1 The In Vitro and In Vivo Antibacterial Activities of Omadacycline, a

27 Pages·2013·0.44 MB·English

AAC Accepts, published online ahead of print on 2 December 2013
Antimicrob. Agents Chemother. doi:10.1128/AAC.01242-13
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
1 The In Vitro and In Vivo Antibacterial Activities of Omadacycline, a Novel
2 Aminomethylcycline
3 Running Title: The In Vitro and In Vivo Activities of Omadacycline
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5 A.B. Macone1, B.K. Caruso1, R.G. Leahy1, J. Donatelli1, S. Weir2, M.P. Draper1, S.K. w
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6 Tanaka1*, S.B. Levy1 a
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7 1Paratek Pharmaceuticals, Inc. 75 Kneeland Street, Boston, MA 02111 fr
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8 2Boston University School of Medicine, Boston, MA 02118 t
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9 *To whom correspondence should be addressed. S.K. Tanaka, Paratek Pharmaceuticals, Inc. 75 a
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10 Kneeland Street, Boston, MA 02111. Telephone: [phone]. FAX: [phone]. E-mail: s
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11 [email protected] r
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12 Omadacycline is the first intravenous and oral 9-aminomethylcycline in clinical development for
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13 multiple infectious disease indications including Acute Bacterial Skin and Skin Structure ril
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14 Infections (ABSSSI), Community-Acquired Bacterial Pneumonia (CABP) and urinary tract ,
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15 infections (UTI). The comparative in vitro activity of omadacycline was determined against a 9

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16 broad panel of gram-positive clinical isolates, including methicillin-resistant Staphylococcus y
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17 aureus (MRSA), vancomycin-resistant Enterococccus (VRE), and Lancefield Groups A and B s
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18 beta-hemolytic streptococci, penicillin-resistant Streptococcus pneumoniae (PRSP), and
19 Haemophilus influenzae (H. influenzae). The MIC s for MRSA, VRE and beta-hemolytic
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20 streptococci were 1.0 µg/ml, 0.25 µg/ml and 0.5 µg/ml, respectively, and the MIC s for PRSP
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21 and H. influenzae were 0.25 µg/ml and 2.0 µg/ml, respectively. Omadacycline was active against
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22 organisms demonstrating the two major mechanisms of resistance, ribosomal protection and
23 active tetracycline efflux. In vivo efficacy of omadacycline was demonstrated using an
24 intraperitoneal infection model in mice. A single intravenous dose of omadacycline exhibited
25 efficacy against Streptococcus pneumoniae, Escherichia coli, and Staphylococcus aureus,
26 including tet (M) and tet (K) efflux containing, and MRSA, strains. The ED s for Streptococcus
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27 pneumoniae obtained ranged from 0.45 mg/kg to 3.39 mg/kg, the ED s for Staphylococcus w
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28 aureus obtained ranged from 0.30 mg/kg to 1.74 mg/kg, and the ED50 for Escherichia coli was a
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29 2.02 mg/kg. These results demonstrate potent in vivo efficacy including activity against strains d

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30 containing common resistance determinants. Omadacycline demonstrated in vitro activity m

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31 against a broad range of gram-positive and select gram-negative pathogens, including resistance ttp
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32 determinant containing strains, and this activity translated to potent efficacy in vivo. a
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33 INTRODUCTION m
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34 Widespread resistance to antibiotics, including resistance to the older tetracyclines (tetracycline, /
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35 doxycycline, and minocycline), has limited their usefulness in recent years (1-2). New A
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36 generations of tetracycline derivatives that inhibit resistant organisms have been approved or are il
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37 in development including the glycylcyclines, specifically tigecycline, and fluorocyclines, 2
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38 including eravacycline (TP-434) both of which have potent gram-positive and gram-negative in 9

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39 vitro activity (3-6). The discovery of the 9-aminomethyl class of tetracyclines has led to the g
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40 identification of omadacycline (PTK 0796) that is poised to begin Phase 3 clinical trials in s
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41 ABSSSI, CABP and UTI with both an intravenous and oral tablet formulation. Omadacycline,
42 (4S,4aS,5aR,12aS)-4,7-bis(dimethylamino)-9{[(2,2-dimethylpropyl)amino]methyl}-3,10,12,12a-
43 tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide), contains a
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44 four-ring carbocyclic skeleton and is a semi-synthetic compound prepared by chemical
45 modification of minocycline (Fig 1) (7-8).
46 Omadacycline is unique from older generation tetracyclines because it demonstrates in vitro
47 activity against a relatively broad spectrum of organisms including gram-positive, gram-
48 negative, anaerobic and atypical pathogens, but demonstrates similar in vitro activity against D
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49 pathogens that express not only tetracycline resistance, but other antibiotic resistance, including
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50 methicillin, vancomycin, erythromycin, and ciprofloxacin (9-20). This broad in vitro activity has a
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51 been confirmed in various in vivo models of infection (21-24). Omadacycline is bioavailable in
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52 humans by both oral and intravenous routes and does not demonstrate significant gastrointestinal

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53 side effects (25-28). The targeted indications encompass acute bacterial infections where a broad p
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54 spectrum antibiotic with activity against the most prevalent community-acquired multi-drug a
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55 resistant organisms is desired. This report is the initial description of the in vitro spectrum and in s
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56 vivo efficacy of omadacycline. The in vitro activity of omadacycline translates into potent in r
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57 vivo efficacy in a lethal infection model, suggesting that the pharmacodynamic requirements n

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58 necessary for human clinical trial investigation can be achieved. p
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59 MATERIALS AND METHODS ,
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60 Bacterial strains. Routine clinical isolates were obtained from the following sources: b
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61 Children’s Hospital, Boston, MA, Channing Laboratories, Boston, MA. Clinical Microbiology gu
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62 Institute, Wilsonville, OR, Glaxo Smith Kline, Collegeville, PA, Tufts Medical Center, Boston, t
63 MA, University of California at Los Angeles Medical Center, Los Angeles, CA, University of
64 Wisconsin Hospitals and Clinics, Madison, WI. For testing, isolates were chosen randomly so
65 that all sites would be represented. All isolates were stored frozen at –80°C in tryptic soy broth
66 or Mueller Hinton broth (Northeast Laboratories, Waterville, Maine) plus 20% glycerol (Becton
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67 Dickinson, Sparks MD). Horse or sheep blood supplementation was used for fastidious
68 organisms. Isolates were subcultured twice onto appropriate solid media (tryptic soy agar with
69 5% sheep blood or chocolate agar, Becton Dickinson, Sparks MD), prior to MIC testing. Quality
70 control isolates were obtained from American Type Culture Collection (ATCC, Manassas, VA).
71 For the in vivo experiments, the bacterial strains were obtained from the following sources: S. D
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72 pneumoniae 700905, S. aureus 29213 and the clinical isolate S. aureus USA300 were obtained n
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73 from American Type Culture Collection (Manassas, VA) (ATCC 700905, ATCC 29213, and CA d
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74 USA300 FPR3757/ATCCBAA 1556, respectively). Tetracycline-sensitive S. pneumoniae f
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75 PBS1339 (GSK1629) was obtained from GlaxoSmithKline, Philadelphia, PA. S. pneumoniae
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76 157E-2 was derived from passing S. pneumoniae 157E (originally called GSK 157E and :
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77 obtained from GlaxoSmithKline) twice through mice. S. aureus USA400 is a clinical isolate c
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78 (CA USA400 REF 571) obtained from Paul Fey, University of Nebraska Medical Center, m
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79 Omaha, NE. S. aureus MRSA5 was obtained from the University of Maryland, College Park, rg
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80 MD. E. coli PBS1478 (also referred to as SC8294) was originally obtained from Bristol Meyers n

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81 Squibb, Fort Devens, MA. r
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82 Antibiotics and in vitro susceptibility testing. Omadacycline was synthesized at Paratek 2
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83 Pharmaceuticals, Inc. Antibiotic comparators used for the in vitro studies were obtained from 9

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84 Sigma-Aldrich, St. Louis, MO. For the in vivo studies, tigecycline was obtained from Bosche g
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85 Scientific, New Brunswick, NJ. Doxycycline was obtained from Hovione, East Windsor, NJ. s
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86 Linezolid and levofloxacin was purchased from Sequoia Research, Pangbourne, UK.
87 Vancomycin HCl and ceftriaxone was purchased from Sigma, Atlanta, GA. Daptomycin was
88 obtained from Cubist Pharmaceuticals, Inc., Lexington, MA. Microdilution broth MICs were
89 performed according to CLSI (formerly NCCLS) guidelines (18).
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90 PCR for detection and identification of tetracycline resistance genes. The presence of the
91 efflux genes tet(K), tet(L), as well as tet(A), tet(B) and genes of the ribosomal protection (RP)
92 family (tet(M), tet(O), and tet(S)) were assessed by multiplex PCR (29).
93 Systemic intraperitoneal (IP) challenge model. Six-week-old, specific-pathogen-free, male
94 CD-1 mice, weighing 18-30g (Charles River, Wilmington, MA) were used for all experiments. D
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95 Animals were acclimated for 1 week following delivery. Mice were allowed food and water ad n
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96 libitum and kept at a constant 12 h light/dark cycle. Bacterial cultures were grown by either d
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97 streaking frozen colonies onto trypicase soy agar II plates with 5% sheep’s blood (Northeast f
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98 Laboratories, Waterville, Maine) and incubating them overnight in a CO2 enriched environment h
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99 at 37ºC (S. pneumoniae) or growing frozen isolates in a 37ºC shaker at 180 rpm in Mueller :
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100 Hinton Broth (Northeast Labs, Waterville, Maine) (S. aureus, E. coli). For S. pneumoniae, c
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101 following the overnight incubation, the colonies were aseptically collected off 2-3 agar plates m
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102 and resuspended in 3 ml of sterile Phosphate Buffered Saline (PBS) (Fisher Scientific, Boston, rg
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103 MA) for a final concentration of approximately 1 × 109 colony forming units (CFU)/ml. For S. n

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104 aureus and E. coli strains, an overnight broth was grown to a concentration of approximately 1 × r
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105 109 CFU/ml. Serial dilutions of all bacterial suspensions were performed in sterile PBS to obtain 3,

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106 the infectious dose used for individual experiments. Infectious doses used in each experiment 1
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107 were confirmed by plating serial dilutions on trypicase soy agar II plates with 5% sheep’s blood y

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108 and incubating overnight (in a CO enriched environment for S. pneumoniae) at 37ºC and the e
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109 bacterial colonies were then enumerated. Septicemia was induced by infecting mice
110 intraperitoneally (IP) with 500 µls containing a mean ± standard deviation of 6.85 ± 1.58 × 102
111 CFU of S. pneumoniae PBS1339, 1.07 ± 1.15 × 106 CFU of S. pneumoniae 700905, 1.02 ± 1.22
112 × 105 CFU of S. pneumoniae 157E-2, 7.13 ± 3.31 × 107 CFU of S. aureus USA300, 6.40 ± 1.53
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113 × 106 CFU of S. aureus 29213, 1.08 ± 0.43 × 103/ml CFU of S. aureus USA400, 1.06 ± 0.56 ×
114 108 CFU of S. aureus MRSA5, and 6.60 ± 2.34 × 106 CFU of E. coli PBS1478 in an autoclaved
115 4.5% bacteriological mucin (VWR Scientific, Pittsburg, PA) suspension. Mice were infected
116 using a 3 ml lock top sterile syringe with a sterile 25 gauge × 5/8 inch needle (Becton Dickinson,
117 Franklin Lakes, NJ). At 1 h post infection (p.i.), mice were dosed intravenously (IV) with
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118 omadacycline or comparator compounds of interest, dissolved in sterile saline for injection at a w
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119 volume of 10 ml/kg. All drug doses were formulated fresh immediately prior to administration a
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120 and adjusted to account for percent activity. A minimum of 4 dose levels were tested per d

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121 experiment with n=5 mice/group. The typical doses tested ranged from 0.11 to 18 mg/kg with m

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122 exceptions for comparators that required significantly higher or lower doses to achieve 50% ttp
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123 efficacy (dose range minimum-maximum, 0.08-54 mg/kg). Each study also included an untreated a
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124 control group. Mice were housed in filter topped cages in an isolated room and monitored for as
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125 morbidity at least every 24 h for 7 days. Efficacy was determined by calculating the 50% o
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126 effective dose (ED ) for all drugs tested. The ED is defined as the dose required to achieve o
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127 50% survival at 7 days p.i and was estimated when possible using the formula y = 1/1+10(log(k)- p
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128 log(×)*4.2) where k=0.5, by non-linear regression analysis with Prism 3.0 software. All animal 1
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129 protocols were critically reviewed and approved by the Paratek Pharmaceuticals, Inc. 0
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130 Institutional Animal Care and Use Committee. b
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131 RESULTS e
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132 MICs of omadacycline, tetracycline and doxycycline on characterized tetracycline resistant
133 gram-positive and gram-negative bacteria. Omadacycline demonstrated activity against the
134 gram-positive pathogens, S. aureus, E. faecalis, E. faecium, S. pneumoniae, and beta hemolytic
135 streptococci, carrying ribosomal protection (tet(M), tet(O), and tet(S)) and efflux (tet(K) and
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136 tet(L)) tetracycline resistance genes (Table 1). The concentration of omadacycline required to
137 inhibit growth of several strains of E coli carrying efflux genes (tetA) was also reduced
138 compared to conventional tetracyclines.
139 Omadacycline demonstrates in vitro activity against a broad panel of clinically relevant
140 gram-positive and gram-negative bacterial strains. The comparative in vitro activity of D
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141 omadacycline was assessed against a broad panel of clinically significant gram-positive and n
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142 gram-negative bacteria. Omadacycline was as active as comparators against susceptible S. d
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143 aureus and was more active than most comparators against MRSA, most of which were resistant f
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144 to more than one comparator antibiotic (Table 2). These results indicate that omadacycline
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145 specifically overcomes the problem of tetracycline, doxycycline, and minocycline resistance in S. :
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146 aureus. c
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147 One of the more difficult to treat pathogens, and the pathogen that has been the most .
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148 problematic in terms of resistance to antibiotics, is the genus Enterococcus, including both /
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149 Enterococcus faecalis and Enterococcus faecium. Isolates of both species have acquired A
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150 mechanisms for resistance to vancomycin and such strains present a difficult therapeutic il
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151 challenge. Omadacycline is active against both species and is equally active against vancomycin 2
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152 susceptible and resistant isolates (Table 2). Omadacycline is also equally active against 9

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153 tetracycline-resistant and susceptible isolates of E. faecalis and E. faecium. g
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154 S. pneumoniae is an important respiratory pathogen in the hospital and community. Of
155 particular concern in the community are isolates resistant to accepted oral antibiotics, particularly
156 penicillins and cephalosporins, macrolides, and tetracyclines. Omadacycline exhibits activity
157 against all S. pneumoniae isolates tested regardless of resistance to these agents and even when
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158 isolates are resistant to multiple antibiotics (tetracycline plus penicillin plus azithromycin) (Table
159 2).
160 Omadacycline also exhibits in vitro activity against other streptococci. S. pyogenes
161 (Lancefield Group A, β-hemolytic streptococcus) and S. agalactiae (Lancefield Group B, β-
162 hemolytic streptococcus) are susceptible to omadacycline (Table 2). D
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163 Finally, omadacycline exhibits activity in vitro against some gram-negative bacteria lo
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164 including, E. coli, K. pneumoniae, and H. influenzae (Table 3). e
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165

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166 Comparative efficacy studies in the in vivo systemic infection model. The efficacy of :/
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167 omadacycline was tested in a systemic infection model to determine if omadacycline has c
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168 potential as a clinical therapy in humans. The IP challenge model is a standard in vivo model of m
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169 systemic infection commonly used as a basic screening tool to evaluate the antibiotic potential of g
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170 novel therapies (30).
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171 Omadacycline has demonstrated favorable pharmacokinetics intravenously in multiple il 1
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172 species and has demonstrated good intravenous and oral bioavailability in humans (27-28, 31). 2
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173 Omadacycline is currently being developed as both an intravenous and oral broad spectrum
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174 clinical therapy (25-26, 32). However, the oral bioavailability of omadacycline in rodents is g
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175 significantly lower as demonstrated by pharmacokinetic evaluation and subsequent efficacy t
176 studies (data not shown). Because murine omadacycline bioavailability is particularly poor
177 compared to the good oral bioavailability previously observed in other non-rodent species and
178 humans, in vivo efficacy studies in mice were conducted by administering omadacycline
179 intravenously.
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180 A single IV dose of omadacycline demonstrated potent efficacy against tetracycline-
181 sensitive and tetracycline-resistant strains of S. pneumoniae and S. aureus, as well as proving
182 efficacious against the common gram-negative pathogen, E. coli, in the murine systemic IP
183 challenge model (Table 4). Efficacy was evaluated by determining the ED for omadacycline
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184 and each comparator antibiotic. Against the highly virulent, mucoid, tetracycline-sensitive S.
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185 pneumoniae PBS1339 strain, the ED of 3.34 mg/kg for omadacycline was similar to the w
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186 glycycline, tigecycline (4.13 mg/kg). The ED50 for omadacycline was over 4-5 times more a
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187 efficacious than doxycycline, vancomycin, and levofloxacin, and over 7 times more efficacious d

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188 than linezolid (14.23 mg/kg, 15.7 mg/kg, 19.35 mg/kg, and 24.47 mg/kg, respectively). m

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189 Ceftriaxone and daptomycin were slightly more potent than omadacycline (1.10 mg/kg and 1.43 ttp
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190 mg/kg, respectively). a
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191 Against the tetracycline-resistant, azithromycin-resistant, Tet-M S. pneumoniae 700905 m
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192 strain, the ED50 for omadacycline (0.45 mg/kg) was lower than all the other comparators tested. rg
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193 It was over 30 times more active than linezolid (13.88 mg/kg) and slightly more efficacious than n

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194 vancomycin (0.91 mg/kg) and tigecycline (1.72 mg/kg), Doxycycline failed to demonstrate any r
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195 efficacy even at the highest dose tested (54 mg/kg), thus an ED50 value could not be calculated. 3,
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196 The ED of 1.10 mg/kg for omadacycline was lower than all the other antibiotics tested 9
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197 against the tetracycline-sensitive S. pneumoniae 157E-2 strain. It demonstrated similar efficacy g
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198 as doxycycline (1.55 mg/kg) and tigecycline (1.72 mg/kg), but was over 11 times more active s
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199 than vancomycin (12.32 mg/kg). Linezolid failed to protect the mice at the highest dose tested
200 (27 mg/kg), thus an ED value could not be calculated.
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201 In the tetracycline-sensitive S. aureus 29213 IP challenge model, omadacycline was more
202 than 3-5 times more potent than vancomycin and linezolid (1.74 mg/kg versus 6.09 mg/kg and
203 9.91 mg/kg, respectively) but was slightly less effective than doxycycline and tigecycline (0.91
204 mg/kg and 0.73 mg/kg, respectively).
205 Against the clinical MRSA tet-K, tet-38 S. aureus USA300 strain, the ED of 0.90 D
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206 mg/kg for omadacycline was over 9 times more active than linezolid (8.18 mg/kg). n
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207 Omadacycline was over 4 times more potent than doxycycline (4.13 mg/kg), but slightly less d
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208 potent than tigecycline (0.58 mg/kg). Vancomycin failed at all the doses tested including 18 f
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209 mg/kg, thus an ED50 could not be accurately calculated. h
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210 A single IV dose of omadacycline resulted in an ED50 of 0.45 mg/kg against the //a
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211 tetracycline-sensitive, clinical S. aureus USA400 strain. This was more efficacious than any of .a
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212 the other comparators tested. Omadacycline was twice as active as doxycycline and tigecycline .
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213 (1.12 mg/kg and 1.09 mg/kg, respectively) and 7 and 18 times more effective than vancomycin /
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214 and linezolid (3.29 mg/kg and 8.12 mg/kg, respectively). A
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215 Omadacycline also had a lower ED50 than any of the other comparators tested against the 3
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216 MRSA, Tet-M, S. aureus MRSA5 strain. With an ED of 0.30 mg/kg, omadacycline was over 5 0
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217 times more efficacious than tigecycline (1.74 mg/kg) and over 80 times more active than b
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218 linezolid (24.53 mg/kg). Neither vancomycin nor doxycycline demonstrated efficacy at the u
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219 highest doses tested (18 mg/kg and 54 mg/kg, respectively).
220 With an ED of 2.02 mg/kg, omadacycline also demonstrated in vivo efficacy against the
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221 gram-negative bacteria, tetracycline-sensitive, E. coli PBS 1478. Although it was not as potent as
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