Treatment-Shortening Effect of a Novel Regimen Combining Clofazimine and High-Dose Rifapentine in Pathologically Distinct Mouse Models of Tuberculosis.

Clofazimine and high-dose rifapentine have each separately been associated with treatment-shortening activity when incorporated into tuberculosis (TB) treatment regimens. We hypothesized that both modifications, i.e., the addition of clofazimine and the replacement of rifampin with high-dose rifapentine, in the first-line regimen for drug-susceptible TB would significantly shorten the duration of treatment necessary for cure. We tested this hypothesis in a well-established BALB/c mouse model of TB chemotherapy and also in a C3HeB/FeJ mouse model in which mice can develop caseous necrotic lesions, an environment where rifapentine and clofazimine may individually be less effective. In both mouse models, replacing rifampin with high-dose rifapentine and adding clofazimine in the first-line regimen resulted in greater bactericidal and sterilizing activity than either modification alone, suggesting that a rifapentine- and clofazimine-containing regimen may have the potential to significantly shorten the treatment duration for drug-susceptible TB. These data provide preclinical evidence supporting the evaluation of regimens combining high-dose rifapentine and clofazimine in clinical trials.

Oxazolidinones Can Replace Clarithromycin in Combination with Rifampin in a Mouse Model of Buruli Ulcer.

Rifampin (RIF) plus clarithromycin (CLR) for 8 weeks is now the standard of care for Buruli ulcer (BU) treatment, but CLR may not be an ideal companion for rifamycins due to bidirectional drug-drug interactions. The oxazolidinone linezolid (LZD) was previously shown to be active against Mycobacterium ulcerans infection in mice but has dose- and duration-dependent toxicity in humans. Sutezolid (SZD) and tedizolid (TZD) may be safer than LZD. Here, we evaluated the efficacy of these oxazolidinones in combination with rifampin in a murine BU model. Mice with M. ulcerans-infected footpads received control regimens of RIF plus either streptomycin (STR) or CLR or test regimens of RIF plus either LZD (1 of 2 doses), SZD, or TZD for up to 8 weeks. All combination regimens reduced the swelling and bacterial burden in footpads after two weeks of treatment compared with RIF alone. RIF+SZD was the most active test regimen, while RIF+LZD was also no less active than RIF+CLR. After 4 and 6 weeks of treatment, neither CLR nor the oxazolidinones added significant bactericidal activity to RIF alone. By the end of 8 weeks of treatment, all regimens rendered footpads culture negative. We conclude that SZD and LZD warrant consideration as alternative companion agents to CLR in combination with RIF to treat BU, especially when CLR is contraindicated, intolerable, or unavailable. Further evaluation could prove SZD superior to CLR in this combination.

High-Dose Rifamycins Enable Shorter Oral Treatment in a Murine Model of Mycobacterium ulcerans Disease.

Buruli ulcer (BU), caused by Mycobacterium ulcerans, is a neglected tropical skin and soft tissue infection that is associated with disability and social stigma. The mainstay of BU treatment is an 8-week course of rifampin (RIF) at 10 mg/kg of body weight and 150 mg/kg streptomycin (STR). Recently, the injectable STR has been shown to be replaceable with oral clarithromycin (CLR) for smaller lesions for the last 4 weeks of treatment. A shorter, all-oral, highly efficient regimen for BU is needed, as the long treatment duration and indirect costs currently burden patients and health systems. Increasing the dose of RIF or replacing it with the more potent rifamycin drug rifapentine (RPT) could provide such a regimen. Here, we performed a dose-ranging experiment of RIF and RPT in combination with CLR over 4 weeks of treatment in a mouse model of M. ulcerans disease. A clear dose-dependent effect of RIF on both clinical and microbiological outcomes was found, with no ceiling effect observed with tested doses up to 40 mg/kg. RPT-containing regimens were more effective on M. ulcerans All RPT-containing regimens achieved culture negativity after only 4 weeks, while only the regimen with the highest RIF dose (40 mg/kg) did so. We conclude that there is dose-dependent efficacy of both RIF and RPT and that a ceiling effect is not reached with the current standard regimen used in the clinic. A regimen based on higher rifamycin doses than are currently being evaluated against tuberculosis in clinical trials could shorten and improve therapy of Buruli ulcer.

Impact of Clofazimine Dosing on Treatment Shortening of the First-Line Regimen in a Mouse Model of Tuberculosis.

The antileprosy drug clofazimine was recently repurposed as part of a newly endorsed short-course regimen for multidrug-resistant tuberculosis. It also enables significant treatment shortening when added to the first-line regimen for drug-susceptible tuberculosis in a mouse model. However, clofazimine causes dose- and duration-dependent skin discoloration in patients, and the optimal clofazimine dosing strategy in the context of the first-line regimen is unknown. We utilized a well-established mouse model to systematically address the impacts of duration, dose, and companion drugs on the treatment-shortening activity of clofazimine in the first-line regimen. In all studies, the primary outcome was relapse-free cure (culture-negative lungs) 6 months after stopping treatment, and the secondary outcome was bactericidal activity, i.e., the decline in the lung bacterial burden during treatment. Our findings indicate that clofazimine activity is most potent when coadministered with first-line drugs continuously throughout treatment and that equivalent treatment-shortening results are obtained with half the dose commonly used in mice. However, our studies also suggest that clofazimine at low exposures may have negative impacts on treatment outcomes, an effect that was evident only after the first 3 months of treatment. These data provide a sound evidence base to inform clofazimine dosing strategies to optimize the antituberculosis effect while minimizing skin discoloration. The results also underscore the importance of conducting long-term studies to allow the full evaluation of drugs administered in combination over long durations.

Clofazimine shortens the duration of the first-line treatment regimen for experimental chemotherapy of tuberculosis.

A key drug for the treatment of leprosy, clofazimine has recently been associated with highly effective and significantly shortened regimens for the treatment of multidrug-resistant tuberculosis (TB). Consequently, we hypothesized that clofazimine may also shorten the duration of treatment for drug-susceptible TB. We conducted a controlled trial in the mouse model of TB chemotherapy comparing the activity of the 6-mo standard regimen for TB treatment, i.e., 2 mo of daily rifampin, isoniazid, pyrazinamide, and ethambutol followed by 4 mo of rifampin and isoniazid, with a 4-mo clofazimine-containing regimen: 2 mo of daily rifampin, isoniazid, pyrazinamide, and clofazimine followed by 2 mo of rifampin, isoniazid, and clofazimine. Treatment efficacy was assessed on the basis of Mycobacterium tuberculosis colony counts in the lungs and spleens during treatment and on the proportion of mice with culture-positive relapse 6 mo after treatment cessation. No additive effect of clofazimine was observed after the first week of treatment, but, by the second week of treatment, the colony counts were significantly lower in the clofazimine-treated mice than in the mice receiving the standard regimen. Lung culture conversion was obtained after 3 and 5 mo in mice treated with the clofazimine-containing and standard regimens, respectively, and relapse-free cure was obtained after 3 and 6 mo of treatment with the clofazimine-containing and standard regimens, respectively. Thus, clofazimine is a promising anti-TB drug with the potential to shorten the duration of TB chemotherapy by at least half (3 mo vs. 6 mo) in the mouse model of TB.

Clofazimine has delayed antimicrobial activity against Mycobacterium tuberculosis both in vitro and in vivo.

OBJECTIVES: The anti-leprosy drug clofazimine has been shown to have antimicrobial activity against Mycobacterium tuberculosis and has been associated with treatment-shortening activity in both clinical and preclinical studies of TB chemotherapy. However, a reported lack of early bactericidal activity (EBA) in TB patients has raised questions regarding the usefulness of clofazimine as an anti-TB drug. Our objective was to systematically evaluate the EBA of clofazimine in vitro and in vivo to provide insight into how and when this drug exerts its antimicrobial activity against M. tuberculosis. METHODS: We evaluated the 14 day EBA of clofazimine (i) in vitro at concentrations ranging from 4 times below to 4 times above the MIC for M. tuberculosis and (ii) in vivo in infected BALB/c mice at doses ranging from 1.5 to 100 mg/kg/day, and serum clofazimine levels were measured. In both experiments, isoniazid was used as the positive control. RESULTS: In vitro, clofazimine, at any concentration tested, did not exhibit bactericidal activity during the first week of exposure; however, in the second week, it exhibited concentration-dependent antimicrobial activity. In vivo, clofazimine, at any dose administered, did not exhibit bactericidal activity during the first week, and limited antimicrobial activity was observed during the second week of administration. While serum clofazimine levels were clearly dose dependent, the antimicrobial activity was not significantly related to the dose administered. CONCLUSIONS: Our data suggest that clofazimine's delayed antimicrobial activity may be due more to its mechanism of action rather than to host-related factors.

Clofazimine Contributes Sustained Antimicrobial Activity after Treatment Cessation in a Mouse Model of Tuberculosis Chemotherapy.

Experimental and clinical studies have indicated that the antileprosy drug clofazimine may contribute treatment-shortening activity when included in tuberculosis treatment regimens. Clofazimine accumulates to high levels in tissues, has a long half-life, and remains in the body for months after administration is stopped. We hypothesized that in tuberculosis treatment, accumulated clofazimine may contribute sustained antimicrobial activity after treatment cessation, and we used the BALB/c mouse model of chronic tuberculosis chemotherapy to address this hypothesis. Mycobacterium tuberculosis-infected mice were treated for 4 weeks or 8 weeks with either isoniazid alone, clofazimine alone, the first-line regimen rifampin-isoniazid-pyrazinamide-ethambutol, or a first-line regimen where clofazimine was administered in place of ethambutol. To evaluate posttreatment antimicrobial activity, bacterial regrowth in the lungs and spleens was assessed at the day of treatment cessation and 2, 4, 6, and 8 weeks after treatment was stopped. Bacterial regrowth was delayed in all mice receiving clofazimine, either alone or in combination, compared to the mice that did not receive clofazimine. This effect was especially evident in mice receiving multidrug therapy. In mice not receiving clofazimine, bacterial regrowth began almost immediately after treatment was stopped, while in mice receiving clofazimine, bacterial regrowth was delayed for up to 6 weeks, with the duration of sustained antimicrobial activity being positively associated with the time that serum clofazimine levels remained at or above the 0.25-µg/ml MIC for M. tuberculosis Thus, sustained activity of clofazimine may be important in the treatment-shortening effect associated with this drug.

Characterization of mouse models of Mycobacterium avium complex infection and evaluation of drug combinations.

The Mycobacterium avium complex is the most common cause of nontuberculous mycobacterial lung disease worldwide; yet, an optimal treatment regimen for M. avium complex infection has not been established. Clarithromycin is accepted as the cornerstone drug for treatment of M. avium lung disease; however, good model systems, especially animal models, are needed to evaluate the most effective companion drugs. We performed a series of experiments to evaluate and use different mouse models (comparing BALB/c, C57BL/6, nude, and beige mice) of M. avium infection and to assess the anti-M. avium activity of single and combination drug regimens, in vitro, ex vivo, and in mice. In vitro, clarithromycin and moxifloxacin were most active against M. avium, and no antagonism was observed between these two drugs. Nude mice were more susceptible to M. avium infection than the other mouse strains tested, but the impact of treatment was most clearly seen in M. avium-infected BALB/c mice. The combination of clarithromycin-ethambutol-rifampin was more effective in all infected mice than moxifloxacin-ethambutol-rifampin; the addition of moxifloxacin to the clarithromycin-containing regimen did not increase treatment efficacy. Clarithromycin-containing regimens are the most effective for M. avium infection; substitution of moxifloxacin for clarithromycin had a negative impact on treatment efficacy.

Pharmacokinetics and pharmacodynamics of clofazimine in a mouse model of tuberculosis.

The antileprosy drug clofazimine has shown potential for shortening tuberculosis treatment; however, the current dosing of the drug is not evidence based, and the optimal dosing is unknown. Our objective was to conduct a preclinical evaluation of the pharmacokinetics and pharmacodynamics of clofazimine in the mouse model of tuberculosis, with the goal of providing useful information on dosing for future studies. Pharmacokinetic parameters were evaluated in infected and uninfected BALB/c mice. Pharmacodynamic parameters were evaluated in Mycobacterium tuberculosis-infected mice that were treated for 12 weeks with one of six different clofazimine dosing regimens, i.e., doses of 6.25, 12.5, and 25 mg/kg of body weight/day and 3 regimens with loading doses. Clofazimine progressively accumulated in the lungs, livers, and spleens of the mice, reaching levels of greater than 50 µg/g in all tissues by 4 weeks of administration, while serum drug levels remained low at 1 to 2 µg/ml. Elimination of clofazimine was extremely slow, and the half-life was dependent on the duration of drug administration. Clofazimine exhibited dose-dependent tissue and serum concentrations. At any dose, clofazimine did not have bactericidal activity during the first 2 weeks of administration but subsequently demonstrated potent, dose-independent bactericidal activity. The antituberculosis activity of clofazimine was dependent on neither the dose administered nor the drug concentrations in the tissues, suggesting that much lower doses could be effectively used for tuberculosis treatment.

Contribution of moxifloxacin or levofloxacin in second-line regimens with or without continuation of pyrazinamide in murine tuberculosis.

RATIONALE: High-dose levofloxacin (L) (1,000 mg) was as active as moxifloxacin (M) (400 mg) in an early bactericidal activity trial, suggesting these fluoroquinolones could be used interchangeably. Whether pyrazinamide (Z) contributes sterilizing activity beyond the first 2 months in fluoroquinolone-containing second-line regimens remains unknown. OBJECTIVES: We compared the efficacy of M and high-dose L alone or in combination with ethionamide (Et), amikacin (A), and Z given for 2 or 7 months. METHODS: A pharmacokinetic study was performed to determine the L dose equivalent to 1,000 mg in humans. Treatment started 2 weeks after aerosol infection with Mycobacterium tuberculosis H37Rv. Mice received M or L alone or in combination with 2 months of EtZA followed by 5 months of Et or EtZ. MEASUREMENTS AND MAIN RESULTS: After 2 months of treatment, lung colony-forming unit (CFU) counts were similar in mice receiving either fluoroquinolone alone, but, after 4 and 5 months, CFU counts were 2 log10 lower in mice receiving M. Mice receiving 2MEtZA/3MEt and 2LEtZA/3LEt had 1.0 and 2.7 log10 lung CFUs, respectively. When Z was given throughout, both regimens rendered mice culture negative by 5 months, and most mice did not relapse after 7 months of treatment, with fewer relapses observed in the M group after 6 and 7 months of treatment. CONCLUSIONS: In murine tuberculosis, M had superior efficacy compared with L despite lower serum drug exposures and may remain the fluoroquinolone of choice for second-line regimens. Z contributed substantial sterilizing activity beyond 2 months in fluoroquinolone-containing second-line regimens, largely compensating for L's weaker activity.

Assessment of clofazimine activity in a second-line regimen for tuberculosis in mice.

RATIONALE: Although observational studies suggest that clofazimine-containing regimens are highly active against drug-resistant tuberculosis, the contribution of clofazimine for the treatment of this disease has never been systematically evaluated. OBJECTIVES: Our goal was to directly compare the activity of a standard second-line drug regimen with or without the addition of clofazimine in a mouse model of multidrug-resistant tuberculosis. Our comparative outcomes included time to culture conversion in the mouse lungs and the percentage of relapses after treatment cessation. METHODS: Mice were aerosol-infected with an isoniazid-resistant (as a surrogate of multidrug-resistant) strain of Mycobacterium tuberculosis. Treatment, which was administered for 5 to 9 months, was initiated 2 weeks after infection and comprised the following second-line regimen: daily (5 d/wk) moxifloxacin, ethambutol, and pyrazinamide, supplemented with amikacin during the first 2 months. One-half of the mice also received daily clofazimine. The decline in lung bacterial load was assessed monthly using charcoal-containing agar to reduce clofazimine carryover. Relapse was assessed 6 months after treatment cessation. MEASUREMENTS AND MAIN RESULTS: After 2 months, the bacillary load in lungs was reduced from 9.74 log10 at baseline to 3.61 and 4.68 in mice treated with or without clofazimine, respectively (P < 0.001). Mice treated with clofazimine were culture-negative after 5 months, whereas all mice treated without clofazimine remained heavily culture-positive for the entire 9 months of the study. The relapse rate was 7% among mice treated with clofazimine for 8 to 9 months. CONCLUSIONS: The clofazimine contribution was substantial in these experimental conditions.

Improving existing tools for Mycobacterium xenopi treatment: assessment of drug combinations and characterization of mouse models of infection and chemotherapy.

BACKGROUND: Mycobacterium xenopi is a common agent of non-tuberculous mycobacterial lung diseases in Europe. However, an optimal treatment regimen for M. xenopi infection has not yet been established. Appropriate in vitro and in vivo model systems are needed for characterization of the activity of potential drugs and drug combinations against M. xenopi. METHODS: We utilized three experimental platforms to analyse the anti-M. xenopi activity of single and combination drug regimens. First, we determined the bacteriostatic and bactericidal activities of drugs alone and in combination in vitro. Second, we used serum from treated mice to evaluate drug activities ex vivo. Third, we analysed M. xenopi growth in four strains of mice (BALB/c, C57BL/6, beige and athymic nude) and developed a mouse model of chemotherapy for this infection. RESULTS: Two-drug combinations of ethambutol with rifampicin, rifapentine or moxifloxacin, and of clarithromycin with moxifloxacin were bactericidal in vitro, and the combination of ethambutol and rifampicin with either clarithromycin or moxifloxacin showed significant bactericidal activity ex vivo. Nude mice were the most susceptible strain to M. xenopi infection, and in this model amikacin-containing regimens were the most effective against M. xenopi. No difference in activity was found between regimens containing clarithromycin and moxifloxacin in vivo. CONCLUSION: The ethambutol/rifampicin combination with clarithromycin or moxifloxacin had significant bactericidal activity against M. xenopi. The nude mouse, being highly susceptible to M. xenopi, can be utilized for in vivo chemotherapy studies for this infection.

Dose-ranging comparison of rifampin and rifapentine in two pathologically distinct murine models of tuberculosis.

In previous experiments, replacing the 10-mg/kg of body weight daily dose of rifampin with 7.5 to 10 mg/kg of rifapentine in combinations containing isoniazid and pyrazinamide reduced the duration of treatment needed to cure tuberculosis in BALB/c mice by approximately 50% due to rifapentine's more potent activity and greater drug exposures obtained. In the present study, we performed dose-ranging comparisons of the bactericidal and sterilizing activities of rifampin and rifapentine, alone and in combination with isoniazid and pyrazinamide with or without ethambutol, in BALB/c mice and in C3HeB/FeJ mice, which develop necrotic lung granulomas after infection with Mycobacterium tuberculosis. Each rifamycin demonstrated a significant increase in sterilizing activity with increasing dose. Rifapentine was roughly 4 times more potent in both mouse strains. These results reinforce the rationale for ongoing clinical trials to ascertain the highest well-tolerated doses of rifampin and rifapentine. This study also provides an important benchmark for the efficacy of the first-line regimen in C3HeB/FeJ mice, a strain in which the lung lesions observed after M. tuberculosis infection may better represent the pathology of human tuberculosis.

Rifapentine is not more active than rifampin against chronic tuberculosis in guinea pigs.

Rifamycins are key sterilizing drugs in the current treatment of active tuberculosis (TB). Daily dosing of rifapentine (P), a potent rifamycin with high intracellular accumulation, in place of rifampin (R) in the standard antitubercular regimen significantly shortens the duration of treatment needed to prevent relapse in a murine model of active TB. We undertook the current study to compare directly the activities of human-equivalent doses of P and R in a guinea pig model of chronic TB, in which bacilli are predominantly extracellular within human-like necrotic granulomas. Hartley strain guinea pigs were aerosol infected with ~200 bacilli of Mycobacterium tuberculosis H37Rv, and treatment given 5 days/week was initiated 6 weeks later. R at 100 mg/kg of body weight and P at 100 mg/kg were given orally alone or in combination with isoniazid (H) at 60 mg/kg and pyrazinamide (Z) at 300 mg/kg. Culture-positive relapse was assessed in subgroups of guinea pigs after completion of 1 and 2 months of treatment. Human-equivalent doses of R and P showed equivalent bactericidal activity when used alone and in combination therapy. In guinea pigs treated with rifampin, isoniazid, and pyrazinamide (RHZ) or PHZ, microbiological relapse occurred in the lungs of 8/10 animals treated for 1 month and in 0/10 animals treated for 2 months. Substitution of P for R in the standard antitubercular regimen did not shorten the time to cure in this guinea pig model of chronic TB. Data from ongoing clinical trials comparing the activity of these two drugs are awaited to determine the relevance of the guinea pig TB model in preclinical drug screening.

Treatment of tuberculosis with rifamycin-containing regimens in immune-deficient mice.

RATIONALE: Daily rifapentine plus isoniazid-pyrazinamide in mice infected with Mycobacterium tuberculosis produces cure in 3 months. Whether cure corresponds to latent infection contained by host immunity or true tissue sterilization is unknown. OBJECTIVES: To determine the length of treatment with rifapentine-isoniazid-pyrazinamide or rifampin-isoniazid-pyrazinamide needed to prevent relapse in immune-deficient mice. METHODS: Aerosol-infected BALB/c and nude mice were treated 5 days per week with either 2 months of the rifapentine-based regimen followed by rifapentine-isoniazid up to 12 months or the same regimen with rifampin instead of rifapentine. Cultures of lung homogenates were performed during the first 3 months and then every 3 months. Relapse rates were assessed after 3, 6, 9, and 12 months of treatment in BALB/c (± 1 mo of cortisone) and nude mice. MEASUREMENTS AND MAIN RESULTS: All rifapentine-treated mice were lung culture-negative at 3 months but 13% of BALB/c that received cortisone and 73% of nude mice relapsed. After 6, 9, and 12 months of treatment no mouse relapsed. Rifampin-treated BALB/c mice remained culture positive at 3 months. All were culture negative at 6, 9, and 12 months. None, including those receiving cortisone, relapsed. Rifampin-treated nude mice harbored more than 4 log(10) lung cfu at Month 2 and approximately 6 log(10) cfu with isoniazid resistance at Month 3. A supplementary experiment demonstrated that 7 days a week treatment did not prevent isoniazid resistance, whereas addition of ethambutol did. CONCLUSIONS: In nude mice, sterilization of tuberculosis is obtained with rifapentine-containing treatment, whereas failure with development of isoniazid resistance is obtained with rifampin-containing treatment.

Activity of 5-chloro-pyrazinamide in mice infected with Mycobacterium tuberculosis or Mycobacterium bovis.

BACKGROUND & OBJECTIVES: Pyrazinamide is an essential component of first line anti-tuberculosis regimen as well as most of the second line regimens. This drug has a unique sterilizing activity against Mycobacterium tuberculosis. Its unique role in tuberculosis treatment has lead to the search and development of its structural analogues. One such analogue is 5-chloro-pyrazinamide (5-Cl-PZA) that has been tested under in vitro conditions against M. tuberculosis. The present study was designed with an aim to assess the activity of 5-Cl-PZA, alone and in combination with first-line drugs, against murine tuberculosis. METHODS: The minimum inhibitory concentration (MIC) of 5-Cl-PZA in Middlebrook 7H9 broth (neutral pH) and the inhibitory titre of serum from mice that received a 300 mg/kg oral dose of 5-Cl-PZA 30 min before cardiac puncture were determined. To test the tolerability of orally administered 5-Cl-PZA, uninfected mice received doses up to 300 mg/kg for 2 wk. Four weeks after low-dose aerosol infection either with M. tuberculosis or M. bovis, mice were treated 5 days/wk with 5-Cl-PZA, at doses ranging from 37.5 to 150 mg/kg, either alone or in combination with isoniazid and rifampicin. Antimicrobial activity was assessed by colony-forming unit counts in lungs after 4 and 8 wk of treatment. RESULTS: The MIC of 5-Cl-PZA against M. tuberculosis was between 12.5 and 25 µg/ml and the serum inhibitory titre was 1:4. Under the same experimental conditions, the MIC of pyrazinamide was >100 µg/ml and mouse serum had no inhibitory activity after a 300 mg/kg dose; 5-Cl-PZA was well tolerated in uninfected and infected mice up to 300 and 150 mg/kg, respectively. While PZA alone and in combination exhibited its usual antimicrobial activity in mice infected with M. tuberculosis and no activity in mice infected with M. bovis, 5-Cl-PZA exhibited antimicrobial activity neither in mice infected with M. tuberculosis nor in mice infected with M. bovis. INTERPRETATION & CONCLUSION: Our findings showed that 5-Cl-PZA at doses up to 150 mg/kg was not active in chronic murine TB model. Further studies need to be done to understand the mechanism and mode of inactivation in murine model of tuberculosis.

PA-824 exhibits time-dependent activity in a murine model of tuberculosis.

PA-824 is one of two nitroimidazoles in phase II clinical trials to treat tuberculosis. In mice, it has dose-dependent early bactericidal and sterilizing activity. In humans with tuberculosis, PA-824 demonstrated early bactericidal activity (EBA) at doses ranging from 200 to 1,200 mg per day, but no dose-response effect was observed. To better understand the relationship between drug exposure and effect, we performed a dose fractionation study in mice. Dose-ranging pharmacokinetic data were used to simulate drug exposure profiles. Beginning 2 weeks after aerosol infection with Mycobacterium tuberculosis, total PA-824 doses from 144 to 4,608 mg/kg were administered as 3, 4, 8, 12, 24, or 48 divided doses over 24 days. Lung CFU counts after treatment were strongly correlated with the free drug T(>MIC) (R(2) = 0.87) and correlated with the free drug AUC/MIC (R(2) = 0.60), but not with the free drug C(max)/MIC (R(2) = 0.17), where T(>MIC) is the cumulative percentage of the dosing interval that the drug concentration exceeds the MIC under steady-state pharmacokinetic conditions and AUC is the area under the concentration-time curve. When the data set was limited to regimens with dosing intervals of ≤72 h, both the T(>MIC) and the AUC/MIC values fit the data well. Free drug T(>MIC) of 22, 48, and 77% were associated with bacteriostasis, a 1-log kill, and a 1.59-log kill (or 80% of the maximum observed effect), respectively. Human pharmacodynamic simulations based on phase I data predict 200 mg/day produces free drug T(>MIC) values near the target for maximal observed bactericidal effect. The results support the recently demonstrated an EBA of 200 mg/day and the lack of a dose-response between 200 and 1,200 mg/day. T(>MIC), in conjunction with AUC/MIC, is the parameter on which dose optimization of PA-824 should be based.

Activity of the fluoroquinolone DC-159a in the initial and continuation phases of treatment of murine tuberculosis.

DC-159a is a new fluoroquinolone with more potent in vitro activity than available fluoroquinolones against both drug-susceptible and fluoroquinolone-resistant Mycobacterium tuberculosis. Here, we report that DC-159a displays pharmacokinetics similar to those of moxifloxacin yet is more active than moxifloxacin during both the initial and continuation phases of treatment in a murine model. These results warrant further preclinical evaluation of DC-159a in selected drug combinations against drug-susceptible and fluoroquinolone-resistant tuberculosis.

Dose-dependent activity of pyrazinamide in animal models of intracellular and extracellular tuberculosis infections.

Recent in vitro pharmacokinetic data suggest that the currently recommended dose of pyrazinamide may be suboptimal for killing intracellular bacilli in humans. We evaluated a range of pyrazinamide doses against intracellular and extracellular Mycobacterium tuberculosis in chronically infected mice and guinea pigs, respectively. Antibiotics were given five times weekly for 4 weeks beginning 28 days after infection. Human-equivalent doses of isoniazid reduced lung bacterial counts 10-fold in each species. Pyrazinamide given at 1/4 and 1/2 the human-equivalent dose was minimally active, while human-equivalent doses reduced lung bacterial counts by ∼1.0 log(10) in each species. Doubling the human-equivalent dose of pyrazinamide reduced the lung bacillary burden by 1.7 and 3.0 log(10) in mice and guinea pigs, respectively. As in humans and mice, pyrazinamide showed significant synergy with rifampin in guinea pigs. Clinical studies are warranted to investigate the sterilizing activity and tolerability of higher doses of pyrazinamide in combination tuberculosis regimens.

Using bioluminescence to monitor treatment response in real time in mice with Mycobacterium ulcerans infection.

Mycobacterium ulcerans causes Buruli ulcer, a potentially disabling ulcerative skin disease. Only recently was antimicrobial therapy proven effective. Treatment for 2 months with rifampin plus streptomycin was first proposed after experiments in the mouse footpad model demonstrated bactericidal activity. This treatment is now considered the treatment of choice, although larger ulcers may require adjunctive surgery. Shorter, oral regimens are desired, but evaluating drug activity in mice is hampered by the very slow growth of M. ulcerans, which takes 3 months to produce countable colonies. We created a recombinant bioluminescent M. ulcerans strain expressing luxAB from Vibrio harveyi for real-time evaluation of antimicrobial effects in vivo. Mouse footpads were injected with wild-type M. ulcerans 1059 (WtMu) or the recombinant bioluminescent strain (rMu). Two weeks later, mice received rifampin plus streptomycin, kanamycin alone (to which rMu is resistant), or streptomycin alone for 4 weeks and were observed for footpad swelling (preventive model). Untreated controls and kanamycin-treated rMu-infected mice received rifampin plus streptomycin for 4 weeks after developing footpad swelling (curative model). Compared to WtMu, rMu exhibited similar growth and virulence in vivo and similar drug susceptibility. A good correlation was observed between luminescence (measured as relative light units) and number of viable bacteria (measured by CFU) in footpad homogenates. Proof of concept was also shown for serial real-time evaluation of drug activity in live mice. These results indicate the potential of bioluminescence as a real-time surrogate marker for viable bacteria in mouse footpads to accelerate the identification of new treatments for Buruli ulcer.