5 курс / Пульмонология и фтизиатрия / Clinical_Tuberculosis_Friedman_Lloyd_N_,_Dedicoat
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200 Clinical Pharmacology of the Anti-Tuberculosis Drugs
239.Svensson EM, Dooley KE, and Karlsson MO. Impact of lopina- vir-ritonavir or nevirapine on bedaquiline exposures and potential implications for patients with tuberculosis-HIV coinfection.
Antimicrob Agents Chemother. 2014 Nov;58(11):6406–12.
240.Mukherjee T, Boshoff H. Nitroimidazoles for the treatment of TB: Past, present and future. Future Med Chem. 2011 Sep;3(11):1427–54.
241.Stinson K et al. MIC of delamanid (OPC-67683) against Mycobacterium tuberculosis clinical isolates and a proposed critical concentration. Antimicrob Agents Chemother. 2016;60(6):3316–22.
242.Fujiwara M, Kawasaki M, Hariguchi N, Liu Y, and Matsumoto M. Mechanisms of resistance to delamanid, a drug for Mycobacterium tuberculosis. Tuberculosis. 2018 Jan 1;108:186–94.
243.Sasahara K et al. Pharmacokinetics and metabolism of delamanid, a novel anti-tuberculosis drug, in animals and humans: Importance of albumin metabolism in vivo. Drug Metab Dispos Biol Fate Chem. 2015 Aug;43(8):1267–76.
244.Shimokawa Y et al. Metabolic mechanism of delamanid, a new anti-tuberculosis drug, in human plasma. Drug Metab Dispos Biol Fate Chem. 2015 Aug;43(8):1277–83.
245.Gler MT et al. Delamanid for multidrug-resistant pulmonary tuberculosis. N Engl J Med. 2012 Jun 7;366(23):2151–60.
246.Diacon AH et al. Early bactericidal activity of delamanid (OPC67683) in smear-positive pulmonary tuberculosis patients. Int J Tuberc Lung Dis. 2011 Jul;15(7):949–54.
247.Mallikaarjun S et al. Delamanid coadministered with antiretroviral drugs or antituberculosis drugs shows no clinically relevant drug-drug interactions in healthy subjects. Antimicrob Agents Chemother. 2016;60(10):5976–85.
248.Hansen JL, Ippolito JA, Ban N, Nissen P, Moore PB, and Steitz TA. The structures of four macrolide antibiotics bound to the large ribosomal subunit. Mol Cell. 2002 Jul;10(1):117–28.
249.Madsen CT, Jakobsen L, Buriánková K, Doucet-Populaire F, Pernodet J-L, and Douthwaite S. Methyltransferase Erm(37) slips on rRNA to confer atypical resistance in Mycobacterium tuberculosis. J Biol Chem. 2005 Nov 25;280(47):38942–7.
250van der Paardt A-F et al. Evaluation of macrolides for possible use against multidrug-resistant Mycobacterium tuberculosis. Eur Respir J. 2015 Aug 1;46(2):444–55.
251.Brown-Elliott BA, Nash KA, and Wallace RJ. Antimicrobial susceptibility testing, drug resistance mechanisms, and therapy of infections with nontuberculous mycobacteria. Clin Microbiol Rev. 2012 Jul;25(3):545–82.
252 de Carvalho NFG, Pavan F, Sato DN, Leite CQF, Arbeit RD, and Chimara E. Genetic correlates of clarithromycin susceptibility among isolates of the Mycobacterium abscessus group and the potential clinical applicability of a PCR-based analysis of erm(41). J Antimicrob Chemother. 2018 Apr 1;73(4):862–6.
253.Chu SY, Deaton R, and Cavanaugh J. Absolute bioavailability of clarithromycin after oral administration in humans. Antimicrob Agents Chemother. 1992 May;36(5):1147–50.
254.Rodrigues AD, Roberts EM, Mulford DJ, Yao Y, and Ouellet D. Oxidative metabolism of clarithromycin in the presence of human liver microsomes. Major role for the cytochrome P4503A (CYP3A) subfamily. Drug Metab Dispos Biol Fate Chem. 1997 May;25(5):623–30.
255.Ferrero JL et al. Metabolism and disposition of clarithromycin in man. Drug Metab Dispos Biol Fate Chem. 1990 Aug;18(4):441–6.
256.Rodvold KA. Clinical pharmacokinetics of clarithromycin. Clin Pharmacokinet. 1999 Nov;37(5):385–98.
257.Honeybourne D, Kees F, Andrews JM, Baldwin D, and Wise R. The levels of clarithromycin and its 14-hydroxy metabolite in the lung. Eur Respir J. 1994 Jul;7(7):1275–80.
258.Fish DN, Gotfried MH, Danziger LH, and Rodvold KA. Penetration of clarithromycin into lung tissues from patients undergoing lung resection. Antimicrob Agents Chemother. 1994 Apr;38(4):876–8.
259.Pasipanodya JG et al. Systematic review and meta-analyses of the effect of chemotherapy on pulmonary Mycobacterium abscessus outcomes and disease recurrence. Antimicrob Agents Chemother. 2017 Nov;61(11).
260.Pasipanodya JG, Ogbonna D, Deshpande D, Srivastava S, and Gumbo T. Meta-analyses and the evidence base for microbial outcomes in the treatment of pulmonary Mycobacterium avium- intracellular complex disease. J Antimicrob Chemother. 2017 Sep 1;72(Suppl 2):i3–19.
261.Griffith DE et al. An official ATS/IDSA statement: Diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Am J Respir Crit Care Med. 2007 Feb 15;175(4):367–416.
262van Haarst AD et al. The influence of cisapride and clarithromycin on QT intervals in healthy volunteers. Clin Pharmacol Ther. 1998 Nov;64(5):542–6.
263.Zhou S et al. Mechanism-based inhibition of cytochrome P450 3A4 by therapeutic drugs. Clin Pharmacokinet. 2005;44(3):279–304.
264.Sedlmayr T, Peters F, Raasch W, and Kees F. Clarithromycin, a new macrolide antibiotic. Effectiveness in puerperal infections and pharmacokinetics in breast milk. Geburtshilfe Frauenheilkd. 1993 Jul;53(7):488–91.
265.Gopal P, and Dick T. The new tuberculosis drug Perchlozone® shows cross-resistance with thiacetazone. Int J Antimicrob Agents. 2015 Apr;45(4):430–3.
266.Holdiness MR. Clinical pharmacokinetics of the antituberculosis drugs. Clin Pharmacokinet. 1984 Dec;9(6):511–44.
267.Peloquin CA, Nitta AT, Berning SE, Iseman MD, and James GT. Pharmacokinetic evaluation of thiacetazone. Pharmacotherapy 1996 Oct;16(5):735–41.
268.Okwera A et al. Randomised trial of thiacetazone and rifampicincontaining regimens for pulmonary tuberculosis in HIV-infected Ugandans. The Makerere University-Case Western University research collaboration. Lancet 1994 Nov 12;344(8933):1323–8.
269.Nunn P et al. Cutaneous hypersensitivity reactions due to thiacetazone in HIV-1 seropositive patients treated for tuberculosis. Lancet Lond Engl. 1991 Mar 16;337(8742):627–30.
270.Cordillot M et al. In vitro cross-linking of Mycobacterium tuberculosis peptidoglycan by L,D-transpeptidases and inactivation of these enzymes by carbapenems. Antimicrob Agents Chemother. 2013 Dec;57(12):5940–5.
271.Hugonnet J-E, Tremblay LW, Boshoff HI, Barry CE, and Blanchard JS. Meropenem-clavulanate is effective against extensively drug-resistant Mycobacterium tuberculosis. Science 2009 Feb 27;323(5918):1215–8.
272.Hugonnet J-E, and Blanchard JS. Irreversible inhibition of the Mycobacterium tuberculosis beta-lactamase by clavulanate. Biochemistry (Mosc) 2007 Oct 30;46(43):11998–2004.
273.Davies Forsman L, Giske CG, Bruchfeld J, Schön T, Juréen P, and Ängeby K. Meropenem-clavulanate has high in vitro activity against multidrug-resistant Mycobacterium tuberculosis. Int J Mycobacteriol. 2015;4(Suppl 1):80–1.
274.Nilsson-Ehle I, Hutchison M, Haworth SJ, and Norrby SR. Pharmacokinetics of meropenem compared to imipenem-cilas- tatin in young, healthy males. Eur J Clin Microbiol Infect Dis. 1991 Feb;10(2):85–8.
275.Mouton JW, Touzw DJ, Horrevorts AM, and Vinks AA. Comparative pharmacokinetics of the carbapenems: Clinical implications. Clin Pharmacokinet. 2000 Sep;39(3):185–201.
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Key sources not specifically cited 201
276.Conte JE, Golden JA, Kelley MG, and Zurlinden E. Intrapulmonary pharmacokinetics and pharmacodynamics of meropenem. Int J Antimicrob Agents 2005 Dec;26(6):449–56.
277van Hasselt JGC et al. Pooled population pharmacokinetic model of imipenem in plasma and the lung epithelial lining fluid. Br J Clin Pharmacol. 2016;81(6):1113–23.
278.Modai J, Vittecoq D, Decazes JM, and Meulemans A. Penetration of imipenem and cilastatin into cerebrospinal fluid of patients with bacterial meningitis. J Antimicrob Chemother. 1985 Dec;16(6):751–5.
279.Lu C et al. Population pharmacokinetics and dosing regimen optimization of meropenem in cerebrospinal fluid and plasma in patients with meningitis after neurosurgery. Antimicrob Agents Chemother. 2016 Oct 21;60(11):6619–25.
280.England K et al. Meropenem–Clavulanic acid shows activity against Mycobacterium tuberculosis in vivo. Antimicrob Agents Chemother. 2012 Jun;56(6):3384–7.
281.Tiberi S et al. Comparison of effectiveness and safety of imipenem/ clavulanateversus meropenem/clavulanate-containing regimens in the treatment of MDRand XDR-TB. Eur Respir J. 2016;47(6): 1758–66.
282.Sauberan JB, Bradley JS, Blumer J, and Stellwagen LM. Transmission of meropenem in breast milk. Pediatr Infect Dis J. 2012 Aug;31(8):832–4.
KEY SOURCES NOT SPECIFICALLY
CITED
Drugbank, PubChem, Summaries of Product Characteristics, FDA Label, and WHO MDR-TB Companion.
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11
New Developments in Drug Treatment
ALEXANDER S. PYM, CAMUS NIMMO, AND JAMES MILLARD
Introduction
Finding New drugs
Improving existing drugs
Reusing old drugs
Host-direct therapy
New regimens
Conclusions
References
INTRODUCTION
Short-course chemotherapy (SCC) for tuberculosis (TB), using four drugs (isoniazid, rifampicin, pyrazinamide and ethambutol) over 6 months is low cost, highly efficacious, and generally well tolerated. In clinical trial settings, it dependably delivers a cure rate approaching 95% for drug-susceptible TB.1 Apart from high early mortality rates, good clinical outcomes can also be obtained for HIV co-infected TB patients with 6 months treatment when used with antiretroviral therapy.2 However, the duration of treatment has proved an insurmountable obstacle to effective implementation of SCC in many areas of the world. Failure of TB programs to ensure patient adherence to therapy and insufficiently optimized regimens has resulted in poor clinical outcomes, ongoing infectiousness, and selection of antibiotic resistance. An ultra-short course of chemotherapy is therefore an urgent priority. While stratification of lowrisk patients may allow some reductions in treatment duration3 to shorten treatment to weeks, new drugs are needed that more effectively target the tiny subpopulations of bacteria that survive the first few bactericidal weeks of SCC, usually referred to as “sterilizing” ability. However formidable challenges remain, as demonstrated by the failure of treatment shortening attempts using fluoroquinolones,4 as we still do not know the biological basis of persistent organisms in the face of SCC and have no in vitro or animal models that confidently replicate persistent organisms.
In contrast to a radical shortening of TB therapy, developing a new generation of drugs for multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) is more straightforward. While treatment for MDR-TB traditionally required a minimum of 20 months’ therapy, the World Health Organization (WHO) drug-resistant TB 2019 guidelines have now incorporated a shorter 9–12-month regimen, which already has been adopted in many countries.5–7 These regimens consist
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of repurposed drugs or those rejected for drug-susceptible TB because of their poor efficacy or intolerable toxicity. Traditional MDR-TB regimens are poorly effective, rarely delivering programmatic success rates above 60%, whilst the outcomes of the new shorter regimens, although effective in clinical trial settings, still require further programmatic data.6,8 Hence, the introduction of several new drugs would have an immediate impact. In fact with the increasing use of bedaquiline since its approval by the US Food and Drug and Administration (FDA) in December 2012, the identification of two other novel classes of anti-TB drugs (DprE1 and MmpL3 inhibitors), appreciation of the mycobacterial action of existing drug classes (oxazolidinones and nitroimidazoles) and better use of currently available drugs (fluoroquinolones and riminophenazines) there is a high probability that significant improvements in MDR-TB and XDR-TB will be achieved relatively soon. This chapter reviews these tangible advances in the development of drugs that are in or shortly to enter clinical development.
FINDING NEW DRUGS
Respiratory chain inhibitors
Diarylquinolines (DARQs), a new class of anti-TB compounds were identified in 1996, from a whole-cell screen of 70 000 compounds using Mycobacterium smegmatis, a fast-growing nonpathogenic surrogate of M. tuberculosis.9 DARQs are structurally and mechanistically different from fluoroquinolones and other quinolines. Subsequent structure−activity studies on leads from the screens resulted in bedaquiline,10 formerly known as TMC207 and R207910. Bedaquiline is highly active against M. tuberculosis, with a minimum inhibitory concentration (MIC) in the range of 0.03–0.12 µg/mL, as well as against a broad range of other
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mycobacteria.10 Its mechanism of action was elucidated using whole genome sequencing of drug-resistant mutants that localized resistance to atpE. This gene encodes a protein that is part of the F1F0 proton ATP synthase, a trans-membrane protein complex that generates ATP from proton translocation. Further functional studies have identified a binding site of drug to ATP synthase, although importantly human mitochondrial ATP synthase is more than 10,000-fold less sensitive than its mycobacterial equivalent, critical for a good safety profile.11 However, resistant mutants have been identified that do not have any mutations in atpE,12 and atpE mutations appear rare in clinical isolates. The majority of bedaquiline resistance in clinical isolates to date has been related to mutations in the Rv0678 gene, which encodes a negative transcriptional regulator of the MmpL5 efflux pump, which can cause cross-resistance to bedaquiline and clofazimine.194,195 Loss of function mutations in pepQ, which encodes a cytoplasmic peptidase, have also been proposed to caused bedaquiline and clofazimine cross-resistance, although the importance of this in human clinical isolates has yet to be demonstrated.66
ATP synthase is an attractive drug target because it is essential, and ATP is required even in persistent bacterial states.13 Therefore it was not surprising to find that bedaquiline is active on dividing and non-dividing bacteria14 and displays time-dependent bactericidal activity both in vitro and in vivo. It has been extensively studied in the murine model and has emerged as a potential key component of new drug regimens.15 It is highly active given as mono-therapy and can substitute for any of the three first-line drugs,10,16 exhibits strong synergy with PZA17 and can shorten MDR-TB treatment.18 With the aim of developing a universal regimen, active against drug-susceptible and -resistant organisms various combinations of pretomanid (PA-824), clofazimine, sutezolid, bedaquiline, rifapentine and pyrazinamide were evaluated in a long-term mouse model. In terms of relapse-free cure after SCC, only regimens with bedaquiline were successful indicating its importance to new regimens.15
In December 2012, the FDA gave expedited approval of bedaquiline for the treatment of MDR-TB in adults with limited options. A phase II clinical study (C208) was conducted in treatment naive MDR-TB patients over two stages in which participants received either placebo or bedaquiline in combination with standard MDR-TB treatment. The first stage involved 2 months of bedaquiline treatment (n = 47) and the second stage 6 months of treatment (n = 160). For both stages, the time to sputum culture conversion, and proportion of patients culture negative were significantly improved in the bedaquiline arms.19 In the 6-month stage, the median time to culture conversion was 12 weeks and 18 weeks and the proportion culture converted was 78.8% and 57.6%, in the bedaquiline and placebo arms, respectively. This ingenious two-stage design also allowed for intensive pharmacokinetic analysis in stage I to validate the dosing regimen (400 mg daily for 2 weeks, and then 200 mg thrice weekly) for the larger stage 2 to ensure drug accumulation of bedaquiline (a drug with an extremely long half-life) did not occur.
A follow-on trial (C209) was an open-label single-arm study (n = 233) to assess safety in a larger number of patients. It confirms that bedaquiline is generally well tolerated. Of concern is the cumulative effect on the QTc interval, a potentially serious cardiac arrhythmogenic condition. For bedaquiline, relative to the placebo,
there was a modest increase of approximately 12 ms, but this was more than doubled in patients also taking clofazimine. Other drugs such as fluoroquinolones and pretomanid also increase the QTc interval, which might place limitations on combining these drugs. In addition, there was a non-significant increase in mortality in the bedaquiline arm of C208, but this occurred late after bedaquiline had finished, and was not related to cardiac causes of death. While safety data from further phase III studies such as STREAM stage 2 (NCT02409290) are awaited, to date, severe QTc prolongation appears to be an infrequent cause of bedaquiline interruption.20 Meanwhile, further promising data continue to emerge about the use of bedaquiline for drug-resistant TB in programmatic set- tings21–23 and from the Nix-TB clinical trial for XDR-TB treatment.24
The imidazopyridine telacebec (Q203) also targets the mycobacterial respiratory chain, inhibiting cytochrome bc1 by binding to its QcrB subunit, leading mycobacteria to switch to the use of cytochrome bd. This induces less efficient respiration and is associated with ATP depletion. Telacebec was discovered using highthroughput phenotypic screening of over 120,000 compounds for activity against M. tuberculosis in infected macrophages. The compound has an M. tuberculosis MIC <2.5 µg/mL (including MDR strains) and at a dose of 10 mg/kg has an efficacy in a mouse model comparable to isoniazid or bedaquiline over 28 days. In a similar manner to bedaquiline, activity in the murine model is not immediate and this is presumably due to a requirement for cumulative respiratory inhibition to reach a threshold for cell death. Resistance conferring mutations occur in the qcrB gene which encodes the cytochrome bc1 complex.25,26 The drug has progressed through a phase 2a EBA study and provisional results are available. The drug was tested in doses up to 300 mg and was well tolerated with dose-dependent EBA. Telacebec will now be tested in the adaptive design phase 2c STEP trial in combination with various doses of rifampicin and pyrazinamide.
Mycobacterial membrane protein, large 3 (MmpL3) inhibitors
Although SQ109 is indisputably an anti-TB compound with a new mode of action, it was identified through a program to modify ethambutol, the weakest of the four drugs used in SCC.27,28 Ethambutol was a rational choice as historically its structure− activity relationship had not been exhaustively evaluated. Also there was evidence from early clinical trials that its efficacy was markedly superior when used at high doses, subsequently abandoned because of associated ocular toxicity.29,30 Using a library of over 60,000 combinatorial compounds, based on the 1,2-ethyl- enediamine active pharmacophore from ethambutol, researchers identified a diethylamine with promising in vitro activity against M. tuberculosis.31 It has an MIC range of 0.11–0.64 µg/mL against M. tuberculosis, including MDR-TB strains resistant to ethambutol.31 It inhibits growth of M. tuberculosis in macrophages to a similar extent as isoniazid and to a greater extent than ethambutol.32
Ethambutol inhibits the synthesis of arabinogalactan and lipoarabinomannan33 and probably interacts with three arabinosyltransferases EmbA, EmbB and EmbC. Initial studies showed SQ109 induced a different transcriptional signature from ethambutol in drug-stressed bacteria, indicating a distinct mode of action.34 Although SQ109-resistant mutants have not been
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Improving existing drugs 205
reported, strains resistant to closely related ethylene diamine compounds have been characterized, are cross resistant to SQ109 and have mutations in mmpl3,35 an essential gene.36 This encodes a transporter of the RND family that is required for the export of trehalose monomycolate (TMM), an essential component of the cell wall,36 and is compatible with the observation that TMM accumulates in cells treated with SQ109.35 Intriguingly, other screens have independently selected compounds that appear to target MmpL3. These include AU1235, an adamantyl derivative with structural similarity36 and two other compounds; a pyrrole derivative,37,38 and a benzimidazole39 whose structures bear no resemblance to SQ109. Whole cell screens are not unbiased and perhaps they preferentially select for compounds active against MmpL3. Nevertheless the essentiality of MmpL3, and its key role in cell wall assembly means compounds targeting this transporter could be active clinically, and ultimately it will be interesting to see the head to head comparison of all MmpL3 inhibitors with SQ109 (the only MmpL3 inhibitor in clinical development).
Further characterization of SQ109 has demonstrated it could potentially be combined with a wide range of anti-TB drugs. In vitro, at sub-MIC concentrations, SQ109 demonstrates synergy with rifampicin and isoniazid.40 In murine models of TB the 25 mg/kg dose of SQ109 was found to be equivalent to the 100 mg/kg of ethambutol.31 This superiority of SQ109 over ethambutol at standard doses was also seen when the two drugs were compared in combination with rifampicin, isoniazid and pyrazinamide in a 2-month treatment model.41 Pharmacokinetics of SQ109 studied in the mouse suggest the rapid tissue distribution that results in sustained concentrations in lungs (at least 40-fold above the MIC) and spleen may be important for the promising activity seen in the murine model.32
SQ109 has also been favorably combined with second-line antiTB drugs as well as some investigational agents. Activity in vitro was either synergistic or additive with a wide range of MDR-TB treatment drugs and the pharmacokinetics in mice of SQ109 were not affected by moxifloxacin.27 Combinations of SQ109 with bedaquiline were additive with a new oxazolidinone, sutezolid, when tested in vitro. Both improved the rate of M. tuberculosis killing over individual drugs.42,43 In a whole blood assay both drugs were found to have an additive affect, but some antagonism with pretomanid was detected.44 Preliminary studies have recently shown that SQ109, bedaquiline and pyrazinamide combined in vivo in a mouse model of TB induced a durable cure of M. tuberculosis, and suggested at least equivalence to standard regimens.27
In humans, SQ109 appears safe over 14 days’ therapy, but the phase 2 trial did not find significant activity as a sole agent or any enhancement of rifampicin efficacy at 150 or 300 mg doses.45 The SQ109 arms of a multi-arm, multi-stage open-label random- ized-controlled trial of rifampicin-susceptible TB substituting ethambutol for SQ109 were stopped early due to failure to meet prespecified efficacy thresholds.46 However, a phase 2b study in MDR-TB showed an improvement in 6-month culture conversion,47 suggesting that this drug may still be a useful addition to the armamentarium, at least for drug-resistant TB.
Dpre1 inhibitors
Parallel drug discovery efforts have also converged on another essential step in the complex assembly of the mycobacterial cell
wall, DprE1, a flavoenzyme responsible for the epimerization of ribose to arabinose. This pathway provides a unique source of arabinose for both lipoarabinomannan and arabinogalactan, building blocks of the cell wall. The benzothiazinone compound BTZ043 was the first from this group to undergo significant development.48 After nitroreduction by DprE1, BTZ043 covalently binds to a cysteine residue within the active site of DprE1, irreversibly inactivating the enzyme.49–51 Laboratory-selected spontaneous resistant mutations have been localized to dprE1 and were uncommon, arising at a frequency of 10−8.48 Encouragingly, it has a very low MIC (2.3 nM against H37Rv) including against MDR strains.52 It is principally active against dividing organisms in line with its mode of action against the cell wall, and in short-term mouse studies (28 days of monotherapy) has activity similar to rifampicin.48 Combination studies conducted in vitro show it has synergistic activity against M. tuberculosis with bedaquiline and at least additive effects with a range of other firstand second-line anti-TB drugs.53
To date 15 new classes of DprE1 inhibitor with antimycobacterial activity have been identified. Six, including BTZ043, form covalent adducts with cysteine 387 in the active site of DprE1, while the other nine are competitive non-covalent inhibitors. Macozinone (previously known as PBTZ169) is another covalent inhibitor that arose from a lead optimization program of BTZ043 and has a very low MIC of 0.6 nM against H37Rv. Like BTZ043, it acts synergistically with bedaquiline54 as well as clofazimine, delaminid, and sutezolid.55 It is currently in a phase Ib clinical trial (NCT03776500) by Innovate Medicines for Tuberculosis (Lausanne, Switzerland). It was investigated in parallel in a phase 2a trial (NCT03334734) by Nearmedic Plus in Russia, although this was terminated early due to slow recruitment. Results of this have not been published although the company indicated that it had demonstrated good safety and statistically significant early bactericidal activity (EBA) in seven patients.56
From the non-covalent inhibitors identified, the most promising is TCA1 which was identified by screening for inhibitors of M. tuberculosis biofilm formation. It is active against two different enzymes: DprE1 and MoeW, which are involved in molybdenum cofactor biosynthesis and are essential for the hypoxic and nitricoxide stress response. TCA1 resistant mutants remain susceptible to BTZ043 suggesting that it has a different DprE1 binding mechanism. Interestingly, TCA1 also downregulates fdxA, a gene associated with persistence, and unlike BTZ is active in vitro against both replicating and non-replicating mycobacteria.57
The diversity of lead compounds increases the chances of developing a clinically viable drug against the promising DprE1 target.
IMPROVING EXISTING DRUGS
Oxazolidinones
The oxazolidinones are one of a few structurally new classes of antibiotics to be approved in the last 30 years. Currently there is only one oxazolidinone licensed by the US FDA; linezolid, approved in 2000, for the treatment of Gram-positive infections. Many compounds in the class have been evaluated since oxazolidinones were first patented in 1978,58 the success of linezolid has stimulated the development of newer members of the class, with
206 New Developments in Drug Treatment
the aim of improving the safety profile or extending the antibacterial profile.58 These include three new agents with promising antiTB activity that have completed at least phase I trials and remain under development; sutezolid (PNU-100480), delpazolid (LCB01– 100480) and posizolid (AZD5847).
Linezolid has been recommended for the treatment of all cases of drug-resistant TB,7 although it is not yet licensed for TB treatment. Its mechanism of action is common to all oxazolidinones but unique amongst anti-TB drugs, ensuring no cross-resistance. It inhibits protein synthesis by binding to 23 s rRNA in the 50 s ribosomal subunit, which is also the site for drug resistance conferring mutations, although other loci might be involved and spontaneous mutations are thought to occur less frequently than for other drugs. Linezolid has high oral bioavailability and an MIC for M. tuberculosis of 0.125–0.5 µg/mL.59 Its activity evaluated in mouse studies is modest.60 At 100 mg/kg once daily (a dose equivalent to 600 mg orally in humans) it appeared to be bacteriostatic or weakly bactericidal, causing an approximately 1–1.5 log reduction in bacterial counts over 28 days.61
Nonetheless, linezolid has clear clinical efficacy and has become an important anti-tuberculous drug, particularly in the treatment of drug-resistant TB.62–65 In a recent individual patient data meta-analysis of 12 030 patients with drug-resistant TB, which led directly to the prioritization of linezolid for drugresistant TB treatment, linezolid was strongly associated with improved outcomes and reduced mortality.23 However, the use of linezolid for TB treatment is limited by toxicity, principally high rates of myelosuppression and neuropathies.65,24 Toxicity is thought to be cumulative and side effects are reduced at lower doses, hence doses lower than the licensed 600 mg BD dose are used in many settings, but the implications of these strategies on efficacy and avoidance of the development of resistance are not well characterized.
Results from the open-label Nix-TB trial; combining linezolid with bedaquiline and pretomanid, for patients with dif- ficult-to-treat MDR TB and XDR-TB, demonstrated cure by 6 months in almost all surviving patients, with very low rates of relapse 6 months post treatment completion.24 The Zenix trial now aims to identify the optimal dose and duration of linezolid within the Nix-TB regimen (NCT03086486). Linezolid also now forms part of treatment shortening trial regimens for drug-sen- sitive TB (NCT03474198), and drug-resistant TB (NCT02754765, NCT02454205, NCT02589782).
Sutezolid is structurally highly similar to linezolid, differing only by a single sulfur atom, but has superior anti-TB activity.60 Its MIC is approximately 2−4-fold lower than linezolid for a range of clinical isolates,67 and in the mouse model it is bactericidal and more active than linezolid.60,68 Combined with first-line therapy it was able to accelerate the time to culture conversion in the lungs of mice, confirming that it has sterilizing activity.69 Combining sutezolid with bedaquiline, clofazimine, and pretomanid in a murine model resulted in a regimen that was highly active and achieved low relapse rates after only 3 months of therapy.15 There was no evidence of antagonism between these agents and this suggests an oxazolidinone could become a key part of a new universal regimen. Sutezolid has now demonstrated EBA over the course of 14 days in humans, a result that was not previously seen with
linezolid and appears to confirm the improved antimycobacterial activity of sutezolid over linezolid. Rates of mitochondrial inhibition are likely to be lower with sutezolid and this is reflected in lower rates of clinical toxicity in limited duration human studies, however, these results need to be replicated over longer treatment periods.70,71 A phase IIb dose-finding study is now planned (NCT03959566).
Delpazolid (LCB01-0371) appears more potent than linezolid for a range of Gram-positive infections72 and thus is an attractive candidate for assessment of its potential to treat TB. It has demonstrated activity against clinical drug-resistant Mycobacterium tuberculosis isolates in vitro, albeit with a 2-fold higher MIC90 than linezolid.73 The drug has now gone forward for testing in an EBA study, interim results of which suggest that the drug is well tolerated, with modest EBA.74–76 Posizolid (AZD5847) appears to have a similar or higher range of Mycobacterium tuberculosis MICs to delpazolid, but improved extra-, and particularly, intracellular killing of Mycobacterium tuberculosis compared to linezolid.77,78 In a murine chronic infection model, posizolid activity was intermediate between linezolid and sutezolid.79 However, in a murine model of non-replicating TB, posizolid activity was minimal compared to linezolid and sutezolid,80 This observation, combined with the relatively high MIC and less favorable pharmacokinetics of posizolid compared to sutezolid and linezolid,81 may explain the modest activity, which was inferior to that previously described for linezolid, in a recent EBA study.82 It is unlikely that posizolid will advance further.
Nitroimidazoles
The development of nitroimidazoles to the point where there are two currently in clinical use and clinical trials; delamanid (OPC67683)83 and pretomanid (PA-824),84 is an example of drug development by chemical modification of an existing structure.85,86 The first clinically active nitroimidazole was metronidazole (MTZ) discovered in the mid-1950s in a screen for drugs active against trichominiasis. MTZ was subsequently found to be effective therapy against a wide range of anaerobes. Since M. tuberculosis anaerobically adapts to survive in hypoxic granulomas,87 it was rational to try and engineer nitroimidazoles to be more active against mycobacteria. MTZ itself has a maximal effect on M. tuberculosis under anaerobic conditions in vitro,88 but it exhibits inconsistent activity in animal models of TB.87,89–91 Some early attempts to develop a new generation of nitroimidazoles were thwarted by problems of mutagenesis.92 But two independent initiatives succeeded by sidechain modification of the nitroimidazole structure, resulting in a nitroimidazo-oxazine (pretomanid) and a nitroimidazo-oxazole (delamanid). Both of these compounds have good activity against M. tuberculosis grown aerobically and anaerobically, and are active in the mouse and guinea-pig models of TB when given as monotherapy or in combination with other anti-TB drugs.
Pretomanid and delamanid are both pro-drugs, which require nitroreductive activation. The mechanisms of activation, action and resistance are very similar for both compounds. Activation for both drugs is by F(420)-deazaflavin-dependent nitroreductase (Ddn),98,99 and in the case of pretomanid the active des-nitro- imidazole has been shown to act by generating reactive nitrogen
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Improving existing drugs 207
species such as nitric oxide (NO).100 In addition there appears to be a mode of action against mycolic acid synthesis,84 which probably accounts for the aerobic activity of the drug and has also been seen for delamanid.83 Spontaneous resistance to pretomanid occurs at a relatively high frequency, similar to that of isoniazid (approximately 1 × 10−6), in vitro. Mechanisms of resistance have not yet been fully studied in clinical settings.101 But mutations in fgd1 (a glucose-6-phosphate dehydrogenase), fbiA, fbiB, and fbiC (cofactor biosynthesis proteins), genes required for cofactor F(420) synthesis needed for a functional nitroreductase as well as in ddn that encodes the nitroreductase itself have all been documented in pretomanidand delamanid-resistant strains102 selected for in vitro.103,104 However, there is some evidence that cross-resistance between nitroimidazoles may not be complete.105 The number of potential drug-resistant conferring genetic loci for pretomanid may partially explain the high spontaneous mutation rate. Thus far, mutations in fbiA and fgd1 have been identified in phenotypically delamanid-resistant clinical isolates.106,107
Both pretomanid and delamanid are in clinical development, but currently under different trajectories. Otsuka (https://www. otsuka.co.jp/en/), has focused on evaluating delamanid as a drug for MDR-TB, in contrast to the Global Alliance for Tuberculosis Drug Development (http://www.tballiance.org/), which aims to position pretomanid in an optimized regimen for drug susceptible and now, drug-resistant TB.
Two EBA studies have been completed for delamanid, only one of which has been published to date.108 It was a 14-day dose ranging study of 100–400 mg given as monotherapy and demonstrated drug activity. The mean fall in bacterial load was less than that seen for rifampicin or isoniazid in a reference study.109
The results of a phase IIb study in MDR-TB patients has shown that this modest activity in terms of EBA can translate into significant benefit in terms of culture conversion.110 In a randomized, multinational clinical trial, 481 patients with pulmonary MDR-TB received either delamanid (at 100 mg or 200 mg twice daily) or placebo for 2 months in combination with a WHO-approved background MDR-TB regimen. The proportion of patients with sputum culture conversion at 2 months was significantly increased in the patients receiving delamanid (41.9% in the 200 mg group in contrast to 29.6% in the placebo group). A proportion of the patients were rolled over to receive a further 6 months of openlabel delamanid. Overall, favorable outcomes (a combination of cure and completed treatment) were significantly increased in patients in the long-term (≥6 months) treatment group (74.5%), as compared to those in the short-term (≤2 months) delamanid treatment group (55%). On this basis, the European Medicines Agency (EMA) recommended conditional marketing authorization for delamanid in adults with MDR-TB without other treatment options. Results for the long-awaited phase III, randomized, placebo-controlled trial of delamanid for MDR TB have recently been released.111 Five hundred and eleven participants were randomized in a 2:1 ratio to either delamanid or placebo in addition to an optimized background regimen. The primary outcome was time to sputum culture conversion over 6 months and hence, only participants who were culture positive at baseline (64%) were assessed in the modified intention-to-treat analysis, leading to imbalances between the delamanid and placebo groups, with
higher rates of bilateral cavitation and resistance beyond MDR in the delamanid group. Even accounting for this, the results were disappointing, no difference in time to stable culture conversion and 87.6 versus 86.1% culture conversion at 6 months in the delamanid and placebo arms, respectively (RR 1.017; 95% CI 0.927–1.115). There were no differences in rates of clinical success or mortality. Whilst these results suggest that delamanid-based regimens will not allow treatment-shortening for MDR TB, it will likely retain a role when resistance or intolerance to other agents precludes building an adequate regimen.107 Delamanid is known to cause QTc prolongation and hence a key question for its future use has become whether it can safely be used in combination with bedaquiline in order to increase the options for treating MDR TB. A recent phase II trial randomized patients with MDR TB to receive delamanid, bedaquiline, or both as part of their regimen for the first 24 weeks and showed that QTc prolongation from baseline was modest in all arms and was no more than additive when bedaquiline and delamanid were used together. This provides reassurance that these drugs could safely be used together.112
In contrast to delamanid, pretomanid has been extensively evaluated in murine models of TB to determine which are its optimal companion drugs. Initial studies demonstrated it had dose-dependent bactericidal activity during both the initial phase and continuation phases of therapy equivalent to rifampicin and isoniazid113 and could successfully replace isoniazid in combination therapy without showing potential to shorten therapy.114 However, a series of subsequent studies has suggested pretomanid in various combinations, such as with moxifloxacin−pyrazinamide (PaMZ),115 moxifloxacin−pyrazinamide−bedaquiline (BPaMZ),116 bedaquiline−pyrazinamide (BPaZ),117 bedaquiline− linezolid (BPaL)118 or bedaquiline−sutezolid15 could shorten therapy. The latter combination is particularly attractive as it contains no existing firstor second-line drugs, and therefore represents a universal therapy for all forms of susceptible and drug-resistant TB. More recent work has suggested that the addition of pretomanid to the BPaL or BPaMZ regimens independently increased bactericidal activity, prevented emergence of bedaquiline resistance and shortened the duration of treatment needed for relapse-free cure. This suggests that pretomanid may be central to the apparent utility of these regimens.119
Experience with rifapentine and fluoroquinolones (described later) suggests that enhanced sterilizing activity seen in the mouse model does not always translate clinically and studies have been initiated to evaluate pretomanid in clinical trials. Two EBA studies with dose ranging pretomanid monotherapy have showed bactericidal activity reaches a plateau above 200 mg daily.120,121 Using this dose of pretomanid in combination with pyrazinamide and moxifloxacin (PaMZ), it was possible to achieve bactericidal activity over 14 days, although not significantly so given the small numbers of patients.122 Similarly, in an 8-week phase IIb study of this regimen in participants with both drug-sensitive and MDR TB, bactericidal activity was superior in those with drug-sensitive TB and at least as good in those with MDR TB compared to the RHZE control regimen in drug-sensitive TB patients.123 The BPaZ and BPaZM regimens were tested amongst patients with drugsensitive and drug-resistant TB, respectively, in an EBA study (NCT02193776).124 Validating the murine studies, BPaMZ led to
208 New Developments in Drug Treatment
significantly faster mycobacterial clearance than BPaZ and both were superior to the standard regimen. This led to a 8-week phase IIb study, again of BPaZ (with either standard or daily dosing of bedaquiline) in drug-sensitive and BPaZM in drug-resistant TB. Mycobacterial clearance was significantly faster in all investigational arms compared to standard treatment, although higher rates of discontinuation were noted in these arms.125 The PaMZ regimen was utilized in a phase III treatment shortening trial (NCT02342886) for drug-sensitive and MDR TB. However, this trial had to be stopped early as a result of an unexpectedly high rate of hepatotoxicity. The BPaMZ regimen is also currently being tested in a phase III treatment shortening trial (NCT03338621). Both of these trials aim to reduce drug-sensitive and MDR TB treatment to 4 and 6 months, respectively. The BPaL regimen was assessed in the Nix TB study (see earlier) and this has now led to FDA approval for pretomanid,24 but only as part of this regimen for XDR TB and difficult to treat MDR TB.126
REUSING OLD DRUGS
Fluoroquinolones
Quinolones are synthetic antibiotics originally identified as a by-product of chloroquine synthesis.127 Since the introduction of nalixidic acid for the treatment of urinary tract infections in 1967, quinolones have been extensively developed to improve their pharmacokinetics, and broaden their spectrum of activity.128 Lately this focused on developing fluoroquinolones for the treatment of respiratory tract infections caused by Streptococcus pneumoniae and other pathogens. Some of these fluoroquinolones were found to have good activity against M. tuberculosis and are established as one of the most important drugs in regimens to treat MDR-TB, being consistently and strongly associated with successful treatment outcomes in meta-analyses.8,23 They now form a core part of WHO-recommended MDR-TB treatment regimens.7 Their excellent oral bioavailability and bactericidal action, lack of crossresistance with existing TB drugs and favorable safety profile has also resulted in them being evaluated in the treatment of drugsusceptible TB.
The 8-methoxy-fluoroquinolones, gatifloxacin and moxifloxacin, are two quinolones with the most potent anti-TB activity and were evaluated for their ability to shorten drug-sensitive TB treatment from 6 to 4 months. Compared to the earlier fluoroquinolones such as ciprofloxacin and ofloxacin, gatifloxacin and moxifloxacin have lower MICs129 and superior activity against non-replicating M. tuberculosis in vitro,130 although gatifloxacin was withdrawn from the market in 2008 as it was associated with a high rate of dysglycemic events.131 In addition, at clinically recommended doses they have better bioavailability132 resulting in superior pharmacodynamic parameters such as AUC24/MIC90 ratio. This prompted researchers to evaluate whether a fluoroquinolone, such as moxifloxacin, substituted or added to first-line treatment regimens, could shorten therapy by 2 months. Despite impressive results from long-term experiments in the murine model,133,134 this did not translate to humans in three large phase III clinical trials.135–137 Although the fluoroquinolone-containing regimens
reported more culture negative results at 2 months, they had higher rates of unfavorable outcome at 18 months largely due to a higher relapse rate. Fluoroquinolones therefore appear not to have sufficient activity against persister bacteria to reduce treatment to less than 6 months and are now only being evaluated as part of treatment regimens for TB resistant to first-line drugs. Current work is now focusing on whether stratification of patients from these trials is able to identify an easy-to-treat phenotype that may be amenable to a 4-month regimen.3
The use of fluoroquinolones could be compromised in the future by the emergence of drug resistance. The fluoroquinolones inhibit bacterial topoisomerases, enzymes that regulate DNA coiling. In M. tuberculosis, their drug target is DNA gyrase, encoded by gyrA and gyrB, and resistance conferring mutations often occur in these two genes,138 although drug efflux is also a mechanism.139 The widespread use of fluoroquinolones to treat respiratory infections means patients with undiagnosed TB are being treated with fluoroquinolone monotherapy which can select for drug resistance even after limited drug exposure,140 which was reported soon after the introduction of fluoroquinolones for TB.141 Among 40 countries with a high TB or MDR-TB burden, fluoroquinolone resistance was detected in 20% of isolates tested.142 This underlines the importance of developing mycobacterial specific drugs. 8-Methoxy-fluoroquinolones are excellent TB drugs and form a core part of current MDR-TB treatment and are under evaluation in a variety of other combinations (see New Regimens section).
Rifamycins
The use of various rifamycins is currently being reassessed to determine their optimal usage. All rifamycins target the beta subunit of RNA polymerase thereby preventing transcription, and the principal mechanism of resistance is through mutations in rpoB that encodes the polymerase. Unlike cell wall inhibitors whose mode of action is dependent on cell division, transcriptional inhibitors should be active even in quiescent states as some degree of transcription will be required for cell viability. It is this activity that is thought to underpin the sterilizing activity that effectively resulted in the shortening of TB therapy from 18 to 9 months with the introduction of rifampicin. Interest has therefore focused on trying to extract the maximal activity out of rifamycins.
In the case of rifampicin, the currently used maximal dose of 600 mg/10 mg/kg per day was introduced for safety and cost reasons and is not at the limit of the dose-response curve.143 In murine models, higher dose rifampicin regimens demonstrate dose-dependent increases in bactericidal activity and clearance of putative persister organisms, allowing treatment shortening and the prevention of relapse after treatment.144,145 Several clinical studies have taken these observations forward in an attempt to identify an optimized rifampicin dose that increases efficacy whilst minimizing any additional toxicity and hence may lead to treatment shortening. HIGHRIF 1 compared safety and EBA up to 14 days with doses of rifampicin up to 35 mg/kg and demonstrated no difference in toxicity but improved bactericidal activity at higher doses.146 HIGHRIF 2 went on to assess 600, 900 and 1200 mg daily dosing alongside standard doses of other first-line drugs during the first 2 months of therapy in a double-blind,
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Reusing old drugs 209
randomized, placebo-controlled, phase II clinical trial. Again, this study demonstrated that higher doses of rifampicin are safe but without any statistically significant difference in several measures of antimycobacterial activity between the arms.147 Similarly, the RIFATOX trial was a non-blinded, randomized, phase II trial comparing 10, 15 and 20 mg/kg rifampicin in combination with standard-dose first-line drugs during the first 4 months of treatment, with no increase in adverse events or efficacy as defined by 2-month culture conversion.148 The HRIF trial was a randomized phase II trial of doses of 10, 15 and 20 mg/kg rifampicin alongside standard first-line therapy over the course of the first 8 weeks of treatment. A statistically significant improvement in CFU count decline was seen at the 20 mg/kg dose.149 This is the only one of these studies to examine the effect of rifampicin exposure rather than just dose (defined by area under the time–concentration curve [AUC]) on mycobacterial clearance and identified a faster elimination rate with increased rifampicin exposure in the perprotocol analysis. In the MAMS-TB-01 trial, 20 and 35 mg/kg of rifampicin were combined with various first-line drugs, moxifloxacin and SQ109. There was no difference in adverse events between the arms and only the arm with 35 mg/kg rifampicin had accelerated culture conversion (median 48 vs. 62 days, p = 0.003) compared to standard therapy, although this was only seen for liquid and not solid media.46 Taken together, these results confirm that higher doses of rifampicin, up to 35 mg/kg, are safe, but without clear evidence that this dose of rifampicin will enable treatment shortening. It has been suggested that this dose is still too low and there is evidence that the top of the exposure−response curve has not been obtained at these doses; indeed the lowest dose to achieve relapse-free, shortened treatment in the murine model was 80 mg/kg.145 To this end, doses of 40 and 50 mg/kg have been recently investigated, with initial reports that the 50 mg/kg dose is associated with unacceptable rates of adverse events, suggesting that 40 mg/kg may be the maximum tolerated dose.150 Doses of 1200 and 1800 mg of rifampicin are currently being tested as part of an open-label phase III trial aiming to shorten treatment to 4 months (NCT02581527). Higher rifampicin doses may also have a particular role in special situations, for instance, TB meningitis where optimization of CSF exposure may lead to mortality benefits.151 This was not demonstrated in a recent randomizedcontrolled trial, although again the rifampicin dose (15 mg/kg) may have been too low to show benefit.152,153 A potential limitation of this strategy, yet unstudied in detail, are the management of drug−drug interactions (DDIs) that result from cytochrome P450 induction and would likely be increasingly problematic at higher doses of rifampicin.
An alternative approach to increasing rifamycin exposure is through the use of rifapentine, which has a longer half-life of 14–18 hours as well as a lower TB MIC154 generating a better AUC24/MIC90 ratio. Evaluation of rifapentine treatment in combination with moxifloxacin in the murine model suggested this combination given either intermittently or daily could be used to shorten therapy.155,156 However, the phase III RIFAQUIN study combining twice weekly rifapentine at standard dosing and moxifloxacin for 4 months did not achieve adequate cure rates to allow treatment shortening. Daily dosing of rifapentine has also been assessed. A head to head comparison of daily rifapentine
and rifampicin, both at 10 mg/kg showed no difference in terms of culture conversion at 2 months,157 Daily 7.5 mg/kg rifapentine and moxifloxacin (replacing rifampicin and ethambutol, respectively) reduced time to stable culture conversion over 8 weeks in liquid (but not solid) media but not rates of culture conversion, compared to standard therapy.158 Interestingly, in this study moxifloxacin exposures were much lower than those seen in studies combining intermittent rather than daily rifapentine with moxifloxacin and like rifampicin, studies with rifapentine suffer the same limitations in terms of DDIs. Both of these studies of daily rifapentine were initiated on the basis of promising results in mouse models, which failed to translate to findings in humans.155,156,159 The TBTC study 31 is ongoing and will also test rifapentine with or without moxifloxacin at the higher dose of 1200 mg for the duration of treatment in an attempt to shorten treatment length to 4 months (NCT02410772). In addition to the potential for treatment shortening, these studies are important because if successful they would provide a strong rationale for developing a newer generation of potent and tolerable RpoB inhibitors.
β-Lactams
β-Lactam antibiotics form a large bactericidal class, that include penicillins, cephalosporins, monobactams, and carbapenems, whose mode of action is inhibition of transpeptidases which are required for crosslinking (transpeptidation) of the peptidoglycan layer. They have been of little relevance to mycobacterial therapeutics because M. tuberculosis has a single, highly active class A β-lactamase, BlaC. This enzyme hydrolyzes a broad spectrum of β-lactams; conferring resistance. Genetically knocking out blaC that encodes the β-lactamase,160 or chemically inhibiting BlaC with the β-lactam inhibitor clavulinic acid does reduce the MIC, but in the case of amoxicillin and ampicillin the MIC remains relatively high in reference strains.160 For this reason the combination of amoxicillin with clavulinic acid is not classified as a TB drug by WHO, being of questionable benefit.7
Carbapenem β-lactam antibiotics, imipenem, ertapenem and meropenem, in combination with the β-lactamase inhibitor clavulinic acid offer a better alternative. Carbapenems bind highmolecular weight penicillin-binding proteins, inactivating the l,d-transpeptidases that form cell wall crosslinks161 and are less susceptible to BlaC. For example, the MIC for meropenem−clavulinic acid has been reported in a range from 0.32 to 1.28 µg/mL for drug-susceptible reference strains of M. tuberculosis.162,163 There have been some favorable clinical reports with carbapenems164 but the results from mouse studies have been equivocal with the meropenem−clavulinic acid combination.165,166 A fall in bacterial counts in treated mice was only seen in one of the studies where the carbapenem was given at a dose of 300 mg/kg. Recently, the currently available formulation of ceftazidime, an older cephalosporin, in combination with avibactam, a potent inhibitor of BlaC, was identified to have anti-mycobacterial activity in an in vitro screen which was followed by further evaluation in the hollow fiber system. It demonstrated good sterilization of drug-resis- tant isolates at clinically achievable concentrations and may offer another option where alternatives are limited.167