1 Genesoft Pharmaceuticals, 7300 Shoreline Ct., South San Francisco, CA 94080, USA; 2 British Biotech plc, Watlington Road, Oxford OX4 6LY, UK
Received 12 September 2003; returned 3 November 2003; revised 3 December 2003; accepted 8 December 2003
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Abstract |
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Methods: MICs were determined by standard NCCLS broth microdilution. Selectivity of metalloenzyme inhibition was determined with a limited panel of enzymes via standard biochemical assays. Profiling of the pharmacokinetics and select tissue disposition in mice was determined and compared with that of the macrolide, azithromycin. In vivo murine efficacy studies using Streptococcus pneumoniae were conducted using a peritonitis model, as well as lung and thigh burden models of infection.
Results: BB-81384 selectively inhibited PDF with an IC50 10 nM and with MICs < 0.5 mg/L against most S. pneumoniae pathogens. Pharmacokinetic analysis revealed good oral bioavailability and moderate clearance and volume of distribution. BB-81384 partitioning to lung tissue was similar in terms of magnitude and kinetics to that of the plasma compartment. Single-administration oral efficacy in a mouse peritonitis model was evident with an ED50 of 30 mg/kg. BB-81384 reduced the bacterial load by
5 and 3 log units in organ-burden models of lung and thigh infection, respectively.
Conclusion: BB-81384, a novel PDF inhibitor with good activity against S. pneumoniae in vitro, was the first compound of this class to be profiled for oral pharmacokinetics and tissue disposition and to demonstrate oral anti-pneumococcal efficacy in mice.
Keywords: peptide deformylase, efficacy, pharmacokinetics
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Introduction |
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By targeting a unique mechanism for antibacterial activity, such an agent would be expected to lack cross-resistance to existing classes of antibiotics. Indeed, PDF inhibitors have shown potent in vitro and in vivo activity against Gram-positive organisms, including drug-resistant isolates.1014 For the reasons highlighted above, PDF inhibition is an attractive approach for antibacterial chemotherapy. In fact, a first-generation inhibitor, BB-83698, has been advanced into clinical trials for hospitalized community-acquired pneumonia (CAP) (www.britbio.com, product portfolio).
Discovery of PDF inhibitors for the treatment of respiratory tract infections (RTIs) in which Streptococcus pneumoniae represents the most frequently isolated organism15 has been undertaken. Despite numerous advances in PDF inhibitor discovery oral anti-pneumococcal efficacy in vivo has not yet been published. Moreover, the only other N-formylhydroxylamine-type inhibitor previously described (BB-3497)11 exhibited poor activity against S. pneumoniae. Identification of P2' and P3' substituents that substantially improve anti-pneumococcal activity relative to BB-3497, has been a required step in therapeutic optimization for respiratory infections.11,12
A potent, orally active PDF inhibitor for RTI would meet a great clinical need and represent an attractive development opportunity. We set out to identify and characterize orally active, novel PDF inhibitors with potential use against CAP. Demonstrated herein is the anti-pneumococcal oral efficacy of a novel PDF inhibitor (BB-81384) in multiple animal models of infection and, for the first time for any PDF inhibitor, details of its pharmacokinetics and tissue disposition.
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Materials and methods |
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The in vitro activity of BB-81384 against PDF and the related metalloenzymes collagenase (MMP-1), 72 kDa gelatinase (MMP-2) and angiotensin I-converting enzyme was determined as previously described.11 Briefly, fluorescent-labelled peptide substrates with purified recombinant human enzymes were used with fluorescence as the primary endpoint.
Bacterial strains and susceptibility testing
The in vitro bacterial susceptibility was determined by standard NCCLS broth microdilution.16,17 Activity was evaluated against a broad range of organisms including the following subset of strains: methicillin-susceptible Staphylococcus aureus (MSSA) ATCC 29213; penicillin-resistant S. pneumoniae (PRSP) ATCC 9061; penicillin-susceptible S. pneumoniae (PSSP) ATCC 6301; penicillin-susceptible S. pneumoniae (PSSP) ATCC 6303; Haemophilus influenzae (ATCC 31517, 107022); Moraxella catarrhalis (ATCC 37054); Bacillus cereus (ATCC 11778); and Streptococcus pyogenes (ATCC 49399).
Killing kinetics
S. pneumoniae ATCC 6301 and 6303 were grown on trypticase soy agar (TSA) with 5% sheep blood (BD Biosciences, Sparks, MD, USA) at 37°C in 5% CO2. From the overnight growth plate, several colonies were cultured into 3 mL of cation-adjusted MuellerHinton broth (CAMHB) with 5% lysed horse blood. The broth subcultures were incubated for 2 h at 37°C in 5% CO2. The 2 h culture of S. pneumoniae 6301 was adjusted to an OD600 of 0.3 and diluted 1:50 in 5 mL of CAMHB to yield a final inoculum of 5 x 105 cfu/mL into test tubes containing 1x, 2x or 4x the MIC of antibiotic. A growth control tube without antibiotic was included.
Viable counts were determined by removing 100 µL at intervals of 0, 2, 4, 8 and 24 h. Serial dilutions (1:10) were prepared in 96-well microtitre plates. Ten µL was plated on TSA plates containing 5% sheep blood and incubated overnight at 37°C in 5% CO2. In addition, if tubes were macroscopically clear after overnight incubation, 100 µL was plated on agar and incubated as above. The lower limit of detection for this assay was 10 cfu/mL.
Pharmacokinetics and tissue distribution
The pharmacokinetics of BB-81384 and a reference drug, azithromycin, were characterized in ICR mice. Groups of three female mice per time point were treated via intravenous (iv) bolus (lateral tail vein) or oral gavage. At serial time points (e.g. 5, 20, 60, 120, 300 min), blood was collected in lithium heparin tubes by cardiac puncture and separated by centrifugation. Plasma samples were stored at 80°C until analysis.
Tissue distribution. The disposition of BB-81384 and the reference drug azithromycin to select tissues following a 10 mg/kg oral dose was determined after single administration in female ICR mice over a period of up to 5 h (5, 20, 60, 120 and 300 min). Mice were exsanguinated and organs (lung, thigh muscle) were rapidly removed and flash frozen.
Plasma and tissue samples were analysed internally or at Bay Bioanalytics Laboratory (Hercules, CA, USA) according to the methods described below. Plasma and tissue sample analysis was performed using a high-performance liquid chromatography/mass spectrometry/mass spectrometry (HPLC/MS/MS) method with appropriate quality controls.
Plasma analysis. The sample preparation for BB-81384 consisted of a protein precipitation with acetonitrile (containing diphenhydramine as internal standard), removal of the precipitate by centrifugation, concentration of the extract under a stream of nitrogen and reconstitution in mobile phase. A standard curve in the range 320 000 ng/mL was prepared in blank plasma, and treated as described above. Quality controls (1000, 2000, 5000 ng/mL) were prepared and interspersed throughout the sequence run. Back-calculated standards and quality control samples differed by no more than 11% from nominal values.
For azithromycin, a similar protein precipitation method was used to process plasma samples, and a plasma standard curve with standards in the range 71670 ng/mL was used for quantification.
Tissue analysis. To a 50 mg tissue aliquot, 50 µL of water and 150 µL of ice-cold internal standard solution (0.1 µg/mL diphenhydramine in 1:1 methanol/acetonitrile) were added, followed by homogenization at high speed for 1560 s with a serrated microtip on a Polytron homogenizer (Brinkman, Westbury, NY, USA). The samples were then spun in a microcentrifuge at 14 000 rpm for 5 min. An aliquot (40 µL) was transferred from each well to an autosampler microplate to which 200 µL of 0.2% formic acid was added and the plate was sealed with a cap mat. One hundred microlitres from each well was then analysed by HPLC/MS/MS. Standard curves with standards in the range 25000 ng/mL with appropriate quality controls prepared using blank lung and thigh tissueswere used for quantification of BB-81384 and azithromycin in tissues. Standards and quality controls did not deviate from nominal concentrations by >29%.
Murine pneumococcal peritonitis model
S. pneumoniae ATCC 6301 (PSSP) was used for these experiments. Fresh colonies were grown in brain heart infusion (BHI) broth (International BioProducts, Bothell, WA, USA) at 37°C with gentle agitation, and adjusted to a concentration of 100 cfu/mL by dilution in sterile 1x PBS (Hyclone, Logan, UT, USA). This represented greater than 10x the LD50 inoculum, which corresponded to 100% mortality rate by 48 h in vehicle-treated animals (data not shown).
Groups of five ICR female mice (25 g from Taconic Farms) were inoculated intraperitoneally (ip) with 0.5 mL of the inoculum described above. Groups were treated orally with vehicle, positive control (i.e. azithromycin, co-amoxiclav) or test agents 1 h post-infection. Clinical observations were made over 5 days with a primary endpoint of survival.
For all in vivo studies described herein, BB-81384 was formulated in a 50 mM citrate buffer containing 140 mM NaCl at pH 4.0. All animal studies strictly adhered to the guidelines set by the US Federal Government and the internal Institutional Animal Care and Use Committee (IACUC) at Genesoft Pharmaceuticals Inc.
S. pneumoniae lung infection
S. pneumoniae ATCC 6303 (PSSP) was grown overnight in MuellerHinton broth (BD Biosciences) containing 5% lysed horse blood to a concentration of 5 x 106 cfu/mL. Mice were anaesthetized with isofluorane (Baxter, Deerfield, IL, USA) and inoculated with 50 µL of overnight broth culture via the intranasal route. One hour post-inoculation, lungs were collected aseptically into 1 mL of sterile PBS to assess bacterial load. Drug treatments were oral once or twice daily for 3 days beginning 1 h post-inoculation. Survival was monitored over 7 days.
At pre-determined time-points post-treatment, lungs were collected aseptically into 1 mL of 1x sterile PBS. Quantification of bacterial load was accomplished by homogenizing lungs with a Polytron homogenizer PT 10/35 (Brinkman) in 1 mL of sterile PBS. Serial dilutions were performed using PBS as diluent. Three dilutions were plated per homogenate (100 µL each) onto TSA plates containing 5% sheep blood (BD Biosciences). Efficacy was determined by comparing bacterial counts of negative control (vehicle) groups to the test compounds. Blood was collected immediately post-mortem and bacterial counts measured by serial dilution in PBS as above.
S. pneumoniae neutropenic mouse thigh infection
S. pneumoniae ATCC 6301 was grown overnight in BHI broth at 37°C with gentle agitation and adjusted to a concentration of 106 cfu/mL by dilution in sterile 1x PBS. The protocol was essentially as described by Craig & Andes13 and Andes et al.18 ICR mice were rendered neutropenic by ip injections of cyclophosphamide 200 mg/kg (Sigma) on days 4 and 1 prior to infection. Groups of three neutropenic mice under isofluorane anaesthesia were inoculated intramuscularly (im) in the anterior thigh (50 µL) with 6.3 x 104 cfu/thigh in both thighs, at time 2 h. At time 0, mice were treated iv or by mouth with test agents, or negative or positive control compounds. At pre-determined time points post-treatment (5 and 24 h), mice were sacrificed and thighs collected aseptically into 10 mL of 1x sterile PBS. Quantification of bacterial load was accomplished as described above for the lung infection model.
Data analysis
The pharmacokinetic analyses were based on the average plasma concentration for three animals per treatment group at each sample time. Non-compartmental modelling was performed using WinNonlin software (Pharsight, Corp., Mountainview, CA, USA). Data analysis for ED50 determination and coefficient of variance was performed with the WinNonlin pharmacodynamic (efficacy) analysis package.
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Results |
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Directed optimization efforts involving medicinal chemistry and preliminary, limited-spectrum MIC activity described previously and extended herein, led to the identification of BB-81384 depicted in Figure 1.11,12,19 Relative to the early prototype BB-3497 (S. pneumoniae MIC 8 mg/L),11 the substituted piperazine moiety at P3' resulted in significantly improved anti-pneumococcal activity as described below.
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BB-81384 exhibited potent inhibition of peptide deformylase from several different bacterial sources, as indicated in Table 1. Also represented is a high degree of selectivity for PDF against a small set of other relevant mammalian metalloenzymes including MMP-1 and -2 and angiotensin I-converting enzyme.
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The MICs of BB-81384 and reference drugs azithromycin and co-amoxiclav are depicted in Table 2. While an insufficient number of different S. pneumoniae strains were screened for initial MIC50 and MIC90 determinations, of eight different strains tested, the MICs of BB-81384 were in the range 0.060.5 mg/L, with only two strains at the less susceptible end. MICs of BB-81384 versus the ATCC strains of H. influenzae were 24 mg/L. In addition, BB-81384 showed impressive potency against S. pyogenes as well as M. catarrhalis, with MICs of 0.25 and 0.125 mg/L, respectively. The compound was less potent against B. cereus (MIC = 4 mg/L).
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The killing kinetics of BB-81384 against S. pneumoniae ATCC 6301 are shown in Figure 2. The viable counts decreased as the antibiotic concentration increased. The difference between the viable count at time 0 and after 24 h at 1x, 2x and 4 x MIC was 0.3, 2.1 and 2.7 log10 cfu/mL, respectively.
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The observed pharmacokinetics of BB-81384 in mice are depicted in Table 3 and Figure 3. BB-81384 administered iv in mice was characterized by a biphasic disposition with a mean terminal elimination half-life of 2.2 h. The clearance of BB-81384 was 1.5 L/h/kg and the volume of distribution at steady state (1.6 L/kg) exceeded total body water in mice. Oral pharmacokinetics were assessed at two dose levels (10 and 50 mg/kg). After oral administration of BB-81384, plasma concentrations increased rapidly and mean maximum plasma concentrations were achieved within 20 min. Maximum plasma concentrations fell steadily in a biphasic manner with a mean terminal half-life of 3.13.7 h. The increase in Cmax and area under the curve (AUC) values appeared to be more than dose proportional, but may be attributed to limited sampling up to 5 h. A preliminary estimate of bioavailability of BB-81384 at 10 and 50 mg/kg dose levels yielded values of 55% and 88%, respectively.
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A screen of the tissue partitioning of BB-81384 and the reference drug, azithromycin, was undertaken to better understand the in vivo pharmacology of BB-81384, including potential comparisons with azithromycin in vivo potency. The data are represented in Figure 4(a and b). Concentrations of BB-81384 in the lung and thigh tissues reached maximum levels within 20 min. Lung and plasma concentrations were comparable, whereas thigh concentrations were approximately one-third of plasma levels. The partitioning of azithromycin into thigh and lung tissue was substantially greater than that of BB-81384.
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BB-81384 exhibited good oral efficacy in a stringent lethal peritonitis model (Table 4). Vehicle-treated control animals all died within 48 h of inoculation. A single oral administration of 30 mg/kg protected 50% of mice from an S. pneumoniae lethal infection. Complete protection from the infection was achieved by a single 90 mg/kg oral dose of BB-81384. In subsequent peritoneal sepsis studies, we found that increasing the oral administration frequency to twice daily on the day of infection (i.e. 1 and 5 h post-infection) reduced the ED50 to 20 mg/kg (data not shown).
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Two dose levels of BB-83184, 30 and 60 mg/kg, were administered orally in mice following thigh infection with S. pneumoniae. The thigh burden results are shown in Table 5. At 5 h post-treatment, BB-81384 reduced the bacterial load relative to the vehicle control by 3.8 log10 cfu/thigh. At 24 h post-treatment, BB-81384 reduced the viable bacteria by 2.6 and 3.3 log10 cfu/thigh for 30 and 60 mg/kg doses, respectively. In the same study, co-amoxiclav at a 10 mg/kg dose lowered the bacterial load in comparison to the vehicle control by 5 log units at 5 and 24 h post-treatment.
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In the S. pneumoniae lung infection model, all vehicle-treated control animals died within 72 h of infection. At the time of death, animals had 5.0 x 107 cfu/lung and 7.9 x 107 cfu/mL in the blood. BB-81384 was tested in a model wherein only robust therapies have proven efficacious and, when administered twice daily for 3 days at 100 mg/kg, resulted in long-term survival of 67% (Table 6). Notably, BB-81384 reduced the bacterial load in the lungs by 5 log units relative to the vehicle control, which was comparable toor better thanthat observed with the reference drugs (Figure 5). The reduction in bacterial load at 22 h was greater than that of co-amoxiclav, albeit at a higher dose. The efficacy of BB-81384 was comparable with that of ofloxacin in the same dosing regimen, although a dose of 100 mg/kg ofloxacin once daily did not provide sustained protection (Table 6).
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Discussion |
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The in vitro susceptibility and timekill profiles were encouraging for potential application to community-acquired pneumonia and possibly even acute exacerbation of chronic bronchitis (AECB). It is not uncommon for antibacterials, even PDF inhibitors, to exhibit both bacteriostatic and bactericidal activities against S. pneumoniae.12,14 In vitro activity against H. influenzae was not as good as desired for AECB, but represented an improvement over the first-generation compound, BB-83698.20,21
The pharmacokinetics of BB-81384 were attractive as clearance and volume of distribution were moderate and oral bioavailability was good (55%88%, depending on the dose). Examination of pharmacokinetic parameters and the efficacy profile suggested that the kinetics were generally dose proportional, although more definitive conclusions about pharmacokinetic dose-linearity would require more extensive testing. Disposition to tissues clinically relevant for Gram-positive infection such as lung and muscle has not been published for related PDF inhibitors and was therefore determined for BB-81384. Distribution was shown to be rapid, with maximum lung and thigh concentrations being either comparable to plasma levels or three- to five-fold lower. BB-81384 cleared from tissues with kinetics similar to elimination from the plasma compartment, which facilitates potential tissue modelling by analysis of the central compartment. Combined with protein binding analysis via an ultra-centrifugation method, which suggested that plasma protein binding for BB-81384 was 74% (data not shown), the pharmacokinetics of BB-81384 were considered favourable.
The ED50 of 30 mg/kg for BB-81384 in an S. pneumoniae lethal peritonitis model confirmed significant oral efficacy. The efficacy was dose proportional and potency generally correlated with in vitro activity (0.5 mg/L for S. pneumoniae 6301). The greater potency of comparator drugs could be attributed to enhanced in vitro potency in the case of co-amoxiclav and similar MICs but high tissue penetration in the case of azithromycin, as further described below (Tables 2 and 4, and Figure 4b). The BB-81384 results are similar to limited efficacy studies described by others, which show altogether good correlation of in vivo efficacy with plasma exposure and in vitro MICs.13,14
Oral administration of BB-81384 also resulted in good efficacy in organ burden models of lung and thigh infection. Long-term survival in the pneumonia model was observed following a 100 mg/kg twice-daily regimen. The reason for the reduced long-term survival for the BB-81384-treated animals relative to the co-amoxiclav-treated animals was unclear, although it may have been related to the superior potency and bactericidal nature of co-amoxiclav against the S. pneumoniae strain used in the lung infection model. Importantly, survival correlated well with lung and blood cfu counts.
Overall, the results of the S. pneumoniae peritoneal, thigh and lung infection studies demonstrated that BB-81384, an orally efficacious PDF inhibitor, had a dose-related activity profile which was both sufficient for proposed indications and comparable with those of reference drugs.
Pharmacokinetic and pharmacodynamic (PK/PD) assessment, performed previously by Craig & Andes13 on the clinical candidate BB-83698, showed that 24 h AUC/MIC appeared to drive efficacy. The similarities between BB-81384 and BB-83698 warranted investigation of preliminary PK/PD correlates for BB-81384. Profiling of in vivo tissue (lung, muscle) disposition was deemed important as it had not been well studied in general and would facilitate PK/PD projections. Accordingly, given thigh and plasma exposures to BB-81384, estimated AUC/MIC ratios for each compartment were derived. In the context of the thigh infection model, significant oral efficacy was observed at plasma or thigh AUC/MIC ratios in the range 2269 and 612 for 30 and 60 mg/kg doses, respectively (extrapolated from 10 and 50 mg/kg plasma AUC and 10 mg/kg tissue disposition data). Should the AUC/MIC ratio be validated for additional PDF inhibitors including BB-81384, then it would be pertinent to identify ratios correlating with significant efficacy in other pneumococcal models. Cmax as a critical determinant would be unlikely as BB-81384 iv and oral potencies were identical (ED50s 30 mg/kg in the peritonitis model). Pharmacokinetic data project minimal AUC differential whereas Cmax values would vary five- to 10-fold. Moreover, demonstration that 50 mg/kg twice daily provides better long-term survival in the lung infection model than 100 mg/kg once daily would further support AUC as opposed to Cmax as a primary PK parameter for BB-81384 in vivo pharmacodynamics.
Notably, azithromycin had superior potency to BB-81384 in the mouse infection models. However, examination of select tissue disposition properties for azithromycin provided an explanation for the improved in vivo potency as compared with BB-81384 given similar in vitro MIC and plasma exposure profiles. Partitioning of azithromycin to peripheral tissue (e.g. lung and thigh) was substantial with a tissue/plasma ratio 70-fold greater than BB-81384 and with very slow apparent decay. The comparison ultimately becomes most relevant in man and would therefore be dependent upon the pharmacokinetic scaling of a compound like BB-81384 in humans.
In summary, BB-81384 has been identified as a potent and selective inhibitor of peptide deformylase with potent anti-pneumococcal activity in vitro against drug-susceptible and -resistant strains. BB-81384 demonstrated good oral bioavailability, oral efficacy in multiple murine models of S. pneumoniae infection, and was well-distributed to peripheral tissues. Such results support continued optimization of orally active PDF inhibitors for advancement as novel clinical therapeutics.
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Acknowledgements |
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Footnotes |
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Present address. Emisphere Technologies, Tarrytown, NY 10591, USA;
¶ Present address. Sunesis Pharmaceuticals, 341 Oyster Pt, South San Francisco, CA 94080, USA.
Corresponding author. Fax: +1-650-827-0479; E-mail kjohnson{at}genesoft.com
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