3/18/16 Successful Use of Repurposed Drugs for MDR TB: Meropenem/Clavulanate and its Newer Rela?ves Maunank Shah MD PhD Assistant Professor Center for TB Research & Center for Clinical Global Health Educaon Johns Hopkins University BalDmore City Health Department When opDons are limited… • 42 y/o from Peru with MDR-TB – Resistant to HRZE, ethionomide • AdmiSed with sepsis and ARDS • Not taking PO medicaons • LFTs in 1000’s, Tbili 12, Cr: 3.6 • REGIMEN: – IV Moxifloxacin, – IV Amikacin, – IV Linezolid – PAS, cycloserine not immediately available – What other drugs to add? 1 3/18/16 Learning ObjecDves • Understand the role of repurposed anDbioDcs and design stronger treatment regimens for paents with MDR and XDR TB • IdenDfy the toxiciDes associated with these medicaons and improve their ability to monitor and detect them • Discuss the strengths and limitaons of incorporang repurposed anDbioDcs into MDR/XDR TB treatment regimens and improve their ability to appropriately use these agents. Outline • Background: – What is a carbapenem? – How do carbapenems work? – What are carabapenems used for? • Carbapenems for the treatment of M. tuberculosis – In vitro data – In vivo data – Clinical data • Summary and outstanding quesDons 2 3/18/16 What is a carbapenem: core structure 1 6 5 2 7 4 3 • Part of the ‘penicillin’ family (Beta-Lactams) • Has extremely broad spectrum of acDvity: • Gram posiDves, Gram negaves and anaerobes • Tends to be acDve even in presence of bacterial “beta-lactamases” Carbapenems: last line of defense • Currently at least 2million illnesses and 23,000 deaths in the US from drug-resistant bacteria – Carbapenems oAen the only drug class with retained sensi?vity – Carbapenems are the drug of choice for ESBL (extended spectrum beta lactamase) producing organisms • Carbapenems also have acvity against mycobacteria hSp://www.cdc.gov/drugresistance/biggest_threats.html 3 3/18/16 How do they work: Carbapenems aack the cell wall of bacteria • The pepdoglycan layer is present in virtually all bacteria. • This layer is essenDal for survival and growth of bacteria. pepDdoglycan Paul & Beveridge: Infect. Immun. 1994. p.1542 M. tuberculosis SEM. JHU EM Core Facility, 2001. Hayhurst et al, PNAS, 2008 How do Carbapenems work against M. tuberculosis Carbapenems can block the 3-3 linkage β-lactam drugs In M. tuberculosis L-Ala D-Ala 4 D-Glu D-Ala 3 3 mDap mDap 3 PepDdoglycan TranspepDdase D-Glu D-Ala layer L-Ala MTB TranspepDdase Membrane Cytosol 4 3/18/16 Carbapenems are not readily impacted by the β-lactamase produced by M. tuberculosis Carbapenem BlaC Beta- BlaC Beta- lactamase lactamase Clavulanic acid M. tuberculosis M. tuberculosis penicillin BlaC Beta- lactamase Hugonnet et al. Science. 2009 Carbapenems covalently bind and inhibit β-lactamase • Meropenem inhibits BlaC in a dose dependent manner • Also seen with doripenem, ertapenem and tebipenem Hugonnet. Science 2009; Tremblay Biochemistry 2010; Hazra Biochemistry 2014 5 Figure S1. Slow-onset inhibition of BlaC with meropenem. (A) Time courses of nitrocefin hydrolysis with increasing concentration of meropenem (top panel). (B) Rate of isomerization (kiso) versus concentration of meropenem (lower panel). 3/18/16 B-Lactamase Inhibitors also bind to BlaC • Clavulanate, Sulbactam, Tazobactam Hugonnet• andClavulanate Blanchard is the only one that irreversibly inhibited PageBlaC 11 TAZOBACTAM NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript SULBACTAM CLAVULANATE Figure 4. Recovery of BlaC activity after incubation with β-lactamase Hugonnetinhibitors Biochemistry 2007 Enzyme (20 μM) was incubated with 100 μM sulbactam (▲), 100 μM tazobactam (■) or 100 μM clavulanate (●), and activity was determined at the indicated times. Currently Carbapenems are listed as Group 5 drugs for the treatment of MDR-TB Step 1: Include any first-line Step 3: Include an drugs to which the isolate is Step 2: Add a fluoroquinolone injectable agent susceptible Fluoroquinolone First-line Drugs Injectables Levofloxacin Kanamycin Ethambutol Moxifloxacin Amikacin Pyrazinamide Gatifloxacin Capreomycin High-dose isoniazid Streptomycin Oral Group 4 Drugs Group 5 Drugs Step 4: Include second-line Ethionamide drugs until you have 4-6 Clofazimine Prothionamide drugs to which the isolate is Clarithromycin Cycloserine/Terizidone susceptible Amoxicillin-clavulanate Para-aminosalicylic acid Consider third-line drugs if Linezolid there are not 4-6 drugs to Thiacetazone which the isolate is Meropenem-clavulanate susceptible Thioridazine Adapted from: Curry International Tuberculosis Center. Drug-resistant tuberculosis: a survival guide for clinicians. Chang KC, et al. Respirology. 2013;18:8-21. 6 Biochemistry. Author manuscript; available in PMC 2008 December 4. 3/18/16 Evidence for using Carbapenems for MTB: Is it Dme to move on from “Group 5”? • In Vitro data • In Vivo data (i.e. animal models) • Clinical data In Vitro Data 7 REPORTS The combination of clavulanate with b- culosis. Thirteen clinical isolates exhibiting the were determined. The susceptibility of these strains lactams, especially meropenem, was also tested XDR phenotype were tested (18). Clavulanate was experimentally indistinguishable from that Kumarfor the et abilityal. to inhibit the growth of extensively was used at a concentration of 2.5 mgml–1,and determinedPage 18 for H37Rv and the Erdman strain, drug-resistant (XDR) clinical strains of M. tuber- the MIC values of these strains for meropenem that is, ≤1 mgml–1 (Table 1). In contrast, sub- 3/18/16 stantial variability in the MIC values to ampicil- lin, amoxicillin, cephalothin, and imipenem was NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript observed for these same strains (table S2). The clavulanate-meropenem combination is thus equal- ly effective against both susceptible and XDR strains. These structural and mechanistic studies of carbapenem interactions with the BlaC b-lactamase ASacking the Cell Wall have revealed properties of specific b-lactam an- tibiotics that can be exploited in the treatment of Altered morphology tuberculosis, including the treatment of multidrug- and extensively drug-resistant strains. The struc- ture of the meropenem-inactivated acyl-enzyme, in combination with our mechanistic proposal for its hydrolysis and the structure of the clavulanate- BlaC complex, provides the information neces- sary to design improved tuberculosis-specific b-lactams that could form longer-lived acyl-enzyme intermediates. Among currently approved b- lactams, however, meropenem is superior on the basis of its poor activity as a substrate for BlaC, ability to transiently inhibit BlaC, and activity against nonreplicating organisms. This activity provides experimental evidence that peptidoglycan CONTROL 1X MIC90 - 10X 1X MIC90 - remodeling occurs in M. tuberculosis in the non- Fig. 2. replicating state, which may be an important SEM images showing effectsMEROCLAV of meropenem exposureMEROCLAV on Mtb cell shape. (A & B)determinant untreated of clinical efficacy. In culture, meropenem+clavulanic acid controls, (C & D) treated with 1X MCA, (E & F) with 10X MCA or (G & H) with 10XTen yearsINH ago, a report on the early bacteri- Leads to cidal (i.e.lyc killing) acDvity against MTB (Bar=1 μm). The lower magnification pictures shown above (A, C, E, G) were capturedcidal activity at of amoxicillin-clavulanate in pa- 8,000 X and corresponding higher magnification pictures shown below (B, D, F, I) were Kumar, Mol Microbio 2012 tients with tuberculosis appeared (19), but no captured at 25, 000 X. additional reports have appeared since then. Our studies reveal that clinical strain-to-strain varia- bility is observed with combinations of clavulanate with penicillins, cephalosporins, and imipenem Fig. 2. (A) Overall structure of BlaC displayed in rainbow from N term (blue) to the C term (red), but not with meropenem. The synergism of the with the meropenem adduct displayed as a surface mesh. (B) Fo–Fc omit density (green) contoured clavulanate-meropenem combination and the at 4.0 s surrounds the covalent meropenem adduct formed at the Ambler active-site residue Ser70. uniform activity against drug-susceptible, labo- Mero/ClavStructure has acDvity on Replicang Bacteria figures were produced using Pymol (www.pymol.org). (C) Proposed chemical mechanism ratory, and XDR clinical strains suggest this for the BlaC-catalyzed reactionin vitro with meropenem. combination could be useful in the treatment of –1 Fig.Killing curves of MTB in aerobic growth aer exposure 3. Killing curves of M. tuberculosis after exposure to b-lactams and meropenem (0.19 to 12.5 mg ml ) in the presence or absence of 2.5 mg clavulanate. (A) Aerobic growth using the microdilution method. Merope- ml–1 clavulanate. Isoniazid (0.16 to 1.0 mgml–1)andmetronidazole(4.6to73 nemto Meropenem and clavulanate were/Clav added at 5 different concentraons at 2 mg ml–1 +1mgml–1 (■), 2 mg ml–1 + mM) served as negative and positive controls, respectively. Survival was 2 mg ml–1 (□), 4 mgmlMol–1 +1Microbiolmg ml. Author–1 (▲), manuscript; and 4 mgml available–1 +2 inm PMCg ml –20131 (D ),Octoberdetermined 01. by measurement of ATP amounts in surviving bacteria during Hugonnet. Science 2009 respectively, for 5 consecutive days. (B) Meropenem is cidal for non- aerobic outgrowth of 100-fold diluted cells at either 1 week (white bars) or 2 replicating anaerobic M. tuberculosis. Hypoxically adapted M. tuberculosis weeks (shaded bars) of treatment or by enumeration of CFUs (inset) after 2 H37Rv was treated under anaerobic conditions with twofold dilutions of weeks of compound exposure. www.sciencemag.org SCIENCE VOL 323 27 FEBRUARY 2009 1217 8 3/18/16 AcDvity of Carbapenems against M. tuberculosis and XDR-TB strains One representa?ve study NOTE: Wide range of MICs in other studies --methodologic study differences (in defining MIC) MIC range in this study: 0.23-1.25 Hugonnet et al. Science. 2009 AddiDon of B-Lactamase reduces MIC Higher MICs found in this study Differences in MICs Drop in half of MIC with AddiDon of clavulanic acid Newer carbapenems appear To have lower MICs (and some are oral!) Kaushik et al, AAC, 2015. 9 Zhang et al.