ETX5 restoration of sulbactam activity against multidrug resistant Acinetobacter baumannii correlates with β-lactamase inhibition in vitro and in vivo Alita Miller, PhD Head of BioScience April, 07 ECCMID 07 Vienna, Austria
Avibactam and other DABCOs have broader spectra of inhibition than older β-lactamase inhibitors but Class D activity is still a gap in coverage β-lactamases use serine- or zinc-mediated hydrolysis to inactivate β-lactams,9 enzymes identified: Class A (,55); Class B (3); Class C (93); Class D (0) Clavulanic acid Sulbactam Tazobactam Serine Enzymes b-lactamases Metallo Enzymes Class A Class C Class D Class B Emerging threat KPC Carbapenemase OXA Avibactam Relebactam Vaborbactam Major new opportunity Acinetobacter & Pseudomonas ETX5 http://www.bldb.eu/ (5 March 07)
Log(CFU/g) Log(CFU/g) Sulbactam activity restored by ETX5 in vivo in MDR A. baumannii infection models Sulbactam:ETX5 dose response (IV, : ratio) 0 Thigh 0 Lung 9 9 9.0 7.3.03.0.7 Stasis 7 7.0.0.03.3.9 Stasis 5 3.39. 3.97.0.07 5 3.5..9 Pretreatment Pretreatment Vehicle.5 / 0.5 5 /.5 0 /.5 0 / 5 30 / 7.5 0 / 0 0 / 0 sulbactam/etx5 (mg/kg) q3h Vehicle.5 / 0.5 5 /.5 0 /.5 0 / 5 30 / 7.5 0 / 0 0 / 0 sulbactam/etx5 (mg/kg) q3h ARC3 (TEM-, ADC-30, OXA-, OXA-7) Sulbactam alone MIC 3 mg/l Sulbactam+ETX5 MIC = 0.5 mg/l How well does β-lactamase inhibition by ETX5 correlate to sulbactam antibacterial activity in vitro and efficacy in vivo? 3
S U L : E T X 5 M I C ( m g / L ) Relative β-lactamase expression vs. ETX5 restoration of sulbactam activity in vitro MIC (mg/l) A. baumannii Strain ID sulbactam sulbactam + ETX5 ( mg/l) Relative β-lactamase activity* (no drug) 3 ARC3 3 0. ARC507 3 0. ARC507.0 ARC50 0. ARC5955.5 ARC5950.3. ARC5077 0. ARC595 3.53 ARC599 0.75 *nitrocefin cleavage assay using log phase cells Efficacy vs. strains with higher MICs? 0. 5 0. 0 0. 5. 0. 5. 0. 5 r e l a t i v e b L A ( i n u n t r e a t e d b a c t e r i a ) Relatively weak correlation between MIC and β-lactamase activity in vitro
β-lactamase content and relative expression in vitro RT-PCR on log phase MDR A. baumannii grown in MHBII (untreated) A R C 5 9 5 5 - ; -3 0 ; O X A - 3 ; O X A - A R C 5 9 5 0 - ; O X A - 3 ; O X A - 9 A R C 5 0 7 7-3 0 ; O X A -7 A R C 5 9 5-3 0 ; - ; O X A - 3 0 0 S U L -E T X 5 M IC = m g /L 0 0 S U L -E T X 5 M IC = m g /L 0 0 S U L -E T X 5 M IC = m g /L 0 0 S U L -E T X 5 M IC = m g /L 0 0 0 0 O X A 3 O X A / 0 /7 O X A 5-9, 3 O X A 3 O X A / 0 /7 O X A 5-9, 3 O X A 3 O X A / 0 /7 O X A 3 O X A / 0 /7 O X A 5-9, 3 Relative β-lactamase expression is variable and strain dependent 5
In itia l ra te o f n itro c e fin h y d r o ly s is ( 9 0 n m ) In itia l ra te o f n itro c e fin h y d r o ly s is ( 9 0 n m ) In itia l ra te o f n itro c e fin h y d r o ly s is ( 9 0 n m ) In itia l ra te o f n itro c e fin h y d r o ly s is ( 9 0 n m ) Relative β-lactamase activity and inhibition by ETX5 in vitro A R C 5 9 5 5 - ; -3 0 ; O X A - 3 ; O X A - A R C 5 9 5 0 - ; O X A - 3 ; O X A - 9 A R C 5 0 7 7-3 0 ; O X A -7 A R C 5 9 5-3 0 ; - ; O X A - 3 0 0 S U L -E T X 5 M IC = m g /L 0 0 S U L -E T X 5 M IC = m g /L 0 0 S U L -E T X 5 M IC = m g /L 0 0 S U L -E T X 5 M IC = m g /L 0 0 0 0 O X A 3 O X A / 0 /7 O X A 5-9, 3 O X A 3 O X A / 0 /7 O X A 5-9, 3 O X A 3 O X A / 0 /7 O X A 3 O X A / 0 /7 O X A 5-9, 3 β-lactamase (nitrocefin cleavage) assay for log phase MDR A. baumannii grown in MHBII -/+ ETX5 5 0 5 0 5 0 5 0 0 0 0 0 3 0 3 0 3 0 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 m g /L E T X 5 m g /L E T X 5 m g /L E T X 5 m g /L E T X 5 Exposure to ETX5 in vitro results in inhibition of β-lactamase activity of diverse enzymes in all four strains tested
How do these results translate to in vivo efficacy of sulbactam-etx5? Q: Can we measure β-lactamase in vivo? Q: Are in vivo β-lactamase levels different than in vitro? Q3: Can we detect inhibition of β-lactamase in vivo? Q: Can we correlate in vivo inhibition of β-lactamase to in vivo efficacy? 7
Detection of β-lactamase expression and activity in murine models of A. baumannii infection neutropenic 0 cfu X flush = brochoalveolar lavage (BAL) (N = 3) RNA (RT-PCR) β-lactamase (nitrocefin cleavage assay) Harvest lung cfu counts T 0 T hr T hr 0 cfu neutropenic N = 3, SC dosing Q3H Vehicle control Sulbactam alone Sulbactam + increasing ETX5 ETX5 alone Harvest both thighs (N = ) RNA (RT-PCR) β-lactamase (nitrocefin cleavage assay) Remaining tissue cfu counts
Different β-lactamase expression patterns observed in vitro vs. in vivo Results from RT-PCR on A. baumannii ARC5955 grown in MHBII or harvested from lung or thigh infections MHBII (mid-log, no drug) Untreated Lung T hr Untreated Thigh T hr 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 O X A 3 O X A / 0 O X A 5-9, 3 O X A 3 O X A / 0 O X A 5-9, 3 O X A 3 O X A / 0 O X A 5-9, 3 9
In itia l ra te o f n itro c e fin h y d r o ly s is ( 9 0 n m ) log 0 cfu/g β-lactamase activity in BAL vs. thigh homogenates from ARC5955-infected mice 0 0 0 0 0 0 0 0 0 0 0 0 0 V e h ic le E T X 5 (5 0 ) Lung L u n g Thigh ETX5 significantly inhibits β-lactamase activity in both lung and thigh models of A. baumannii infection with minimal corresponding reduction in bacterial burden T h ig h 0
In itia l ra te o f n itro c e fin h y d r o ly s is ( 9 0 n m ) log 0 cfu/g β-lactamase inhibition by ETX5 alone vs. four MDR A. baumannii strains in vivo 0 0 0 0 0 0 0 0 0 0 0 0 0 V e h ic le E T X 5 (5 0 ) A R C 5 9 5 5 A R C 5 9 5 0 A R C 5 0 7 7 A R C 5 9 5 T hr (thigh model) 50 mg/kg ETX5 SC q3h
L o g c fu /g tis s u e L o g c fu /g tis s u e L o g c fu /g tis s u e L o g c fu /g tis s u e In vivo efficacy of sulbactam-etx5 in murine thigh model of MDR A. baumannii infection 0 A R C 5 9 5 5 - ; -3 0 ; O X A - 3 ; O X A - S U L -E T X 5 M IC = m g /L -.9 0 A R C 5 0 7 7-3 0 ; O X A -7 S U L -E T X 5 M IC = m g /L -.9 s ta s is sta sis P r e -tr e a tm e n t V e h ic le S U L a lo n e S U L + 5 (.5 ) S U L + 5 (5 0 ) S U L + 5 ( 0 0 ) E T X 5 (5 0 ) 0 A R C 5 9 5 0 - ; O X A - 3 ; O X A - 9 S U L -E T X 5 M IC = m g /L -. P r e -tr e a tm e n t V e h ic le S U L a lo n e S U L + 5 (.5 ) S U L + 5 (5 0 ) S U L + 5 ( 0 0 ) E T X 5 (5 0 ) 0 A R C 5 9 5 - ; -3 0 ; O X A - 3 S U L -E T X 5 M IC = m g /L -.7 sta sis sta sis P r e -tr e a tm e n t V e h ic le S U L a lo n e S U L + 5 (.5 ) S U L + 5 (5 0 ) S U L + 5 ( 0 0 ) A dose-dependent reduction in bacterial burden is observed when ETX5 is administered in combination with sulbactam but not when either agent is given alone Including strains with elevated sulbactam-etx5 MICs E T X 5 (5 0 ) P r e -tr e a tm e n t V e h ic le S U L a lo n e S U L + 5 (.5 ) S U L + 5 (5 0 ) S U L + 5 ( 0 0 ) E T X 5 (5 0 )
Summary and Conclusions To our knowledge, demonstration of ETX5 activity in this study represents the first example of direct detection of β-lactamase inhibition in bacteria harvested from infected mice ETX5 is a potent, broad-spectrum inhibitor of Class A, C and D β-lactamases in vitro and in vivo β-lactamase inhibition by ETX5 restores sulbactam activity vs. MDR A. baumannii both in vitro and in vivo» Including strains with elevated MICs 3
Acknowledgements