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Successful Use of Repurposed Drugs for MDR TB: Meropenem/Clavulanate and its Newer Relaves

Maunank Shah MD PhD Assistant Professor Center for TB Research & Center for Clinical Global Health Educaon Johns Hopkins University Balmore City Health Department

When opons are limited… • 42 y/o from Peru with MDR-TB – Resistant to HRZE, ethionomide • Admied 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?

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Learning Objecves

• Understand the role of repurposed anbiocs and design stronger treatment regimens for paents with MDR and XDR TB

• Idenfy the toxicies associated with these medicaons and improve their ability to monitor and detect them

• Discuss the strengths and limitaons of incorporang repurposed anbiocs 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 quesons

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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 acvity: • Gram posives, Gram negaves and anaerobes • Tends to be acve 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 oen the only drug class with retained sensivity – Carbapenems are the drug of choice for ESBL (extended spectrum beta lactamase) producing organisms

• Carbapenems also have acvity against mycobacteria

hp://www.cdc.gov/drugresistance/biggest_threats.html

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How do they work: Carbapenems aack the cell wall of bacteria

• The pepdoglycan layer is present in virtually all bacteria.

• This layer is essenal for survival and growth of bacteria. pepdoglycan

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 Pepdoglycan Transpepdase D-Glu D-Ala layer

L-Ala MTB Transpepdase

Membrane

Cytosol

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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 k(iso) 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 me 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 Aacking 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) acvity 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 acvity 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

Acvity of Carbapenems against M. tuberculosis and XDR-TB strains One representave 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

Addion of B-Lactamase reduces MIC

Higher MICs found in this study

Differences in MICs Drop in half of MIC with Addion of clavulanic acid

Newer carbapenems appear To have lower MICs (and some are oral!)

Kaushik et al, AAC, 2015.

9 Zhang et al.

TABLE 1 The ␤-lactams and ␤-lactamase inhibitors used in this study the reagents were purchased from Sigma-Aldrich Chemical Company Agent Classification Generation (St. Louis, MO, USA). MIC. The MICs of MDR-TB isolates against ␤-lactams were deter- Amoxicillin Penicillin mined by using a microplate alamarBlue assay as previously reported (19). Cephalothin Cephalosporin and cephamycin First generation The inoculum was prepared from the 4-week-old cultures grown in LJ Cefaclor Second generation medium. The turbidity of the cultures was adjusted to a 1.0 McFarland Downloaded from Cefoxitin Second generation standard. Then, the 1.0-McFarland standard cell suspension was di- Ceftriaxone Third generation luted to 1:20 in Middlebrook 7H9 broth containing 10% oleic acid- Cefepime Fourth generation albumin-dextrose-catalase (OADC), and 100 ␮lofthisinoculumwas Imipenem Carbapenem added to the wells of the 96-well plate. Following 7 days of incubation, Meropenem 70 ␮lofalamarBluesolutionwasaddedtoeachwell,wasincubatedfor Biapenem 24 h at 37°C, and was assessed for color development. A change from Clavulanate ␤-Lactamase inhibitor blue to pink indicated bacterial growth. MIC was defined as the lowest Tazobactam concentration of antibiotic that prevented the color change from blue Sulbactam

to pink. Final drug concentrations were 0.125 to 512 ␮g/ml. M. tuber- http://aac.asm.org/ culosis H37Rv (ATCC 27249) was tested in each round as a quality control strain. All experiments were done in duplicate to access reproducibility. ␤-lactams and three ␤-lactamase inhibitors, which belong to dif- The ratio of the MIC of the test isolate against a ␤-lactam in combi- ferent subgroups in this study. The broth dilution method based nation with a ␤-lactamase inhibitor compared to a ␤-lactam without a ␤-lactamase inhibitor was calculated. A synergistic effect was declared if on Middlebrook 7H9 broth was used to evaluate the in vitro activ- the ratio was less than 0.25, indicating that the MIC of the ␤-lactam ity of ␤-lactams in combination with ␤-lactamase inhibitors showed at least a 4-fold decrease in combination with a ␤-lactamase in- against MDR-TB isolates. Our aim was to provide more potential hibitor. ␤-lactam–␤-lactamase inhibitor options for the clinical treatment DNA extraction and sequencing of the blaC, dacB2, and ldtMt1 on January 16, 2016 by WELCH MEDICAL LIBRARY - John Hopkins U of MDR-TB cases. genes. Genomic DNA was extracted from freshly cultured bacteria as previously reported (2). The bacterial cells were transferred into a 1.5-ml MATERIALS AND METHODS microcentrifuge tube with 500 ␮l Tris-EDTA (TE) buffer. This was fol- Bacterial strains and culture conditions. All of the MDR-TB strains were lowed by centrifugation at 13,000 rpm for 2 min. After the supernatant isolated from smear-positive tuberculosis patients registered at local TB was discarded, the pellet was resuspended in 500 ␮l TE buffer and then dispensaries in Chongqing as previously reported (18). Drug susceptibil- heated in a 95°C water bath for 1 h. The cellular debris was separated by ity for isoniazid, rifampin, ethambutol, streptomycin, kanamycin, and centrifugation, and the DNA in the supernatantCarbapenems was and used Rifampin for Synergize the PCR ofloxacin was tested with the proportion method endorsed by WHO (3). amplification. Mycobacterium species identification was performed by growth test on a The blaC, dacB2, and ldtMt1 genes were amplified by PCR, respectively. The primers used in this procedure are listed in Table S1 in the supple- 3/18/16 TABLEmedium 6 containingGrowth profilep-nitrobenzoic of rifampin acid monoresistant (PNB) and 2-thiophenecarboxy- clinical isolate of M. tuberculosis in the presence of rifampin and (carba)penemsa lic acid hydrazide (TCH). Prior to performing phenotypic drug suscepti- mental material. The genomic DNA prepared above was used as the tem- bility testing, the strains wereGrowth recovered profile on with Lowenstein-Jensen rifampin at (␮g/ml): (LJ) me- plate to perform PCR amplification. The PCR mixtureGrowth was of control prepared in a Drugdium (concn for 4 weeks [␮g/ml]) at 37°C. 1.0 0.5 0.25 0.12volume 0.063 of 50 ␮l containing 0.031 25 ␮l2ϫ 0.016PCR mixture, Positive 5 ␮l of DNA template,Negative ␤-Lactams and ␤-lactamase inhibitors. A total of nine ␤-lactams and and 0.2 ␮M (each) primer set. PCR products were purified using a Mag- Meropenem (5) Ϫ Ϫ Ϫ Ϫ Ϫ ϩ ϩϩ ϩϩϩ three ␤-lactamase inhibitors were selected to perform the MIC experi- Bind PCR purification kit (CWBio, Beijing, China) and were then sent to

Imipenem (40) Ϫ Ϫ Ϫ Ϫ ϩ ϩϩ ϩϩϩ ϩϩϩ Downloaded from ments. The nine ␤-lactams were from three different classes: penicil- Qingke Company (Beijing, China) for DNA sequencing services. DNA Doripenem (1.25) ϪϪϪϪϪϪϩ lin, cephalosporin and cephamycin, and carbapenem. Out of these, sequences were aligned with the homologous sequences of the reference Biapenem (1.56) Ϫ Ϫ Ϫ Ϫ ϩ ϩϩ ϩϩϩ Ϫ amoxicillin (AMX) belonged to the penicillin group. Five other ␤-lac- M. tuberculosis H37Rv strains using multiple sequence alignments (http: Faropenem (0.63) ϪϪϪϪϪϪϩ Ϫ tams represented different generations of cephalosporin and cepha- //www.ncbi.nlm.nih.gov/BLAST). No (carba)penem ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ mycin group. In addition, the most frequently used carbapenems in Data analysis. A chi-square test was used to compare the proportion a clinicalFor thisB-lactamase Inhibitor Dose effect on MIC strain, practice, the rifampin including MIC90 imipenemis Ͼ1 ␮g/ml. Ϫ (IMI),represents meropenem no visible growth, (MERO), and ϩϩϩofrepresents synergistic growth effect equivalent among to that different of the positive␤-lactam– control (M.␤-lactamase tuberculosis grown inhibitor in brothand biapenemalone). (BIA), were enrolled in this study. For the ␤-lactamase combinations. Statistical analysis was performed in SPSS 11.5 (SPSS Inc., inhibitors, clavulanate (CLAV), tazobactam (TAZ), and sulbactam USA). Differences with a P value of Յ0.05 were considered statistically (SUB) were included in the drug panel for each strain (Table 1). All of significant. observe any growth in plates containing 0.6 ␮g/ml rifampin and results. In addition, for M. abscessus, we repeated the MIC90 as- http://aac.asm.org/ 5.0 ␮g/ml faropenem. sessment using broth microdilution assay in 96-well format per Rifampin/(carba)penem combinations are effective against Clinical and Laboratory Standards Institute guidelines (14). Data TABLE 2 Antimicrobial activity of different ␤-lactam–␤-lactamase inhibitor combinations against five MDR-TB isolates drug-resistant clinical isolates of M. tuberculosis. We evaluated from these independent repeat assessments were highly consis- the efficacy ofMIC rifampin range (␮ ing/ml) combination of drug in combination with meropenem, with: imi- tent. We observed that meropenem in solution is unstable and penem, doripenem,Clavulanate biapenem, (␮g/ml) or faropenem for theirTazobactam activities (␮g/ml)loses potency even after aSulbactam single freezing (␮g/ml) and thawing process. against four clinical isolates of M. tuberculosis: one rifampin Drug 0 1.25 2.5 5 10 0 1.25Therefore, 2.5 5 only previously 10 0 unthawed 1.25 stock 2.5 solutions 5 of mero- 10 monoresistant, one isoniazid monoresistant, and two multidrug- penem were used in this study. It was beyond the scope of current Amoxicillin 32–128 16–128 8–64 2–64 2–64 32–128 16–64 8–64 2–32 2–32 32–128 16–64 2–32 2–16 2–16 resistant strains (Tables 6 to 8; also see Table S1 in the supplemen- study or available resources to assess MBC, activity in combina- on January 16, 2016 by WELCH MEDICAL LIBRARY - John Hopkins U Imipenem 4–64 2–64 1–32 1–16 1–16 4–64 4–64 2–64 2–64 2–64 4–64 4–32 2–32 1–32 1–32 tal material). Rifampin and (carba)penem combinations were tion with rifampin, and frequency of resistant mutants for all growthMeropenem inhibitory 4–32 against 2–32 all 1–16 strains 0.25–4 at sub-MIC 0.25–4 levels 4–32 of the4–32 2–32 2–32 2–16 4–32 2–16 2–16 1–16 1–16 Biapenem 4–64 2–32 1–16 0.5–16 0.5–16 4–64 4–64commercially 2–64 2–64 available 2–64 (carba)penems. 4–64 2–64 We 2–32 also used 1–32 only a single1–32 drugs. The MIC of rifampin for rifampin monoresistant strains Cephalothin 64–25690 64–256 64–256 32–256 32–256 64–256 64–256concentration 32–256 32–128 of clavulanic 32–128 64–256 acid, 5 64–256␮g/ml, an 64–256 optimal 32–256 concentra- 32–256 isCefaclor 0.063 ␮g/ml 64–256 when 32–256 combined 32–256 with 32–256 5 ␮g/ml 32–128 meropenem. 64–256 For 64–256tion 32–256 derived 32–128 from 32–128our titration 64–256 of this 32–256 agent 32–256 with (carba)penems 32–256 32–128 multidrug-resistantCefoxitin 32–256 strains, 32–256 we 32–256 assessed 32–256 the MIC 32–12890 of rifampin32–256 32–256 at for 32–256 activity 32–128 against 16–128M. tuberculosis 32–256 32–256. This32–256 concentration 32–128 is 32–128 also 0.25Ceftriaxone to 0.5 ␮g/ml 32–256 when 32–256 combined 32–256 with 32–128 5 ␮g/ml 32–128 meropenem. 32–25632–256 Of within 32–256 one 32–128 dilution 32–128 of the concentration 32–256 32–256 determined 32–256 32–128 in a previous 16–128 the (carba)penems that we evaluated, faropenem afforded the Cefepime 32–256 32–256 32–256 32–128 16–128 32–256 32–256study 32–128 (5). 16–128 16–128 32–256 32–256 16–128 16–64 16–64 most potent synergy with rifampin: when combined with 0.63 With the exception of panipenem, activities of all (carba)pen- Quesons that need to be answered␮g/ml faropenem, only 0.063 to 0.031 ␮g/ml of rifampin: What’s the opmal dose of Beta-lactamase inhibitor? was re- ems evaluated in this study are therapeutically relevant, as their quired to inhibit growth of both rifampin monoresistant and mul- 394 aac.asm.org Antimicrobial AgentsMIC and Chemotherapyrange from 1.25 to 40 ␮g/ml.January (Carba)penems 2016 Volume are 60 known Number to 1 tidrug-resistant strains (Tables 6 and 7; also see Table S1). 90 be either slow substrates or inhibitors of BlaC, a ␤-lactamase of M. DISCUSSION tuberculosis (2, 5, 27–30). AZhang, AAC 2016 2- to 4-fold reduction in MIC90 was ␤-Lactams, one of the most widely used classes of antibiotics, are seen for all (carba)penems except faropenem when supplemented known to inhibit bacterial D,D-transpeptidases (also known as with 5 ␮g/ml clavulanic acid. The MIC90 of doripenem and biap- penicillin-binding proteins) (17, 18). It has been demonstrated enem against M. tuberculosis decreased 2-fold when the inoculat- that ␤-lactams interfere with peptidoglycan biosynthesis and me- ing dosage was split into two halves. As (carba)penems are known tabolism in multiple ways and eventually results in cell death (17, to exhibit time-dependent activity, our data suggest that a proper 19). Among ␤-lactams, carbapenems and penems are able to bind dosing interval for doripenem and biapenem has the potential to and inhibit activities of D,D-transpeptidases, L,D-transpeptidases, further optimize activities of these drugs. Doripenem, biapenem, and D,D-carboxypeptidases (6, 20–26). This versatile property of faropenem, and tebipenem exhibit bactericidal activity against M. (carba)penems to target multiple enzymes prompted the current tuberculosis within therapeutically achievable concentrations. Due study to test the clinical utility of commercially available (carba) to the poor oral bioavailability of ertapenem, meropenem, imi- penems in M. tuberculosis andSynergy with Rifampin? M. abscessus isolates from patients. penem, and doripenem, these drugs generally are administered

All MIC90 assessments were repeated at least twice to verify intravenously, a route that is not feasible for treatment of TB pa-

TABLE 7 Growth profile of multidrug-resistant clinical isolate 2 of M. tuberculosis in the presence of rifampin and (carba)penemsa Growth profile with rifampin at (␮g/ml): Growth of control Drug (concn [␮g/ml]) 1.0 0.5 0.25 0.12 0.063 0.031 0.016 Positive Negative Meropenem (5) Ϫ Ϫ ϩ ϩϩ ϩϩ ϩ ϩϩ ϩϩϩ Imipenem (40) Ϫ Ϫ ϩ ϩϩ ϩϩ ϩϩϩ ϩϩϩ ϩϩϩ Doripenem (1.25) Ϫ Ϫ Ϫ ϩϩ ϩϩ ϩϩϩ ϩϩϩ Biapenem (1.56) Ϫ Ϫ Ϫ Ϫ ϩϩ ϩϩ ϩϩϩ Ϫ Faropenem (0.63) Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ ϩϩ Ϫ No carba(penem) ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩϩ a For this strain, the rifampin MIC90 is Ͼ1 ␮g/ml and the isoniazid MIC90 is Ͼ0.1 ␮g/ml. Ϫ represents no visible growth, and ϩϩϩ represents growth equivalent to that of the positive control (M. tuberculosis grown in broth alone).

October 2015 Volume 59 Number 10 Antimicrobial Agents and Chemotherapy aac.asm.org 6565

Kaushik. AAC 2015

10 Downloaded from http://aac.asm.org/ on January 16, 2016 by WELCH MEDICAL LIBRARY - John Hopkins U ). M. 397 19 sul- -lac- -lac- ␤ ␤ resistance -lactamase -lactamase ␤ ␤ aac.asm.org -lactamase in- ). On one hand, ␤ 22 ). Considering that -Lactams against MDR-TB ␤ 28 -lactamase inhibitor when in combination ( -lactams– ␤ ). Previous studies have penetration of different ␤ M. tuberculosis 25 . In contrast, the Ser111Arg ,

Activity of 3/18/16 in vivo 24 -lactamase with high potency -lactams and in vivo ␤ ␤ ). Therefore, the relative instabil- against carbapenem and other an- with greater difficulty, resulting in -lactams. Clavulanate is a mecha- ). In this study, we found that 27 In Vitro ␤ 26 ). The protein structure analysis of M. tuberculosis , -lactams biochemical evidence that Ser111 is in- -lactamase inhibitors show species-spe- 29 ␤ 25 BlaC. Therefore, we hypothesized that the ␤ ( M. tuberculosis 6 days) ( ). Ͼ 0.1 mM), while a previous article by Horita in vitro 10 Ͻ M. tuberculosis M. tuberculosis in vivo that the is combinations provide one strategy to overcome i due to the addition of meropenem may increase the is more hydrophilic than sulbactam, we hypothesized K served as the more effective M. tuberculosis -lactams, which confirms our findings ( gene attracts our attention because it may be associated ␤ half-life of clavulanate (1.4 days) is significantly lower than -lactam antibiotics. Hence, the increased permeability of -lactamase wild type reveals that the serine at position 111 is located BlaC polymorphisms enhance Mero/MIC values of this study were determined by the broth dilu- Clav Inhibitor We further analyzed the genetic mutations associated with of clavulanate will result in the loss of active drug during incu- ␤ ␤ -lactamase-mediated resistance ( -lactamase inhibitors on its efficacy in combination with inhibitors, including the aminoT237 of acids R220, A244, S130, and volved in substrate binding as partgion. of Further the molecular carboxylate-binding analysis re- willactive provide sites new of insight BlaC on in substitution the in BlaC seems to haveMDR-TB no strains against role imipenem in due the to susceptibility the of supplement of cla- (typically The that of sulbactam ( bactam against MDR-TB isolateswhen in than combination with clavulanatenism-based and inhibitor to tazobactam class A the near the binding motif of BlaC with substitution of Ser111changes in by BlaC, thereby Arg111 influencing inhibitorthere introduced activity. is However, no conformational direct and colleagues reveals thatthan sulbactam harbors clavulanate higher against potency with bation, thereby limiting the anti-TB activity of synergetic the ity tams. One the other hand, efficient penetration of the inhibition of biosynthesis of thepermeability cell of wall, the thereby bacterial envelope. enhancingbeen This the considered slow a major penetration determinant has of to tuberculosis susceptibility of tibiotics. In agreement with ourmeropenem hypothesis, and the amoxicillin-clavulanate has synergy been between pronouncedrecent in literature ( tion method, which requires 7 days to reach the endpoint ( demonstrated cific differences in activitycribable against to microorganisms, the which classification is andof the as- three-dimensional structure ␤ clavulanate tams for combating TB. the synergistic effect between hibitors. Interestingly, one nonsynonymousblaC mutation in the with an increased susceptibility ofpenem MDR-TB and isolates to amoxicillin mero- binding in affinity the between presence amajorly of ligand dependent and on clavulanate. its themolecular The receptor structural interaction complement protein ( and is inter- that clavulanate might penetrateouter surface the of thick layeran of unsatisfactory lipid inhibitory effect. on Furtherinvestigate the study the is potential warranted to role␤ of inhibitor through theantibacterial cell activity envelope of is essential to enhance the

acvity Agents and Chemotherapy Antimicrobial

TABLE 7 Relationship between synergistic effect and blaC and dacB2 mutation Amino No. of Ratio rangeb Sequence acid isolates Locus change change (%)a AMX-CLAV AMX-SUB AMX-TAZ IMI-CLAV IMI-SUB IMI-TAZ MERO-CLAV MERO-SUB MERO-TAZ BIA-CLAV BIA-SUB BIA-TAZ BlaC AGT333AGG Ser111Arg 4 (3.3) 0.13–0.25 0.13 0.25 1 1 0.5–1 0.031 0.5 0.5 0.25 0.12–0.25 0.13–0.25 ATC786ATT Ile262Ile 9 (7.4) 0.5–1 0.13 0.25–0.5 1 0.5–1 0.5–1 0.25–1 0.5 0.5 0.25–0.5 0.13–0.5 0.13–0.5 DacB2 CTG659CAG Leu220Gln 8 (6.6) 0.13–1 0.13 0.25–0.5 1 0.5–1 0.5–1 0.25–1 0.5 0.5 0.25–0.5 0.13–0.5 0.13–0.5 a A total of 121 isolates were used. b Ratio represents the MIC value with ␤-lactamase inhibitor divided by the MIC value without ␤-lactamase inhibitor. January 2016 Volume 60 Number 1

Zhang, AAC 2016

Summary of in vitro data

• Carbapenems are able to target the MTB cell wall

• MICs are ‘likely’ clinically achievable, especially with addion of B-lactamase inhibitors

• Possibly synergisc effect with rifampin

11 3/18/16

In Vivo studies

2818 CHAMBERS ET AL. ANTIMICROB.AGENTS CHEMOTHER. Downloaded from

FIG. 1. Mean burdens of M. tuberculosis strain H37Rv as log10 CFU/g in spleen and lung tissues. The numbers of mice are as follows: for untreated mice (black diamonds), 14, 19, and 14 on days 0, 14, and 28, respectively; for imipenem-treated mice (open circles), 22 and 15 for days 14 and 28, respectively; and for isoniazid-treated (black squares), 14 and 15 for days 14 and 28, respectively. The standard errors of the means are indicated by the bars.

imipenem in combination with two or more other agents. The approximately 0.35 log10 CFU/ml/week (Fig. 3). Patients 7 and

mean age (Ϯ standard deviation) was 45.3 Ϯ 12.8 years (range 10 had only qualitative cultures performed. Patient 7, with 40 http://aac.asm.org/ of 26 to 61 years). Eight patients were foreign born (five Asians prestudy weeks of documented positive smears and cultures, and three Mexican-Americans), and five were women. Eight became smear and culture negative after 3 months of imi- had radiographic evidence of cavitary disease. The clinical penem plus other agents. Patient 10, with smear-positive and isolates were resistant to 7 Ϯ 2 antituberculous agents (range, culture-positive tuberculosis, with ulcerations and perforation 5 to 10) (Table 1); four isolates were fluoroquinolone resistant. of the stomach and duodenum, had healing documented by Early data showed improved survival (35% vs 70% mortality) With the exceptions that it produced rash (two patients) and follow-up endoscopy after 6 months of imipenem combination occasional diarrhea, the imipenem combination regimen was therapy. Smears and cultures of the duodenal biopsy specimen well tolerated. were negative for M. tuberculosis. on January 16, 2016 by guest Eight patients (95% confidence interval, 50 to 100%) ini- Two patients, patients 4 and 9, each of whom had an isolate and reducon in spleen/lung log CFU/g tially responded with conversion of cultures to negative. Seven that was resistant to 10 drugs, were treatment failures. In both patients (95% confidence interval, 35 to 100%) remained cul- cases, there were transient reductions in the numbers of or- ture negative off of therapy and were considered cured. Two ganisms in sputa, but these eventually returned to pretreat- patients died. ment levels (Fig. 3A). The 18-week clinical isolate from patient with imipenem vs. control at 28 days…but not as good as INHSix of the eight patients who responded had serial quantita- 4 had acquired secondary resistance to streptomycin during tive cultures performed. These showed a continuous and sus- treatment. This isolate also had acquired resistance to imi- 2818 CHAMBERS ET AL. ANTIMICROB.AGENTS CHEMOTHER. tained reduction of mycobacterial burden in sputa averaging penem, as it was able to grow slowly in the presence of imi- penem at concentrations of 8 and 16 ␮g/ml; growth of the prestudy isolate was inhibited at these concentrations (Fig. 4). Whether resistance was beta-lactamase mediated, was due to Control target alterations, or was a penetration defect was not deter- INH mined. Control Three patients, i.e., patients 7, 8, and 10, relapsed shortly after the discontinuation of imipenem and aminoglycoside or Imipenem capreomycin in the regimen. In patient 7, 3 months after dis- Imipenem continuing imipenem and amikacin, given for 4 months, smears Imipenem and cultures were again positive, and secondary resistance to INH ofloxacin was documented. Imipenem and amikacin were re- instituted, with smears and cultures converting to negative INH after 1 and 2 months, respectively. Imipenem, amikacin, and Control oral agents were administered for another 9 months; patient 7 remained culture negative for the 36-month follow-up period. Patient 8, who had a single positive culture on week 18 of

Downloaded from imipenem therapy, received an initial course of 9 months of imipenem and capreomycin in addition to oral agents. Three FIG. 1. Mean burdens of M. tuberculosis strain H37Rv as log10 CFU/g in spleen and lung tissues. The numbers of mice are as follows: for FIG. 2. Survival curves for untreated mice (black diamonds), imi- months after imipenem and capreomycin were discontinued, untreated mice (black diamonds), 14, 19, and 14 on days 0, 14, and 28, respectively; for imipenem-treated mice (open circles), 22 and 15 for days multiple sputum smears and a single culture were again posi- 14 and 28, respectively; and for isoniazid-treated (black squares), 14 and 15 for days 14 and 28, respectively. The standard errors of the means are penem-treated mice (open circles), and isoniazid-treated mice (black squares). tive. Smears and cultures again converted to negative during a indicated by the bars. Example of data from mouse model Chambers AAC 2005 imipenem in combination with two or more other agents. The approximately 0.35 log10 CFU/ml/week (Fig. 3). Patients 7 and

mean age (Ϯ standard deviation) was 45.3 Ϯ 12.8 years (range 10 had only qualitative cultures performed. Patient 7, with 40 http://aac.asm.org/ of 26 to 61 years). Eight patients were foreign born (five Asians prestudy weeks of documented positive smears and cultures, and three Mexican-Americans), and five were women. Eight became smear and culture negative after 3 months of imi- had radiographic evidence of cavitary disease. The clinical penem plus other agents. Patient 10, with smear-positive and isolates were resistant to 7 Ϯ 2 antituberculous agents (range, culture-positive tuberculosis, with ulcerations and perforation 5 to 10) (Table 1); four isolates were fluoroquinolone resistant. of the stomach and duodenum, had healing documented by With the exceptions that it produced rash (two patients) and follow-up endoscopy after 6 months of imipenem combination occasional diarrhea, the imipenem combination regimen was therapy. Smears and cultures of the duodenal biopsy specimen 12 well tolerated. were negative for M. tuberculosis. on January 16, 2016 by guest Eight patients (95% confidence interval, 50 to 100%) ini- Two patients, patients 4 and 9, each of whom had an isolate tially responded with conversion of cultures to negative. Seven that was resistant to 10 drugs, were treatment failures. In both patients (95% confidence interval, 35 to 100%) remained cul- cases, there were transient reductions in the numbers of or- ture negative off of therapy and were considered cured. Two ganisms in sputa, but these eventually returned to pretreat- patients died. ment levels (Fig. 3A). The 18-week clinical isolate from patient Six of the eight patients who responded had serial quantita- 4 had acquired secondary resistance to streptomycin during tive cultures performed. These showed a continuous and sus- treatment. This isolate also had acquired resistance to imi- tained reduction of mycobacterial burden in sputa averaging penem, as it was able to grow slowly in the presence of imi- penem at concentrations of 8 and 16 ␮g/ml; growth of the prestudy isolate was inhibited at these concentrations (Fig. 4). Whether resistance was beta-lactamase mediated, was due to target alterations, or was a penetration defect was not deter- mined. Three patients, i.e., patients 7, 8, and 10, relapsed shortly after the discontinuation of imipenem and aminoglycoside or capreomycin in the regimen. In patient 7, 3 months after dis- continuing imipenem and amikacin, given for 4 months, smears and cultures were again positive, and secondary resistance to ofloxacin was documented. Imipenem and amikacin were re- instituted, with smears and cultures converting to negative after 1 and 2 months, respectively. Imipenem, amikacin, and oral agents were administered for another 9 months; patient 7 remained culture negative for the 36-month follow-up period. Patient 8, who had a single positive culture on week 18 of imipenem therapy, received an initial course of 9 months of imipenem and capreomycin in addition to oral agents. Three FIG. 2. Survival curves for untreated mice (black diamonds), imi- months after imipenem and capreomycin were discontinued, penem-treated mice (open circles), and isoniazid-treated mice (black multiple sputum smears and a single culture were again posi- squares). tive. Smears and cultures again converted to negative during a In Vivo Activity of Meropenem-Clavulanate against TB

and 6 days (P ϭ 0.01 and 0.001 for meropenem, for example, at 4 and 6 days, respectively). At 6 days, the carbapenems demon- strated 1.5- to 2.0-log reductions in bacterial numbers compared to those of untreated controls, with imipenem and meropenem having the largest effect. For comparison, isoniazid and rifampin controls demonstrated a 2-log kill over the same time period. To examine efficacy in an animal model, we infected 30 Downloaded from C57BL/6 mice with M. tuberculosis H37Rv and allowed the infec- tion to progress to a chronic stage. Three months after infection, the mice were divided into three groups of 10 and therapy was initiated. One group was treated with meropenem alone at 300 mg/kg of body weight by subcutaneous injection twice daily, a second group received meropenem at the same dose but was additionally given twice-daily 50-mg/kg oral doses of clavu- lanic acid, and the final group received vehicle control treat- http://aac.asm.org/ ment (phosphate-buffered saline [PBS]). Five mice from each treatment group were sacrificed 2 weeks later, with the remain- ing five sacrificed at 4 weeks of treatment, and bacterial bur- dens in both lung and spleen were enumerated. The data in Fig. 2 illustrate that meropenem did, in fact, show activity in this FIG 1 Intracellular susceptibility of H37Rv. ␤-Lactams evaluated in combi- nation with 200 ␮M clavulanic acid demonstrated killing of intracellular M. very stringent chronic mouse model. Compared to the control tuberculosis. , untreated controls; , rifampin; Œ, isoniazid; ϫ, meropenem; group, both meropenem alone and meropenem in combina-

छ, faropenem; ϩ, doripenem; ૽; ertapenem; Œ, imipenem. Imipenem was tion with clavulanic acid significantly reduced numbers of CFU on July 14, 2015 by WELCH MEDICAL LIBRARY - John Hopkins U most active at killing, with a 2.0-log reduction of bacilli compared to that of in both lung and spleen after 2 weeks of treatment (P ϭ 0.008 untreated controls. By day 2, all of the drug-treated groups were significantly and P ϭ 0.002, respectively, in an analysis of variance different from the control (P Ͻ 0.05, repeated-measures ANOVA). By days 4 3/18/16 and 6, this difference became even more significant (P Ͻ 0.01 and P Ͻ 0.001, [ANOVA]), with a further modest reduction in bacterial bur- respectively, for all drug treatments compared with the control). den observed after 4 weeks of therapy. Clavulanic acid did not show a statistically significant enhancement of killing in the lungs or spleen at either time point. receiving standard therapeutic doses (50 ␮g/ml for meropenem, We also planned to conduct an efficacy study in TB-infected imipenem, and ertapenem, 30 ␮g/ml for doripenem, and 10 New Zealand White rabbits, which develop hypoxic pulmonary ␮g/ml for faropenem). Two control drugs, rifampin and isonia- lesions (16); therefore, we determined pharmacokinetic (PK)

zid, were also dosed once a day at the human-equivalent Cmax (5 parameters and tolerability in uninfected rabbits for mero- ␮g/ml) and also had their medium replaced on days 2 and 4. penem-clavulanate at doses of 75 mg/kg and 125 mg/kg, re- Treated macrophages were lysed at the indicated times, and serial spectively. Drugs were delivered sequentially by intravenous There is intracellular killing and reducon in bacteria in lung dilutions were plated for enumeration of CFU (Fig. 1). These data (i.v.) bolus in four rabbits. Blood was drawn at scheduled time showed significant growth arrest of intracellular H37Rv by 2 days points, and plasma was obtained for determination of serum and spleen of carbapenem(P ϭ 0.05) and highly significant/clav killing withIn Vivo in a chronic mouse model allActivity carbapenems of Meropenem-Clavulanate by 4 concentrations. against TB Data obtained from the four animals were

and 6 days (P ϭ 0.01 and 0.001 for meropenem, for example, at 4 and 6 days, respectively). At 6 days, the carbapenems demon- strated 1.5- to 2.0-log reductions in bacterial numbers compared to those of untreated controls, with imipenem and meropenem having the largest effect. For comparison, isoniazid and rifampin controls demonstrated a 2-log kill over the same time period. To examine efficacy in an animal model, we infected 30 Downloaded from C57BL/6 mice with M. tuberculosis H37Rv and allowed the infec- tion to progress to a chronic stage. Three months after infection, the mice were divided into three groups of 10 and therapy was initiated. One group was treated with meropenem alone at 300 mg/kg of body weight by subcutaneous injection twice daily, a second group received meropenem at the same dose but was additionally given twice-daily 50-mg/kg oral doses of clavu-

MERO http://aac.asm.org/ lanic acid, and the final group received vehicle control treat- IMI ment (phosphate-buffered saline [PBS]). Five mice from each treatment group were sacrificed 2 weeks later, with the remain- ing five sacrificed at 4 weeks of treatment, and bacterial bur- dens in both lung and spleen were enumerated. The data in Fig. 2 illustrate that meropenem did, in fact, show activity in this FIG 1 Intracellular susceptibility of H37Rv. ␤-LactamsFIG evaluated 2 Mouse in combi-efficacy of the study. Chronically M. tuberculosis-infected mice were treated with 300 mg/kg meropenem, 300 mg/kg meropenem with 50 mg/kg very stringent chronic mouse model. Compared to the control nation with 200 ␮M clavulanicINTRACELLULAR acid demonstrated killingclavulanic of intracellular acid (CA),M. or PBS (control) for 22 weeks (light gray bars) or4 weeks 4 (dark gray bars) weeks. Treatment2 weeks with meropenem4 weeks or meropenem and clavulanic acid for either tuberculosis. , untreated controls; , rifampin; Œ, isoniazid;2 or 4 weeksϫ, meropenem; reduced thegroup, bacterial both burdens meropenem in the lungs alone (P ϭ and0.008) meropenem and spleen in (P combina-ϭ 0.002).

छ, faropenem; ϩ, doripenem; ૽; ertapenem; Œ, imipenem. Imipenem was tion with clavulanic acid significantly reduced numbers of CFU on July 14, 2015 by WELCH MEDICAL LIBRARY - John Hopkins U most active at killing, with a 2.0-log reduction of bacilli compared to that of in both lung and spleen after 2 weeks of treatment (P ϭ 0.008 untreated controls. By day 2, all of the drug-treated groups were significantly and P ϭ 0.002, respectively, in an analysis of variance different from the control (P Ͻ 0.05, repeated-measuresJune ANOVA). 2012 Volume By days 564 Number 6 England, AAC 2012 aac.asm.org 3385 and 6, this difference became even more significant (P Ͻ 0.01 and P Ͻ 0.001, [ANOVA]), with a further modest reduction in bacterial bur- respectively, for all drug treatments compared with the control). den observed after 4 weeks of therapy. Clavulanic acid did not show a statistically significant enhancement of killing in the lungs or spleen at either time point. receiving standard therapeutic doses (50 ␮g/ml for meropenem, We also planned to conduct an efficacy study in TB-infected imipenem, and ertapenem, 30 ␮g/ml for doripenem, and 10 New Zealand White rabbits, which develop hypoxic pulmonary ␮g/ml for faropenem). Two control drugs, rifampin and isonia- lesions (16); therefore, we determined pharmacokinetic (PK) zid, were also dosed once a day at the human-equivalent Cmax (5 parameters and tolerability in uninfected rabbits for mero- ␮g/ml) and also had their medium replaced on days 2 and 4. penem-clavulanate at doses of 75 mg/kg and 125 mg/kg, re- Treated macrophages were lysed at the indicated times, and serial spectively. Drugs were delivered sequentially by intravenous dilutions were plated for enumeration of CFU (Fig. 1). These data (i.v.) bolus in four rabbits. Blood was drawn at scheduled time showed significant growth arrest of intracellular H37Rv by 2 days points, and plasma was obtained for determination of serum (P ϭ 0.05) and highly significant killing with all carbapenems by 4 concentrations. Data obtained from the four animals were

BUT not all data supports carbapenem in vivo…

FIG 2 Mouse efficacy of the study. Chronically M. tuberculosis-infected mice were treated with 300 mg/kg meropenem, 300 mg/kg meropenem with 50 mg/kg clavulanic acid (CA), or PBS (control) for 2 (light gray bars) or 4 (dark gray bars) weeks. Treatment with meropenem or meropenem and clavulanic acid for either 2 or 4 weeks reduced the bacterial burdens in the lungs (P ϭ 0.008) and spleen (P ϭ 0.002).

June 2012 Volume 56 Number 6 aac.asm.org 3385

13 3/18/16

Study showed improved survival in TB infected mice BUT persistent growth in lung and spleen at 28 days 2598 VEZIRIS ET AL. ANTIMICROB.AGENTS CHEMOTHER. Beta-Lactams for TB

TABLE 1. MICs of imipenem, meropenem, and ertapenem alone TABLE 2. Mean CFU counts in mouse lungs after 28 days of a and in the presence of 2.5 mg of clavulanate/liter carbapenem treatmentTABLE (with 3 PK parameters or without for infected clavulanate) mice in the efficacy study c Dose Combination Dose Cmax %TMIC in b ␮ d ␮ e MIC (mg/liter) group DrugMean PK log10 CFUdrug lung count(mg/kg)Ϯ SEM Regimen ( g/ml) Tmax (h) AUC (h · g/ml) Half-life (h) 24 h Treatment 1 Amoxicillin Clavulanate 200 BID, Q12 43.07 1.00 82.12 0.58 33 Treatment a Without With EUCAST susceptibility 1 ClavulanateCFU on day1 Amoxicillin CFU 50 on day 28 BID, Q12 26.39 0.08 15.67 0.21 13 a clavulanate clavulanate breakpoints 2 Meropenem Clavulanate 300 TID, Q8 218.04 0.08 89.65 0.39 25 None 2 Clavulanate5.49 Ϯ 0.53 Meropenem 7.07 75Ϯ 0.3* TID, Q8 24.88 0.08 13.34 0.46 19 Imipenem 16 1 2–8 Isoniazid a The parameters were estimated after the 10th dose.4.34 Ϯ 0.82† Meropenem 8 1 2–8 b BID, twice a day; TID, three times a day; Q12, every 12 h; Q8, every 8 h. Imipenem c 6.66 Ϯ 0.18 Cmax, maximum concentration of drug in plasma. Ertapenem 16 4 0.5–1 Imipenem ϩ clavulanate d 6.36 Ϯ 0.3† Tmax, time to maximum concentration of drug in plasma. e Meropenem Based on in vitro synergy between amoxicillin and7.28 clavulanate,Ϯ 0.21 a 1-␮g/ml concentration was used for %TMIC calculation for clavulanate. Amoxicillin, clavulanate, and meropenem a EUCAST (9). Meropenem ϩ clavulanateare reported to be 80%, 75%, and 98% free in plasma,6.88 respectivelyϮ 0.31 (9, 18). Therefore, the %TMIC values were almost the same for free plasma concentrations. Ertapenm 7.23 Ϯ 0.13

Ertapenem ϩ clavulanate 7.08 Ϯ 0.3 pumps and beta-lactamases (2, 3, 4, 16). We have shown in vitro Downloaded from Clavulanate 7.30 Ϯ 0.52 activity of 3 different classes of beta-lactams, penicillins, cephalo- 1-mg/ml suspension of M. bovis variant BCG and further diluted, with saline, to sporins, and carbapenems, in combination with clavulanate, a 0.2 mg/ml for mouse inoculation. Values indicated by “†” were significantly different from the value indicated against replicating, as well as nonreplicating, M. tuberculosis. by “*”. Antimicrobial agents. Meropenem (AstraZeneca), imipenem (Merck Sharp Downloaded from Cephalosporin-clavulanate or carbapenem-clavulanate showed Dome), ertapenem (Merck Sharp Dome), and isoniazid (Laphal) were pur- lower synergy against replicating M. tuberculosis than the penicil- chased from the manufacturers. Clavulanate was purchased as pure powder from lin-clavulanate combination. This could be due to differences in Beta-Lactams for TB the catalytic efficiencies of M. tuberculosis beta-lactamase in hy- Sigma. Veziris, AAC 2011 drolyzing various classes of beta-lactams (2). Cephalosporins and http://aac.asm.org/ Mice. Swiss mice were purchased from the from the Janvier breeding center mice, one died on day 19 in the imipenem group (probably due carbapenems were reported to have 10- to 100-fold lower K /K a cat m (Le Genest Saint-Isle, France). TABLE 3 PK parameters for infectedto mice a in gavage the efficacy studyaccident), 3 died between days 19 and 23 in mero- ratios for M. tuberculosis beta-lactamase than the penicillin class. MIC measurement. MIC was measured for imipenem, meropenem, and c %T Dose CombinationpenemDose group, and 3 inCmax ertapenem group died between days 19MIC in Carbapenems, like meropenem and faropenem, showed mod- group Drug PK drug (mg/kg) Regimenb ␮ d AUC (h · ␮g/ml) Half-life (h) 24 he ertapenem alone and in the presence of 2.5 mg of clavulanate/liter in Middle- ( g/ml) Tmax (h) erate bactericidal activity against M. tuberculosis ss18b, as reported brook 7H9 medium supplemented with oleic acid-albumin-dextrose-catalase1 Amoxicillin Clavulanateand 22. 200 The addition BID, Q12 of 43.07 clavulanate 1.00 improved 82.12 survival0.58 since no33 previously (12, 17). However, none of the combinations with cla- 1 Clavulanate Amoxicillinmouse 50 died inBID, Q12the imipenem 26.39 0.08 plus clavulanate 15.67 or0.21 the mero-13 vulanate were active against M. tuberculosis under hypoxia. This (OADC) (17). 2 Meropenem Clavulanate 300 TID, Q8 218.04 0.08 89.65 0.39 25 discrepancy in activity against nonreplicating M. tuberculosis in 2 Infection of mice. Ninety female 4-week-old Swiss mice2 were Clavulanate intravenously Meropenempenem 75 plus TID,clavulanate Q8 24.88 groups 0.08 (P ϭ 13.340.01 versus0.46 untreated19 http://aac.asm.org/ a different in vitro models could not be explained. infected in the tail vein with 0.5 ml of the bacterial suspensionThe parameters prepared were estimated as after the 10th dose. on January 16, 2016 by guest b mice), and only one mouse died in ertapenem plus clavulanate 5 BID, twice a day; TID, three times a day; Q12, every 12 h; Q8, every 8 h. We tested 2 combinations for activity against replicating and described above and containing approximately 5 ϫ 10 CFU.c The animal exper- Cmax, maximum concentration of drug in plasma. d group (P ϭ 0.06 versus untreated mice). nonreplicating M. tuberculosis in the mouse TB model to see if the iment guidelines of the Faculte´ de Me´decine Pitie´-SalpeMarginal to no effect with ˆtrieT`remax, time were to maximum followed. concentration of drug in plasma. mero/clav with acute and chronic e in vitro activity could be translated in vivo. The required %TMIC Chemotherapy. On the day after infection, the mice wereBased randomly on in vitro synergy allocated between amoxicillin andSpleen clavulanate, a 1-weights␮g/ml concentration and was grossused for %TMIC lungcalculation lesions. for clavulanate.Compared Amoxicillin, clavulanate, to and the meropenem are reported to be 80%, 75%, and 98% free in plasma, respectively (9, 18). Therefore, the %TMIC values were almost the same for free plasma concentrations. for in vivo efficacy of carbapenems and penicillins against multiple to seven groups. A negative control group consisted of 20 mice infectedinfecon mouse models: that were untreated control, group onlyPK showed 25% T>MIC the mice of isoniazid group had bacterial pathogens has been reported to be 20 to 40% (18). There- not treated. The treated groups consisted of 10 mice each. A positive control reduced spleen weights. All carbapenem-treated mice (alone fore, the dosing regimen for the mouse efficacy study was chosen pumps and beta-lactamases (2, 3, 4, 16). We have shown in vitro Downloaded from group was treated with isoniazid alone. The test groups were treated either with to achieve 20 to 40% T in mouse plasma over a period of 24 h. or in combination withactivity clavulanate) of 3 different classeshad ofsplenomegaly beta-lactams, penicillins, equiv- cephalo- MIC imipenem, meropenem, or ertapenem alone or combined with clavulanate. An sporins, and carbapenems, in combination with clavulanate, Lack of efficacy in both the acute and chronic mouse models may additional control group was treated with clavulanate alone. alent to that of untreatedagainst mice replicating, (data as not well asshown). nonreplicating, M. tuberculosis. suggest that the %TMIC requirement could be significantly higher All of the mice thatCephalosporin-clavulanate died before day 28 or and carbapenem-clavulanate two of the four showed for M. tuberculosis. A similar magnitude of effect was also reported

Isoniazid suspension in gelosed water was prepared weekly and kept at 4°C. on January 16, 2016 by guest previously using a twice-daily regimen for the meropenem-clavu- Beta-lactam solutions were prepared daily in saline according to laboratory lower synergy against replicating M. tuberculosis than the penicil- mice in the negative controllin-clavulanate group combination. sacrificed This could on be day due to 28 differences had in lanate combination (9). However, the efficacy did not improve advising. developed massive grossthe catalytic lung efficiencieslesions of(ϩϩM. tuberculosisto ϩϩϩbeta-lactamase). Com- in hy- even after using a 3-times-daily regimen in this study. Treatment was started the day after infection (preventive model) and admin- pared to the controls,drolyzing the isoniazid-treated various classes of beta-lactams mice (2). had Cephalosporins fewer and http://aac.asm.org/ Considering an average doubling time of 0.5 to 1.0 h for bac- istrated once a day for 5 days a week for 1 month, either by oral gavage carbapenems were reported to have 10- to 100-fold lower Kcat/Km terial pathogens like Staphylococcus aureus, Pseudomonas aerugi- lung lesions (three miceratios with for M. lung tuberculosis lesionbeta-lactamase scores thanof 0, theϩ penicillin, and class. (isoniazid) or subcutaneously (beta-lactams). nosa, or Streptococcus pneumoniae, a 20 to 40% TMIC of beta-lac- Carbapenems, like meropenem and faropenem, showed mod- tams may cover 5 or 6 generations over 24 h. The generation time To match the Cmax obtained in humans (3, 8, 20, 26) and using the available ϩϩ). In contrast, all miceerate bactericidal had severe activity lesions against M. after tuberculosis clavulanatess18b, as reported for M. tuberculosis growing in the infected mouse lung is reported data for mice (2, 25, 29, 34), the following doses were selected: imipenem, treatment or carbapenempreviously treatment, (12, 17). However, even when none of combined the combinations with with cla- meropenem, ertapenem. and clavulanate at 100 mg/kg and isoniazid at 25 mg/kg. vulanate were active against M. tuberculosis under hypoxia. This to vary between 24 h during rapid growth in the acute model to clavulanate. 1,676 h during the slowly growing/nonreplicating phase in the Assessment of infection and treatment. To provide baseline values before discrepancy in activity against nonreplicating M. tuberculosis in 2 Enumeration of CFUdifferent in thein vitrolungs.models could not be explained. chronic model (19, 20). Since the bactericidal effect of beta-lac- initiation of chemotherapy, 10 control mice were killed on day 1 after inocula- The mean CFU count in on January 16, 2016 by guest We tested 2 combinations for activity against replicating and tams is exerted during cell division, poor activity of these beta- tion. To assess the activity of treatment, all of the surviving mice were killed on the lungs was 5.5 log10nonreplicatingCFU amongM. tuberculosis miceinsacrificed the mouse TB model the to day see if the lactams against M. tuberculosis in vivo could be due to an insuffi- day 28. after inoculation (Tablein 2). vitro Inactivity the could untreated be translated controlin vivo.group, The required this %TMIC cient duration of exposure during its growth cycle. The severity of infection and the treatment activity were assessed by deter- for in vivo efficacy of carbapenems and penicillins against multiple Recently, the meropenem-clavulanate combination was value increased by 1.6 log10 CFU to reach 7.1 log10 among the mining the survival rate, spleen weight, gross lung lesion score (0, no lesions; ϩ, bacterial pathogens has been reported to be 20 to 40% (18). There- shown to be efficacious in patients with XDR TB (8) using a FIG 3 Efficacies of meropenem-clavulanate and amoxicillin-clavulanate in four mice surviving onfore, day the dosing 28. The regimen lung for the CFU mouse efficacy counts study were was chosen 3-times-daily (2 g each time) dosing regimen. Based on the re- Ͻ10 tubercles; ϩϩ, 10 to 50 tubercles; ϩϩϩ, Ͼ50 tubercles), and the numbersACUTE MODEL tothe achieve acute (A) 20 to and 40% chronicT (B)in mouse mouse models plasma for over TB. a The period bacterial of 24 load h. at the MICCHRONIC MODEL ported human PK for meropenem (Merrem product information; of CFU in the lungs. Lungs were aseptically removed and homogenized accord- significantly reduced byLackbeginning 1.2 of efficacy log of10 treatment inCFU both (early the in acute control) the and isoniazid is chronic indicated mouse bygroup the models dashed may lines. Solid horizontal lines indicate the mean values. AstraZeneca, 27 February 2007), such a dosing regimen is ex- ing to a standard procedure (30). Enumeration of CFU was done as previously (4.3 log10 CFU, P ϭ 0.002)suggest compared that the %TMIC torequirement the start could of be treatment. significantly higher described (23). Clinical queson: What is the correct dose and frequency? for M. tuberculosis. A similar magnitude of effect was also reported In all of the carbapenem-treatedpreviously using a groups,twice-daily regimen even for with the meropenem-clavu- a clavu- Statistical analysis. Mean CFU counts were compared using a Wilcoxon test. lanate combination, thelanateJune CFU 2013 combination Volume counts 57 (9 Number). increased However, 6 the efficacy by 0.8 did to not 1.8 improve aac.asm.org 2509 The proportions of surviving mice were compared by using a Fisher exact test even after using a 3-times-daily regimen in this study. Solpure, AAC 2013 since the samples were small. Differences were considered significant at the 95% log10 CFU. Compared toConsidering the CFU an counts average doubling in the time four of 0.5 surviving to 1.0 h for bac- level of confidence. untreated mice, thereterial was pathogens a significant like Staphylococcus difference aureus, inPseudomonas favor of aerugi- nosa, or Streptococcus pneumoniae, a 20 to 40% TMIC of beta-lac- treated mice only in thetams imipenem-clavulanate may cover 5 or 6 generations over group 24 h. The (6.4 generation ver- time sus 7.1 log10 CFU, P ϭfor0.007).M. tuberculosis In allgrowing of thein the otherinfected mouse groups, lung is the reported RESULTS to vary between 24 h during rapid growth in the acute model to CFU count at day 28 was1,676 hnot during different the slowlyfrom growing/nonreplicating that of the phase sur- in the MICs. The MICs of carbapenems against M. tuberculosis in viving untreated mice. chronic model (19, 20). Since the bactericidal effect of beta-lac- tams is exerted during cell division, poor activity of these beta- 14 the presence of clavulanate were reduced 4- to 16-fold (Table lactams against M. tuberculosis in vivo could be due to an insuffi- 1). The MICs of the combination were below the EUCAST cient duration of exposure during its growth cycle. DISCUSSIONRecently, the meropenem-clavulanate combination was breakpoint of susceptibility for imipenem and meropenem (1 shown to be efficacious in patients with XDR TB (8) using a FIG 3 Efficacies of meropenem-clavulanate and amoxicillin-clavulanate in mg/liter; critical concentrations, 2 to 8 mg/liter)the acute (9). (A) and chronic (B) mouse modelsWe for TB. have The bacterial shown load at that the 3-times-daily imipenem, (2 meropenem,g each time) dosing and regimen. ertapenem Based on the re- beginning of treatment (early control) is indicated by the dashed lines. Solid ported human PK for meropenem (Merrem product information; Survival rate. Six of ten untreated mice diedhorizontal between lines indicate days the mean values.alone do not prevent mortalityAstraZeneca, of 27 miceFebruary infected 2007), such with a dosingM. regimen tuber- is ex- 18 and 24. All of the isoniazid-treated mice survived. Three culosis reference virulent strain H37Rv. The addition of clavu- clavulanate-treated mice died. Of the carbapenem-treatedJune 2013 Volume 57 Number 6 lanate significantly improved survival since there was noaac.asm.org mor- 2509 3/18/16

Summary of in vivo data

• Evidence of improved survival, especially with B- lactamase inhibitor

• Unclear magnitude of effect on reducing bacterial load

• The dose and dosing frequency relave to the MIC are important for effect

Clinical studies: General Summary Representave data

15 VOL. 49, 2005 IMIPENEM FOR TUBERCULOSIS 2819

is exceedingly difficult. The need for drug combinations in treating active tuberculosis and the lack of a standardized regimen for comparison confounds the3/18/16 assessment of the ac- tivity of any single agent and its contribution to the outcome. The need to control for the many variables known to affect outcome (5, 13, 17), including prior drug-exposure history, duration of disease, the number of drug resistances, prior treatment with or resistance to fluoroquinolones, and the pres- ence of cavitary disease, further complicates study design. Thus, medical therapy of MDR tuberculosis is based almost entirely on noncomparative observational studies, case series, Pharmacodynamics: Imipenem may have and case reports, despite their weaknesses and limitations. With this in mind, the following conclusions about the effi- cacy of imipenem seem warranted. The murine studies indicate

anmycobacterial acvity in humans that imipenem as a single agent has activity against tuberculo- Downloaded from sis: it reduced the numbers of organisms in target organs and improved survival. These results are particularly encouraging • 10 paents with MDR TB on Imipenem 1 G BID (30 mg/kg if < 50 kg), given the brevity of the period of exposure of organisms to imipenem in the infected mice, because of a short half-life and along with other drugs lower serum concentrations in mice compared to humans. Viewed within the context of the animal studies, even though only 10 patients were treated, the data indicate that

imipenem was therapeutically active and useful. There was a http://aac.asm.org/ strong temporal association between the institution of imi- penem combination therapy and the elimination of M. tuber- culosis in sputa as assayed by quantitative culture. While it can be argued0.35 log10 CFU/ml/ that it was other agents, and not imipenem, that were responsible for this, it is likely that imipenem contributed significantlyweek decrease to the overall efficacy. As a group, these patients were at high risk for treatment failure: nine were smear posi-

tive at study entry, eight had cavitary disease, eight had previ- on January 16, 2016 by guest ously received a fluoroquinolone, and four had a fluoroquin- olone-resistant isolate. The prestudy isolate average was seven drug resistances, and no isolate was resistant to fewer than five drugs. Our patients were quite similar to those reported by Goble et al. (5), as were cure rates, i.e., 70% versus 59%. Cure rates (77 to 96%) higher than these have been reported pre- viously (13, 14, 17), but the patients in those studies were Chambers AAC 2005 FIG. 3. Results of quantitative sputum cultures indicating M. tuber- infected by strains resistant to fewer drugs, typically five or culosis burdens, expressed as log10 CFU/ml, over time in sputa of fewer, and fluoroquinolones were used. individual patients (numbers correspond to patient numbers in Table Individual responses further indicate that imipenem had an- 1) (A) and as a calculation of the overall elimination rate by linear timycobacterial activity. Case 1 had been culture positive for 11 regression for those patients who responded to treatment (B). The years, failing pneumonectomy and numerous courses of di- linear regression equation is shown. rectly observed therapy. Compared to the most recent prestudy regimen, imipenem was the only substantive addition, although amikacin was substituted for streptomycin because of second- second 5.5-month course of imipenem and capreomycin in ary resistance. In case 2, conversion of sputum cultures to addition to oral medications, which were given for a total of 18 negative was accomplished with a regimen of only three drugs, Clinical Studies: 8 studies—case reports months after the last positive culture. This patient had no i.e., ofloxacin, imipenem, and amikacin. Case 4 is particularly evidence of active tuberculosis after 2 years of follow-up off of instructive, because microbiologic failure was associated with therapy. the emergence of resistance to imipenem as well as to strep- • Limited data with Patient 10heterogenous relapsed 6 months after the discontinuation usage of tomycin. Ironically, failure associated with emergence of resis- imipenem, which was stopped because of severe rash, and died tance is perhaps the best evidence of drug activity in vivo (10), of disseminated tuberculosis. Patient 2 died of cor pulmonale with the caveat that drug potency is difficult to assess because – Backbone regimenand chronic respiratory, dosing, clavulanic acid, duraon, outcome insufficiency 6 months after completing less-active drugs still will select for resistant mutants. Three therapy; there was no evidence of active tuberculosis. patients, i.e., patients 7, 8 and 10, after initially responding to Drug Studies Dosing Outcome summaryimipenem plus Side Effects amikacin or capreomycin, relapsed when these Meropenem 5 studies: total 59 --Some with Clavulanic acid DISCUSSION ~49 of 59 with cure drugs were discontinuedDiarrhea, GI from the regimen. Two remained paents Proving the--1g TID to 2g TID clinical efficacy of any agent, new or old,or culture conversion for culture negative issues in a for at least 2 years and presumably were cured All MDR or XDR treatment of tuberculosis,--Some with linezolid especially that due to MDR strains, after a secondminority of course of imipenem combination therapy. The paents --Range 67days to 18 months paents Imipenem 3 studies: total 18 --most without clavulanic acid ~14 of 18 with cure 1 drug rash, 1 paents --500mg QID to 1g BID or culture conversion resistance, 2 with All MDR or XDR --6mo to 18 months GI issues paents Ertapenem 2 studies: total 23 -1g daily without clavulanic acid ~22 of 23 with cure 1 allergic paents --77 to 430 days or improvement reacon, 1 l, 2 --in 1 study, all inially --Majority with linezolid GI received mero or imi

16 TUBERCULOSIS S. DE LORENZO ET AL.

TABLE 3 Treatment outcomes of multidrug-resistant tuberculosis (TB) patients, excluding those not treated with linezolid, enrolled in two specialised clinical centres in Italy and the Netherlands after 30, 60 and 90 days of treatment

Variables Italian cohort: cases# Dutch cohort: controls" p-value

Sputum-smear conversion At 30 days of treatment 5/28 (17.9) 9/18 (50.0) 0.02 At 60 days of treatment 17/28 (60.7) 7/16 (43.8) 0.28 At 90 days of treatment 24/28 (85.7) 9/16 (56.3) 0.03 Sputum-culture conversion At 30 days of treatment 9/32 (28.1) 12/29 (41.4) 0.28 At 60 days of treatment 20/32 (62.5) 13/25 (52.0) 0.43 At 90 days of treatment 26/32 (81.3) 15/24 (62.5) 0.12 Days from start of anti-TB therapy to sputum smear 52.5 (42.0–65.0) 46.0 (6.0–157.0) 0.80 TUBERCULOSIS conversion S. DE LORENZO ET AL. Days from start of anti-TB therapy to culture 42.0 (28.0–77.0) 46.0 (13.0–96.0) 0.79 conversion

TABLE 3 Treatment outcomes of multidrug-resistant tuberculosis (TB) patients, excludingData are presented those not as treated n/N (%) with or median linezolid, (interquartile range), unless otherwise stated. #: meropenem–clavulanate3/18/16 containing anti-TB regimen; ": meropenem– enrolled in two specialised clinical centres in Italy and the Netherlands afterclavulanate-sparing 30, 60 and anti-TB90 days regimen. of treatment

Variables Italian cohort: cases# Dutch cohort: controls" p-value

Sputum-smear conversion Italy The Netherlands tolerability in treating MDR- and XDR-TB cases after 3 months a) 100 ¶ At 30 days of treatment 5/28 (17.9) 9/18 (50.0) 0.02 of treatment with second-line drugs. To our knowledge, this is 90 *** At 60 days of treatment 17/28 (60.7) 7/16 (43.8) 0.28 the first large study evaluating the added value of meropenem– At 90 days of treatment 24/28 (85.7)80 9/16 (56.3)# 0.03 clavulanate in managing difficult-to-treat MDR-/XDR-TB cases. Sputum-culture conversion 70 At 30 days of treatment 9/32 (28.1) 12/29 (41.4) 0.28 In spite of the worse initial clinical severity of cases (in terms of At 60 days of treatment 20/32 (62.5)60 13/25 (52.0) 0.43 proportion of re-treatment cases, XDR-TB cases, proportion of At 90 days of treatment 26/32 (81.3)50 15/24 (62.5) 0.12 sputum-smear positive cases and prevalence of resistance to Case-Control study: 98 MDR TB cases treated with linezolid Days from start of anti-TB therapy to sputum smear 52.5 (42.0–65.0) 46.0 (6.0–157.0) 0.80 40 pyrazinamide, fluoroquinolones, ethionamide and cycloserine), conversion meropenem–clavulanate significantly increased the proportion 30 regimens with (N=37)/without (N=61) Days from start of anti-TB therapy to culture 42.0 (28.0–77.0) 46.0 (13.0–96.0)mero/clav 0.79 1 G TID of sputum-smear conversion in the overall sample, including conversion 20 XDR-TB patients, and sputum-culture conversion among MDR- Sputum-smear conversion % 10 TB cases after 90 days of treatment with second-line drugs. Data are presented as n/N (%) or median (interquartile range), unless otherwise stated. #: meropenem–clavulanate containing anti-TB regimen; ": meropenem– clavulanate-sparing anti-TB regimen. 0 Furthermore, although the clinical pattern of cases was 90 DAY OUTCOME, EXCLUDING XDR 30 60 90 significantly worse than that of controls (as previously Time days mentioned), the added value of meropenem–clavulanate has ƒ already been seen after 60 days of treatment with second-line Italy The Netherlands tolerability in treatingb) 100 MDR- and XDR-TB cases after 3 months a) 100 ¶ drugs (with differences not yet significant); although no of treatment with second-line90 drugs. To our knowledge, this is 90 *** § difference was found in the time to microbiological conversion. the first large study evaluating80 + the added value of meropenem– 80 # clavulanate in managing difficult-to-treat MDR-/XDR-TB cases. While a comprehensive assessment of culture conversion was 70 70 performed, unfortunately we could not assess the sputum- In spite of the worse60 initial clinical severity of cases (in terms of 60 proportion of re-treatment cases, XDR-TB cases, proportion of smear conversion in all the individuals. This was probably due 50 sputum-smear positive50 cases and prevalence of resistance to to a lower pulmonary bacillary load in those with a less severe disease (particularly in the control group) and the effect of 40 pyrazinamide, fluoroquinolones,40 ethionamide and cycloserine), meropenem–clavulanate significantly increased the proportion previous treatments patients underwent before being admitted 30 30 of sputum-smear conversion in the overall sample, including to the reference centres. 20 XDR-TB patients, and20 sputum-culture conversion among MDR- Sputum-culture conversion % Sputum-smear conversion % Importantly, meropenem–clavulanate at an i.v. dosage of 1 g 10 TB cases after 90 days10 of treatment with second-line drugs. three times a day was well tolerated and a single episode of 0 Furthermore, although0 the clinical pattern of cases was drug withdrawal was recorded over a median hospital 30 60 90 significantly worse than that30 of controls (as60 previously 90 exposure time of 67 days. Time days mentioned), the added value of meropenem–clavulanateTime days has ƒ The clinician’s decision to interrupt meropenem–clavulanate (a b) 100 already been seen after 60 days of treatment with second-line drugs (with differencesFIGURE 1. nota) Sputum-smear yet significant); conversion although and b) culture no conversion at 90 days drug only administered intravenously and needing inpatient 90 of multidrug-resistant tuberculosis cases, excluding the extensively drug-resistant care) was related to clinical improvement and microbiological § difference was found in theCULTURE Conversion time to microbiological conversion. SMEAR Conversion + 80 tuberculosis patients, enrolled in two specialised clinical centres in Italy and the conversion (e.g. the conditions allowing hospital discharge) While a comprehensive assessment of# culture" conversion+ was 1 e 70 Netherlands. ***: p,0.001. :p50.71; :p50.10; :p50.001; :p50.25; :p50.03. and not to adverse event occurrence in all but one case. BLACK= MERO; GRAY= CONTROL performed, unfortunately we could not assess the sputum- 60 smear conversion in all the individuals. This was probably due 50 to a lower pulmonary1390 bacillary load in thoseDe Lorenzo, with a less severeEurVOLUME Respir 41 NUMBER J 2013 6 EUROPEAN RESPIRATORY JOURNAL 40 disease (particularly in the control group) and the effect of previous treatments patients underwent before being admitted 30 to the reference centres. 20

Sputum-culture conversion % Importantly, meropenem–clavulanate at an i.v. dosage of 1 g 10 three times a day was well tolerated and a single episode of 0 drug withdrawal was recorded over a median hospital 30 60 90 exposure time of 67 days. Time days The clinician’s decision to interrupt meropenem–clavulanate (a FIGURE 1. a) Sputum-smear conversion and b) culture conversion at 90New data days drug only administered intravenously and needing inpatient of multidrug-resistant tuberculosis cases, excluding the extensively drug-resistant care) was related to clinical improvement and microbiological tuberculosis patients, enrolled in two specialised clinical centres in Italy and the conversion (e.g. the conditions allowing hospital discharge) • Observaonal study: 2003-2015 Netherlands. ***: p,0.001. #:p50.71; ":p50.10; +:p50.001; 1:p50.25; e:p50.03. and not to adverse event occurrence in all but one case.

1390 VOLUME 41 NUMBER 6 EUROPEAN RESPIRATORY JOURNAL • Not randomized Culture conversion • 84 MDR-pts with Imipenem – Median # of resistance: 8; 68% XDR TB – Imipenem 500mg 4x/day for median 187 days • 168 ‘controls’ MDR pts without Imipenem – 6% with XDR TB

• 59.7% success rate with Imipenem vs • 85.8% success rate without

Tiberi 2016 CID

17 TUBERCULOSIS | S.P. VAN RIJN ET AL.

180

160 –1 140

120

100

80

60

40

Ertapenem concentration mg·L concentration Ertapenem 20

0 FIGURE 1 Ertapenem plasma 0 3 6 9 12 15 18 21 24 concentration–time curves in 12 Time post dose h patients.

Drug susceptibility of M. tuberculosis to ertapenem All the M. tuberculosis strains appeared susceptible to ertapenem. However, actual determination of the MIC was complicated by the fact that ertapenem itself appeared an instable compound at 37°C [17]. This was confirmed by the fact that after 7 days MIC values were lower than after 14 days. In addition, freshly prepared plates showed lower MIC values compared with plates stored at 4°C. The refrigerator-stored plates showed lower MIC values than plates stored at room temperature. If ertapenem was combined with clavulanic acid, all MIC values were even lower. 3/18/16 Pharmacokinetics and pharmacodynamics The plasma concentration–time curves were obtained in 12 patients with MDR-TB. In the remaining six patients, routine plasma concentrations were collected at a time-point at which they did not yet receive ertapenem or this drug was no longer administered. Three patients had multiple plasma concentration– time curves and these were consistent. The mean curve is shown in figure 1. The mean (range) area under 1 the concentration–time curve up to 24 h (AUC0–24) was 544.9 (309–1130) h·mg·L− . The steady state pharmacokinetic parameters are shown in table 2. Based on a MIC of 0.25 mg·L−1, 11 out of 12 patients exceeded a minimum of 40% tfree>MIC. In nine patients the MDR-TB remained susceptible with a MIC of 1 1 0.5 mg·L− . Only twoPharmacokinecs ( patients exceeded a minimum of 40% tfree>MIC with aertapenem MIC of 1 mg·L− . The ) pharmacokinetic population model (KinPOP) of ertapenem showed a mean (range) clearance of 2.26 • (0.86Retrospecve analysis on 18 MDR-TB paents in the Netherlands –3.19) L·h−1 per 1.73 m2 and a mean (range) volume of distribution of 8.79 (4.76–13.57) L. Safety and tolerability In general, ertapenem was tolerated very well. In three patients, treatment with ertapenem was stopped. One of • theseQueson: What % achieve patients suffered from Crohn’s disease and developed MDR-TBTfree after multiple>MIC of > 40% dosages of infliximab, a (bactericidal level) tumour necrosis factor-α blocker [21, 22]. This patient experienced allergic fever, shortly after administration • ofIf MIC was 0.25: 92% achieved ertapenem and ethambutol. After reintroduction this happened again (Naranjo score=4). Both AEs subsided after withdrawal of the offending drug. In the second patient, ertapenem was stopped after an increase in liver enzymes (aspartate aminotransferase: 109 U·L−1; alanine aminotransferase: 255 U·L−1) after 13 days of • treatmentIf MIC was 1: only 17% achieved this with ertapenem. However, after 2 months, while this patient was still on treatment without ertapenem, liver enzymes remained elevated (Naranjo score=1). In the third patient, kanamycin, linezolid and ertapenem were stopped due to line sepsis. This was considered not to be related to ertapenem. After removal • ofHigh interpaent variability, reduced absorpon, higher clearance the venous access port, the patient recovered. Ertapenem and a new venous access port were not reintroduced, since they were not indicated anymore, due to low bacillary load at that time, and these i.v. drugs couldcompared to healthy be substituted with oral antimycobacterial drugs. None of the patients experienced diarrhoea, vomiting or dizziness.

TABLE 2 Pharmacokinetic parameters of ertapenem

1 1 1 Study AUC0–24 h·mg·L− Cmax mg·L− Half-life h Volume of distribution L Clearance L·h−

1gi.v. in MDR-TB patients 544.9 (309–1130) 127.5 (73.9–277.9) 2.4 (2.047–3.528) 7.3 (2.612–11.1) 2.1 (0.0884–3.231) 1gi.v. in healthy volunteers [18] 572.1 (572–672) 154.9 (145–175) 4 (3.8–4.4) 8.2 1.8

Data are presented as mean (range) and were calculated using KinFIT. AUC0–24: area under the concentration–time curve up to 24 h; Cmax: maximum observed plasma concentration; MDR-TB: multidrug-resistant tuberculosis. Van Rijn, Eur Respir J 2016

4 DOI: 10.1183/13993003.01654-2015

New data: Beta-Lactams Against TB: Teaching a New Trick to an Old Dog • Early bactericidal acvity (EBA) study of Smear-posive pts • 14 days of treatment with: – IV Meropenem (2g) + clavulanic acid – ORAL Faropenem 600mg +clavulanic acid

• Results: “The viable mycobacterial sputum load was significantly reduced with standard treatment (validang the laboratory assays) and with meropenem A/CA but not with faropenem A/CA”

Diacon et al. Abstract CROI 2016

18 3/18/16

Summary of Clinical Data

• Suggests some benefit • Difficult to separate effect from rest of regimen • Limited studies, selecon and publicaon bias • Requires IV treatment, no studies on tebipenem • Concern of keeping T>MIC

Conclusions • Carbapenems have structural basis for acvity against mycobacteria (L-D transpepdase) • In vitro data suggests that carbapenems should have acvity against M. tuberculosis • In vivo data shows improved survival, but mixed efficacy in reducing bacterial load • Limited clinical data suggests there may be a role for carbapenems as part of MDR/XDR TB regimens

19 3/18/16

Quesons to be answered • The opmal place in the TB regimen is sll unclear: – Which Carbapenem should be used? – Can it be used in conjuncon with or as a replacement for other injectables? – What is the ‘order’ of selecon as part of MDR regimens? • Linezolid, Bedaquiline, Delaminid, Carbapenems?

• What is the opmal duraon of Carbapenem usage?

• What is the opmal dosing frequency of carbapenems?

• Should clavulanic acid be used in combinaon with carbapenem, and if so what dose?

Acknowledgements

• Special Thanks to Dr. Devan Jaganath • Gyanu Lamichhane

20 3/18/16

Extra slides

Outcomes - Meropenem Study Paent Type Regimen Duraon of Carb Outcome Adverse Effects Tiberi, et al., MDR/XDR Meropenem 3G TID/ mero/impinem 3 cure GI (n=2) 2016 Pulm TB (N=5) Imipenim 500 mg QID, inpt, average 24.4 days, 1 improved, no Ertapenem 1G daily outpt ertapenem final sputum to average 431.4 classify days 1 did not convert, died Payen, et al., XDR Pulm TB Meropenem 2G TID Unknown 5 culture None reported 2012 (N=6) intensive, 2G BID conversion, one of connuaon. Augmenn 500 which defined as mg/125mg TID cured

Dauby, et al., XDR Pulm TB Meropenem 1.5 G TID, 18 months Cure Linezolid 2011 (N=1) augmenn 1G/200mg TID x associated 8 months, meropenem 2 G peripheral BID, augmenn 500 mg TID x neuropathy 10 months

21 3/18/16

Outcomes - Meropenem

Study Paent Type Regimen Duraon of Carb Outcome Adverse Effects De Lorenzo, et MDR/XDR TB Meropenem/ 67 (46-85) days Smear Diarrhea (n=5) al. 2013 (N=37) Clav 1 G TID with conversion Elevated LFTs linezolid 28/32 (88%) (n=2) containing Culture regimen conversion ( 31/37 (84%) Palmero, et al, XDR/pre-XDR TB Mero 2 G TID At least 6 Sputum 3 GI, 1 hepas, 2015 (N=10) unl cx neg, months conversion: 8/10 1 hematologic, 1 then 1 G TID. 1 failed, 2 deaths polyneuris Augmenn 500/125 TID

Outcomes - Imipenem

Study Paent Type Regimen Duraon of Carb Outcome Adverse Effects Tiberi, et al., MDR/XDR Pulm Meropenem 3G mero/impinem 3 cure GI (n=2) 2016 TB (N=5) TID/Imipenim 24.4 days, 1 improved, no 500 mg QID, ertapenem final sputum to inpt, Ertapenem 431.4 days classify 1G daily outpt 1 did not convert, died Chambers et al., MDR TB, Pulm/ Imipenem 1 G 6 months (range 7 cured, 2 Severe drug rash 2005 GI (N=10) BID (30 mg/kg if 4-9) failure, 3 (n=1) < 50 kg) relapsed, 2 Imipenem deaths (1 TB resistance (n=1) related) Arbex et al., XDR (N=3) Imipenem/Clav 18 months 2 cured, 1 Not reported 2015 unknown dose improved

22 3/18/16

Outcomes - Ertapenem

Study Paent Type Regimen Duraon of Carb Outcome Adverse Effects Van Rijn, et al. MDR TB (N=18) Ertapenem 1G 77 days (range All cure, no Allergic fever 2015 daily 5-210) relapse (n=1), elevated LFTs (n=1) Tiberi, et al., MDR/XDR Pulm Meropenem 3G mero/impinem 3 cure GI (n=2) 2016 TB (N=5) TID/Imipenim 24.4 days, 1 improved, so 500 mg QID, ertapenem final sputum to inpt, Ertapenem 431.4 days classify 1G daily outpt 1 did not convert, died

23