antibacterial activity,18, 19 and that any BPꢀrelated antimicrobial effect is negligible;
therefore, at least partial release of the parent drug is a prerequisite for significant antimicrobial
activity, as observed with 6. This is consistent with the low antimicrobial activity of 11 differing
in its more stable amide linkage, which resulted in 2–64x the concentration of the more labile
carbamateꢀlinked conjugate 6 to achieve the same antibacterial effect in the assay.
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After evaluating the antimicrobial efficacy of 6, we sought to assess the boneꢀbinding
functionality of the BP moiety and found effective adsorption and retention to HA spherules by
the conjugate in a concentrationꢀdependent manner. These results are consistent with previously
reported analogs in this class containing BP moieties with similar bone affinities.13, 19 We then
tested whether activity of 6 would vary in different pH conditions and found a slightly improved
profile in acidic conditions, which may be explained at least partially by the fact that the linker is
more labile at pH 5 than at pH 7.4 thus releasing more ciprofloxacin at the lower pH. This could
be useful for clinical osteomyelitis applications where biofilm pathogens along with host
inflammation and osteoclastogenesis produce an acidic local milieu. Other investigators have
suggested, however, that although acidic pH brought on by infecting organisms and
inflammation could result in some drug release in bone, the efficacy of such a process in
providing a significant concentration of the antimicrobial agent is doubtful, and that prodrug
design, conjugation scheme, and susceptibility to local enzymatic hydrolysis likely have greater
impact on linker cleavability and efficacy.13 Our findings also support such conclusions.
Investigation of timeꢀkill kinetics for 6 demonstrated an efficient rate of bactericidal
activity against tested bacteria with sustained bactericidal activity over 24 hrs, supporting
cleavage activity of the parent antibiotic with a steadily sustained release profile over time. The
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