In Vitro Synergy between Clofazimine and Amikacin in Treatment of Nontuberculous Mycobacterial Disease

DISCUSSION

Significant synergy between clofazimine and amikacin is observed across a range of nontuberculous Mycobacterium species, including both slow and rapid growers. This in vitro observation is most prominent for M. abscessus, MAC, and M. simiae, which are well-known causative agents of human disease. Disease caused by M. abscessus and M. simiae is notorious for the poor outcomes of drug treatment (8, 9, 10, 19). Amikacin plays a central role in the treatment of M. abscessus disease (8), and the combination with clofazimine may improve its efficacy. In contrast to a previous study in Taiwan (17), which applied similar methods to a smaller number of isolates, we did encounter M. abscessus isolates with MICs of ≥1 μg/ml to clofazimine and intermediate susceptibility or resistance to amikacin in which no synergy could be demonstrated. Hence, synergy should be tested, rather than assumed, prior to treatment.

In MAC disease, amikacin is mainly used in the first 2 to 3 months of treatment for moderate or severe disease (8); here, too, a combination with clofazimine may increase the antimycobacterial activity of the intensive phase of treatment. In M. simiae disease, the role of amikacin used to be limited, due to its limited in vitro activity (20). However, if that activity can be as enhanced by clofazimine, as our in vitro evidence suggests, there may be a future role for the amikacin-clofazimine combination in M. simiae disease. The outcome of treatment in M. simiae has been very poor, especially with the rifampin-ethambutol-macrolide regimen (8, 19). This has, in part, been related to the lack of synergistic activity between rifampin and ethambutol against M. simiae (20). Amikacin-clofazimine-based regimens may provide interesting new leads and should be tested in clinical trials. However, the MICs and their interpretation for amikacin and, particularly, clofazimine still await clinical validation (21), and the relationship of MICs to achievable drug serum concentrations in patients and outcome of treatment remains to be established (22); this, too, could be part of the trial design. Despite the use of the synergistic combination of rifampin and ethambutol in MAC lung disease, the treatment outcome of current regimens is suboptimal (8, 22), and the in vitro synergy between clofazimine and amikacin is thus no guarantee for synergy and good outcome of treatment in vivo. It is also important to realize that in one trial of HIV-related disseminated M. avium disease, the use of clofazimine was associated with excess mortality (16). Hence, any new trial of clofazimine therapy should proceed with caution.

Clofazimine is currently registered for use in leprosy only, although it is also used to treat multidrug-resistant tuberculosis and, in some cases, NTM. The drug is not registered for NTM disease, and the production and dissemination of clofazimine are limited. In many settings, specific approval to acquire clofazimine for an individual patient has to be sought. Previously, more active analogs of clofazimine have been produced (14), and the synthesis of novel compounds in this class may help circumvent the limited availability of clofazimine and offer more-active alternatives in the long term.

A biological explanation for this synergistic activity is lacking. Clofazimine is thought to have cell wall-destabilizing properties (3, 4). This may allow increased influx of amikacin, possibly by attaching to clofazimine and using it as a “Trojan horse” (14). The synergistic activity of clofazimine may extend to other drugs with intracellular targets, including macrolides, fluoroquinolones, and rifamycins.

The synergistic activity of clofazimine and amikacin, as well as kanamycin, has been previously noted in mouse models of MAC disease (7, 15). Subsequent researchers suggested that the synergy in vivo may be overestimated and result partly from carryover of clofazimine from tissue samples to culture media, thereby inhibiting growth and, thus, bacterial load assessments (11). The carryover hypothesis was later refuted (14).

This study has three important limitations. The first is that interpretation of clofazimine MICs as susceptible or resistant has not been clinically validated (21). In fact, the clinical efficacy of clofazimine against rapid growers has never been evaluated and its efficacy against MAC has only been demonstrated in an uncontrolled trial (6). The second is the low number of M. abscessus subsp. bolletii isolates. As we were able to demonstrate clofazimine-amikacin synergy in a range of rapidly growing mycobacteria, as well as slowly growing mycobacteria, it is likely that this synergy is relevant to all M. abscessus subspecies and all MAC members. For the rapid growers, the test method probably resulted in an underestimation of the synergistic activity. As most isolates were already susceptible to either 0.5 μg/ml of clofazimine or 2 μg/ml of amikacin, synergy could not be detected; the combination of 0.5 μg/ml of clofazimine and 2 μg/ml of amikacin was the lowest concentration tested. And yet, it is likely that synergy is present at equal levels in those isolates; this, as well as its potential clinical relevance, warrants separate study.

In summary, clofazimine and amikacin show significant synergistic activity against a variety of NTM, including rapid and slow growers; it could be observed in all MAC, M. simiae, and M. chelonae isolates, 84% of M. abscessus isolates, and 1 of 2 M. fortuitum isolates. Therefore, the addition of clofazimine to amikacin-containing regimens should be tested in clinical trials. The activity of clofazimine, in itself, against NTM and the synergy with amikacin may warrant a “renaissance” of clofazimine, or of riminophenazines in the broader sense.