Acceleration of Tuberculosis Treatment by Adjunctive Therapy with Verapamil as an Efflux Inhibitor

Abstract

Rationale: A major priority in tuberculosis (TB) is to reduce effective treatment times and emergence of resistance. Recent studies in macrophages and zebrafish show that inhibition of mycobacterial efflux pumps with verapamil reduces the bacterial drug tolerance and may enhance drug efficacy. Objectives: Using mice, a mammalian model known to predict human treatment responses, and selecting conservative human bioequivalent doses, we tested verapamil as an adjunctive drug together with standard TB chemotherapy. As verapamil is a substrate for CYP3A4, which is induced by rifampin, we evaluated the pharmacokinetic/ pharmacodynamic relationships of verapamil and rifampin coadministration in mice. Methods: Using doses that achieve human bioequivalent levels matched to those of standard verapamil, but lower than those of extended release verapamil, we evaluated the activity of verapamil added to standard chemotherapy in both C3HeB/FeJ (which produce necrotic granulomas) and the wild-type background C3H/HeJ mouse strains. Relapse rates were assessed after 16, 20, and 24 weeks of treatment in mice. Measurements and Main Results: We determined that a dose adjustment of verapamil by 1.5-fold is required to compensate for concurrent use of rifampin during TB treatment. We found that standard TB chemotherapy plus verapamil accelerates bacterial clearance in C3HeB/FeJ mice with near sterilization, and significantly lowers relapse rates in just 4 months of treatment when compared with mice receiving standard therapy alone.Conclusions: These data demonstrate treatment shortening by verapamil adjunctive therapy in mice, and strongly support further study of verapamil and other efflux pump inhibitors in human TB.Keywords: human equivalent doses; verapamil; efflux; Mycobacterium tuberculosis Tuberculosis (TB), a disease affecting millions of people worldwide, requires treatment of patients with multiple drugs for several months. The complexity and length of therapy has led to the emergence of extensively drug-resistant Mycobacterium tuberculosis, which poses a global threat (1). The limited number of new antimicrobials in the TB drug development pipeline further emphasizes the need for fresh approaches in TB therapy. Repurposing existing approved drugs that may serve as treatmentshortening adjuvants is a promising and relatively efficient approach (2). Efflux pump inhibitors (EPIs) are potential agents in this category, and there have been initial promising reports for licensed agents, such as verapamil, reserpine, and piperine (3).Recently, Adams and colleagues (4) showed that the bacterial efflux pump-encoding gene, Rv1258c promotes intracellular bacterial survival, and may mediate drug tolerance. Addition of verapamil reduced tolerance to rifampin in both M. tuberculosis and Mycobacterium marinum, and incubation of M. marinuminfected macrophages with verapamil reduced intracellular bacterial growth. When verapamil was added to isoniazidand rifampin-treated M. marinum-infected macrophages, it restored antibiotic killing by red...

DosS Is Required for the Complete Virulence of Mycobacterium tuberculosis in Mice with Classical Granulomatous Lesions

“…A detailed analysis of the role of the entire DosR regulon in pathogenesis has not been performed in vivo. We hypothesized that C3HeB/FeJ mice, which develop necrotichypoxic TB granulomas (15) and have been used for studying the efficacy of anti-Mtb (11) and host-directed (14) drugs, would be ideally suited to study if the DosR regulon is required for both the establishment as well as the maintenance of infection in vivo.…”

“…C57Bl/6 mice do not faithfully reproduce certain aspects of human TB. In contrast, Mtb-infected C3HeB/FeJ mice display lesions with prominent necrotic degeneration, thus more closely resembling human granulomas (11)(12)(13)(14)(15). Harper and colleagues first demonstrated that tubercle lesions in this model develop hypoxia (15).…”

Latent Tuberculosis Infection: Myths, Models, and Molecular Mechanisms

“…Similarly, induction of the macrophage-induced efflux pump Rv1258c confers M. tuberculosis drug tolerance primarily to rifampin, which is reduced by the efflux pump verapamil. Interestingly, the addition of verapamil to the standard anti-TB regimen accelerates bacillary clearance in C3HeB/FeJ mice and significantly lowers relapse rates following 4 months of treatment compared with the case for mice receiving standard therapy alone (305). Recently, semisynthetic analogs of the protein synthesis inhibitor spectinomycin were found to have potent activity against drugsusceptible and drug-resistant M. tuberculosis, at least in part due to chemical modification, which allows the drugs to evade the Rv1258c efflux pump (306).…”

The antifibrotic drug pirfenidone promotes pulmonary cavitation and drug resistance in a mouse model of chronic tuberculosis

“…As previously described, treatment efficacy was assessed by lung CFU during treatment and relapse rates were measured 3 months after treatment cessation (Table 2) (44).…”

“…At each sacrifice point, at least 3 lungs from each group were examined for gross pathology as previously described (44). For histopathological assessments, at least 3 lungs were fixed in either 10% neutral buffered formalin or 4% paraformaldehyde for 3 days.…”