Alita Miller, PhD. Head of BioScience. April 24, 2017 ECCMID 2017 Vienna, Austria

Transcription

1 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

2 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 (5 March 07)

3 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 Stasis Stasis Pretreatment Pretreatment Vehicle.5 / /.5 0 /.5 0 / 5 30 / / 0 0 / 0 sulbactam/etx5 (mg/kg) q3h Vehicle.5 / /.5 0 /.5 0 / 5 30 / / 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

4 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 ARC ARC ARC507.0 ARC50 0. ARC ARC ARC ARC ARC *nitrocefin cleavage assay using log phase cells Efficacy vs. strains with higher MICs? 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

5 β-lactamase content and relative expression in vitro RT-PCR on log phase MDR A. baumannii grown in MHBII (untreated) A R C ; -3 0 ; O X A - 3 ; O X A - A R C ; O X A - 3 ; O X A - 9 A R C ; O X A -7 A R C ; - ; O X A 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 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

6 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 ; -3 0 ; O X A - 3 ; O X A - A R C ; O X A - 3 ; O X A - 9 A R C ; O X A -7 A R C ; - ; O X A 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 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 -/+ ETX 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

7 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

8 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

9 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 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

10 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 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

11 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 V e h ic le E T X 5 (5 0 ) A R C A R C A R C A R C T hr (thigh model) 50 mg/kg ETX5 SC q3h

12 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 ; -3 0 ; O X A - 3 ; O X A - S U L -E T X 5 M IC = m g /L A R C ; 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 ; 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 ; -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 )

13 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

14 Acknowledgements