Spectinamides are effective partner agents for the treatment of tuberculosis in multiple mouse infection models
Objectives: New drug regimens employing combinations of existing and experimental antimicrobial agents are needed to shorten treatment of tuberculosis (TB) in humans. The spectinamides are narrow-spectrum semisynthetic analogues of spectinomycin, modified to avoid intrinsic efflux by Mycobacterium tuberculosis. Spectinamides, including lead 1599, have been previously shown to exhibit a promising therapeutic profile in mice as single agents. Here we explore the in vivo activity of lead spectinamides when combined with other agents. Methods: The efficacy of 1599 or 1810 was tested in combination in three increasingly advanced TB mouse models. Mice were infected by aerosol and allowed to establish acute or chronic infection, followed by treatment (≤4 weeks) with the spectinamides alone or in two- and three-drug combination regimens with existing and novel therapeutic agents. Bacteria were enumerated from lungs by plating for cfu. Results: Herein we show the following: (i) 1599 exhibits additive or synergistic activity with most of the first-line agents; (ii) 1599 in combination with rifampicin and pyrazinamide or with bedaquiline and pyrazinamide promotes significantly improved efficacy in the high-dose aerosol model; (iii) 1599 enhances efficacy of rifampicin or pyrazinamide in chronically infected BALB/c mice; and (iv) 1599 is synergistic when administered in combination with rifampicin and pyrazinamide in the C3HeB/FeJ mouse model showing caseous necrotic pulmonary lesions. Conclusions: Spectinamides were effective partner agents for multiple anti-TB agents including bedaquiline, rifampicin and pyrazinamide. None of these in vivo synergistic interactions was predicted from in vitro MIC chequerboard assays. These data support further development of the spectinamides as combination partners with existing and experimental anti-TB agents.
Introduction<!>Bacteria<!>Drug compounds<!>Drug preparation for in vivo models<!>Ethics<!>Infection of mice<!>Drug delivery and dose<!>BALB/c mouse high-dose aerosol infection model<!>BALB/c mouse chronic infection model<!>C3HeB/FeJ mouse chronic infection model<!>Histological analysis<!>Enumeration of cfu from lungs<!>Statistical analysis<!>In vitro drug combination chequerboard assays<!><!>In vitro assessment of synergy<!>Discussion<!>
<p>New drug regimens based on combinations of existing and experimental classes of antimicrobial agents are desperately needed to shorten and simplify treatment of TB infection caused by MDR or XDR Mycobacterium tuberculosis. Current TB research has therefore focused on the discovery and development of novel agents that complement and extend the efficacy of both first- and second-line TB agents, as well as TB agents under clinical development.1,2</p><p>The spectinamides are novel, semisynthetic antibiotics with a narrow spectrum of activity against M. tuberculosis and closely related mycobacterial species, lack cross-resistance to existing TB drugs and are active under hypoxic conditions.3 The molecular basis for this improved activity has been attributed to selective ribosomal inhibition, combined with synthetic chemical modifications that allowed evasion of the Rv1258c efflux pump. Efflux by Rv1258c is responsible for the poor intrinsic activity of the parent compound, spectinomycin (Figure S1, available as Supplementary data at JAC Online).3 Lead spectinamides exhibit a long post-antibiotic effect and a short serum half-life owing to extensive renal elimination, and are efficacious in both acute and chronic mouse models of TB.3 Poor oral bioavailability in this series has limited its usage to an injectable form so far; however, owing to their hydrophilic nature, the spectinamides have proven highly effective when administered via inhalation in mouse models.3 We recently explored the potential in vivo benefit for combination of the spectinamides with antibiotics not typically used to treat TB. In this published study, 1599 did not act as a general efflux pump inhibitor and synergy was only observed with non-conventional TB agents that also inhibited protein synthesis.4 Building from these studies, herein we investigated the efficacy of 1599 in combination with conventional anti-TB agents, including agents under late-stage clinical development. Synergistic interactions were assessed by conventional in vitro chequerboard assays and also through a series of short-term (≤4 weeks) acute and chronic murine infection models of TB, including C3HeB/FeJ mice exhibiting advanced, hypoxic, caseating granulomas.5,6 The resulting data revealed appreciable in vivo synergy between lead spectinamides and existing therapeutics, as well as compounds in late-stage clinical development that were not otherwise predicted by in vitro MIC chequerboard testing. Further development of this series and exploration of the mechanisms of synergy observed in these studies, using longer treatment durations in hard-to-treat caseous necrotic pulmonary lesions, may lead to new treatment options for human TB.</p><!><p>M. tuberculosis H37Rv was used for in vitro chequerboard studies. M. tuberculosis Erdman strain7 or Erdman transformed with pFCA-LuxAB8 (Erdman-Lux) was used for drug evaluations in mice. The luciferase signal expressed from Erdman-Lux provided an early estimate of bacterial burden in lung and spleen homogenates of mice, with no appreciable decrease in virulence in conventional mouse infection models of TB. Excluding vector-encoded kanamycin resistance, Erdman and Erdman-Lux have shown equivalent susceptibility to spectinamides and standard TB drugs in vitro. Bacteria were prepared for infection as described previously.5</p><!><p>1599 and 1810 were synthesized as described (Figure S1).3 Isoniazid, rifampicin, pyrazinamide, ethambutol, levofloxacin, kanamycin, amikacin, ethionamide, ofloxacin, cycloserine, rifapentine and clofazimine were purchased from Sigma-Aldrich. Linezolid was purchased from 21CEC. Moxifloxacin was obtained from Bayer. PA-824 (Pretomanid) was obtained from the Global Alliance for TB Drug Development. Bedaquiline (Sirturo) was provided by Johnson and Johnson. SQ109 was synthesized as described.9</p><!><p>Spectinamides were formulated in Plasma-Lyte A (pH 7.4) diluted 5:1 with sterile water. Rifampicin, isoniazid, pyrazinamide, ethambutol, levofloxacin and moxifloxacin were ground by mortar and pestle and dissolved in sterile water. Linezolid was dissolved in 5% (w/v) PEG-200 (Sigma-Aldrich) followed by a 20-fold dilution with 0.5% (w/v) methyl cellulose (Fisher Scientific). PA-824 was formulated in 10% (w/v) 2-hydroxypropyl-β-cyclodextrin (2-HPCD) (Sigma-Aldrich) and 10% (w/v) lecithin (Sigma-Aldrich) as described.7 Bedaquiline was dissolved in 20% (w/v) 2-HPCD and formulated as described previously.10,11</p><!><p>All animal studies were performed at Colorado State University in a certified bio-safety level III animal facility in accordance with the guidelines of the Colorado State University Institutional Animal Care and Use Committee (reference number 13-4477A).</p><!><p>Aerosol infection of mice with M. tuberculosis Erdman or Erdman-Lux employed a Glas-Col inhalation exposure system as described.12</p><!><p>1599 or 1810 was administered in a 0.2 mL volume by subcutaneous injection at 200 mg/kg. Isoniazid, rifampicin, pyrazinamide, ethambutol, linezolid, levofloxacin, moxifloxacin, PA-824 and bedaquiline were administered by oral gavage in a 0.2 mL volume at 25, 10, 150, 100, 100, 100, 100, 50 and 25 mg/kg, respectively. For combination studies, mice were first administered 1599 or 1810 by subcutaneous injection followed by administration of the combination agent(s) by oral gavage. In cases where mice were administered two or more oral drugs, these were combined to limit the total volume of oral administration to 0.2 mL. All treatments were given once daily, 5 days a week (Monday to Friday).</p><!><p>Female pathogen-free BALB/c mice (6–8 weeks old; Charles River) were exposed to high-dose aerosol of M. tuberculosis Erdman or Erdman-Lux from broth culture (optical density at 600 nm of ∼0.8) to achieve deposition of ∼3.5–4.7 log10 cfu in the lungs of each mouse.13,14 Treatment was initiated on day 12 post-aerosol and continued for 3–4 weeks. Groups of five or six mice were sacrificed on day 12, prior to treatment initiation, and 3 days following the last day of treatment, to determine the bacterial loads in the lungs of mice. Mice treated with bedaquiline were sacrificed 5 days following the last day of treatment to allow drug clearance.</p><!><p>Female pathogen-free BALB/c mice (6–8 weeks old; Charles River) were exposed to a low-dose aerosol of M. tuberculosis Erdman-Lux using 2 × 106 cfu/mL to achieve 50–100 cfu in the lungs of each mouse. Treatment was initiated on day 41 post-aerosol and continued for 4 weeks. Groups of three to six mice were sacrificed on day 41, prior to treatment initiation, and 3 days following the last day of treatment, to determine the bacterial loads in the lungs of mice.</p><!><p>Female C3HeB/FeJ mice (6–8 weeks old; Jackson Laboratories) were exposed to a low-dose aerosol of M. tuberculosis Erdman using 1.5 × 106 cfu/mL to achieve ∼50–75 cfu in the lungs of each mouse.5 Treatment was initiated on day 56 at the time when necrotic lesions had already developed6 and continued for 4 weeks. Groups of four or seven mice were sacrificed on day 56, prior to treatment initiation, and 3 days following the last day of treatment to determine the bacterial loads in the lungs of mice.</p><!><p>Lung tissues were collected and processed as described.6,15</p><!><p>The number of viable organisms was determined by serial dilutions of homogenates (Glas-Col, Inc.) from whole lungs (C3HeB/FeJ) or left lung lobes (BALB/c) plated on 7H11-OADC. Lungs from bedaquiline-treated animals were homogenized in phosphate-buffered saline plus 10% (w/v) bovine serum albumin and plated on 7H11-OADC agar plates containing 0.4% (w/v) activated charcoal to prevent drug carry-over.16 Colonies were enumerated after at least 21 days of incubation at 37 °C. In vivo synergy, additivity or antagonism when evaluating the combined effects of two or more drugs was defined as follows: greater than the combined efficacies of each drug alone, synergy; no greater than the combined efficacies of each drug alone, additivity; and less than the efficacies of each drug alone, antagonism.</p><!><p>Data were evaluated by a one-way analysis of variance followed by a multiple-comparison analysis of variance by a one-way Tukey test (SAS software program). Differences were considered significant at the 95% level of confidence.</p><!><p>In vitro synergy was assessed using M. tuberculosis strain H37Rv, which was cultured as described previously.17 Fractional inhibitory concentration index (FICI) scores were calculated as described.18 FICIs were reported as the range of two independent experiments and were interpreted as follows: synergy, ≤0.5; indifference, >0.5–4.0; antagonism, >4.0.18</p><!><p>Evaluation of 1599 in combination with novel anti-TB agents under development in BALB/c mice following high-dose aerosol infection with M. tuberculosis Erdman-Lux. Bars represent mean log10 cfu values ±SEM from the lungs of individual BALB/c mice. Mice were treated for 3 weeks with the following drugs, alone or in various combinations: 1599 at 200 mg/kg by subcutaneous injection or bedaquiline at 25 mg/kg and/or PA-824 at 50 mg/kg by oral gavage. Isoniazid at 25 mg/kg, rifampicin at 10 mg/kg and pyrazinamide at 150 mg/kg were given by oral gavage. Initiation of treatment controls are the number of log10 cfu in lungs at the start of treatment, 12 days post-aerosol infection. BDQ, bedaquiline; INH, isoniazid; PZA, pyrazinamide; RIF, rifampicin; NS, not significant; ***P < 0.001.</p><p>Combination of 1599 with second-line and novel drugs under development in BALB/c mice following high-dose aerosol infection with M. tuberculosis Erdman-Lux</p><p>NA, not applicable; ND, not done.</p><p>D1, D12 and D33 are 1, 12 and 33 days post-aerosol infection.</p><p>Drug treatment started on day 12.</p><p>Drug dose in milligrams per kilogram of body weight.</p><p>Drug treatments Monday to Friday.</p><p>1599 combination studies with rifampicin and pyrazinamide in BALB/c mice following high-dose aerosol infection with M. tuberculosis Erdman. Bars represent mean log10 cfu values ±SEM from the lungs of individual BALB/c mice. Mice were treated for 4 weeks with the following drugs, alone or in various combinations: 1599 at 200 mg/kg by subcutaneous injection, or rifampicin at 10 mg/kg, and pyrazinamide at 150 mg/kg by oral gavage. Initiation of treatment controls are the number of log10 cfu in lungs at the start of treatment, 12 days post-aerosol infection. PZA, pyrazinamide; RIF, rifampicin; *P < 0.05; ***P < 0.001.</p><p>Combination of spectinamides with first-line antitubercular agents in BALB/c mice following high-dose aerosol infection with M. tuberculosis Erdman</p><p>NA, not applicable.</p><p>D1, D12 and D40 are 1, 12 and 40 days post-aerosol infection.</p><p>Drug treatment started on day 12.</p><p>Drug dose in milligrams per kilogram of body weight.</p><p>Drug treatments Monday to Friday.</p><p>1599 combination studies with rifampicin, pyrazinamide or levofloxacin in BALB/c mice chronically infected with M. tuberculosis Erdman-Lux. Mice were treated for 4 weeks with the following drugs, alone or in various combinations: 1599 at 200 mg/kg by subcutaneous injection, rifampicin at 10 mg/kg, pyrazinamide at 150 mg/kg or levofloxacin at 100 mg/kg by oral gavage. The vehicle-only group received Plasma-Lyte by subcutaneous injection. LVX, levofloxacin; PZA, pyrazinamide; RIF, rifampicin; NS, not significant; ***P < 0.001.</p><p>1599 and 1810 combination studies with rifampicin and pyrazinamide in C3HeB/FeJ mice experimentally infected with M. tuberculosis Erdman. Plots represent the log10 cfu values from the lungs of individual mice. The mean log10 cfu value is represented by a horizontal line. The broken line represents the mean log10 cfu value for the untreated control. Fifty-six days following low-dose aerosol infection, mice were treated for 4 weeks with the following drugs, alone or in various combinations: 1599 or 1810 at 200 mg/kg by subcutaneous injection, or rifampicin at 10 mg/kg, and pyrazinamide at 150 mg/kg by oral gavage. The untreated control group received no treatment. PZA, pyrazinamide; RIF, rifampicin.</p><!><p>An interesting result of this study was the lack of correlation between the in vitro synergy and the in vivo synergy. It would be a rational expectation that the in vivo synergy observed when 1599 was paired with rifampicin, pyrazinamide or bedaquiline would be reflective of the in vitro mode of action of each agent. However, when tested in conventional MIC in vitro chequerboard assays, 1599 in combination with rifampicin, pyrazinamide or bedaquiline did not reveal any specific synergies, although none of these interactions was antagonistic either (Table S4). Some synergy (FICI 0.13–0.25) was observed when 1599 was paired with clofazimine or rifapentine in vitro (Table S4); however, these agents have yet to be evaluated for activity in vivo when combined with a spectinamide.</p><!><p>In the studies presented here, we report the in vivo benefit of combining 1599 with existing and novel anti-TB therapies in multiple short-course murine infection models as a means to assess potential mechanistic synergy partners. We demonstrate: (i) mild antagonism with isoniazid, a drug that has been reported before to be weakly antagonistic with rifampicin and pyrazinamide in certain mouse models,28,29 but no interference or adverse effects when 1599 is administered in combination with other agents tested here; (ii) 1599 in combination with rifampicin and/or pyrazinamide was additive or synergistic in three mouse models evaluated (the acute and chronic BALB/c as well as the C3HeB/FeJ model); and (iii) the most effective combination tested was the three-drug regimen combining 1599 with bedaquiline/pyrazinamide. Importantly, none of these synergistic interactions was predicted from in vitro MIC chequerboard assays, suggesting that current in vitro methodologies cannot replace conventional in vivo drug combination studies for TB drug development. Additionally, while appreciable in vivo benefit was observed when moxifloxacin was paired with 1599 in the high-dose aerosol model, this was not similarly the case for a second fluoroquinolone, levofloxacin, when tested in chronically infected mice. Whether this reflects differences in these two models or the superior bactericidal activity of moxifloxacin30 remains to be determined.</p><p>The spectinamide 1599 was highly effective in vivo when paired with rifampicin. Mechanistically, both drugs effect direct inhibition of protein synthesis, albeit at different drug targets (i.e. rifampicin at the level of mRNA synthesis and 1599 as an inhibitor of ribosome translocation). Appreciable synergy was also observed in a previously published study where 1599 was paired with another protein synthesis inhibitor, clarithromycin.4 It is therefore plausible to suggest that inhibition of two different steps within the same pathway could result in a complete shutdown of protein synthesis. Targeting protein synthesis in multiple ways is, however, not a guarantee of enhanced in vivo activity, as this was not observed when 1599 was paired with linezolid, which acts at the initiation phase of protein synthesis.31 No improvement in potency was observed when rifampicin was combined with 1599 in in vitro MIC chequerboard assays either. Alternative explanations for the improved potency of this combination may relate to increased retention or uptake of rifampicin into the bacterium in the presence of 1599 or killing of rifampicin-tolerant subpopulations by 1599. This could be through an alteration of efflux since the intrinsic efflux pump (Rv1258c) avoided by the spectinamides has been shown to contribute to in vivo growth and also macrophage-induced rifampicin tolerance.32 Finally, changes in pharmacokinetic properties upon combining drugs, which were not assessed in these early in vivo studies, will be investigated further.</p><p>Pyrazinamide has previously been shown to be synergistic with several new TB agents in development.33–36 When combined with 1599 herein, it also showed promising synergism, especially in the C3HeB/FeJ model. In our studies, pyrazinamide showed a range in efficacy dependent on the mouse infection model utilized. Reduction in bacterial loads was highest in the acute BALB/c mouse model, modest in the chronic model and lower in C3HeB/FeJ mice. These results are consistent with earlier published data on pyrazinamide in C3HeB/FeJ mice, where the limited potency observed was thought to arise from the lack of a sufficiently acidic environment to support the antimicrobial activity of pyrazinamide within the neutral to slightly basic caseum.22,25,37,38 Pyrazinamide has been shown in earlier work to show some activity at neutral pH under certain in vitro conditions, many of which are in fact present in necrotic lesions in C3HeB/FeJ mice in vivo (e.g. hypoxia8 and nutrient starvation39). A recent study provided more evidence to support this by demonstrating that susceptibility to pyrazinamide and its biologically active metabolite (pyrazinoic acid) in vitro is independent of environmental pH under certain conditions.40 Consequently, the limited activity of pyrazinamide when tested alone in C3HeB/FeJ mice might only be explained by contributions of multiple micro-environmental stressors present in necrotic lesions affecting the metabolic state of bacteria in a profound and specific way, and not by pH alone. Pyrazinamide also exhibits activity with bedaquiline in C3HeB/FeJ mice,38 suggesting that the pyrazinamide potentiating effect may not be specific to the spectinamides, per se. These observations suggest that the synergistic effects of pyrazinamide with companion drugs and the antibacterial activity observed when used as a single drug may be separable mechanisms. Cooperative mechanisms of action,16,35,41 different target subpopulations of metabolically distinct bacteria or pharmacokinetic effects may also contribute to the observed efficacy when used in combination.</p><p>Of all drug regimens evaluated, the most remarkable result in terms of synergy was obtained with the combination of rifampicin, pyrazinamide and a spectinamide (i.e. 1599 or 1810) in C3HeB/FeJ mice. The observed synergy was especially striking given the limited overall activity of each drug alone in this mouse model. The effect is clearly dependent on the addition of a spectinamide since the combined efficacy of all three drugs is significantly higher than the rifampicin/pyrazinamide combination tested without a spectinamide. C3HeB/FeJ mice are known to exhibit advanced lung pathology upon M. tuberculosis infection.42 Compared with that observed in more conventional mouse models, the presence of advanced, hypoxic, caseating lung lesions and the higher ratio of extracellular to intracellular bacilli in C3HeB/FeJ mice has been shown to have a profound effect on treatment outcome with multiple chemotherapeutic agents,6,23–27 including rifampicin and pyrazinamide.5,25,37 Our studies therefore suggest that the combination of 1599 with rifampicin and pyrazinamide is able to significantly enhance drug efficacy of both rifampicin and/or pyrazinamide in this advanced disease model during a 4 week treatment regimen, which warrants further testing in long-term trials.</p><p>In conclusion, the data presented in this study support the further study of the spectinamides as combination partners with existing and experimental classes of anti-TB agents. While synergy between two antibiotics is often demonstrated in vitro, such a demonstration in vivo in complex disease infection models, where the sum of the individual drug response is lower than that observed for the combination, is a rarity. It is not yet known whether the improvements in in vivo activity observed herein result from mechanistic interactions or from alterations in drug exposure, drug uptake or retention. Further studies are clearly warranted to better understand the molecular basis for these interactions and to explore the sterilizing activity of spectinamide/rifampicin/pyrazinamide and spectinamide/bedaquiline/pyrazinamide combinations in mouse relapse models. It is our hope that detailed exploration of the molecular mechanism of synergy between the spectinamides, pyrazinamide and rifampicin, may aid the discovery of novel approaches to treat TB and to shorten therapy in humans.</p><!><p>Click here for additional data file.</p>