Repurposing clinically approved cephalosporins for tuberculosis therapy

Santiago Ramón-García, Rubén González Del Río, Angel Santos Villarejo, Gaye D Sweet, Fraser Cunningham, David Barros, Lluís Ballell, Alfonso Mendoza-Losana, Santiago Ferrer-Bazaga, Charles J Thompson, Santiago Ramón-García, Rubén González Del Río, Angel Santos Villarejo, Gaye D Sweet, Fraser Cunningham, David Barros, Lluís Ballell, Alfonso Mendoza-Losana, Santiago Ferrer-Bazaga, Charles J Thompson

Abstract

While modern cephalosporins developed for broad spectrum antibacterial activities have never been pursued for tuberculosis (TB) therapy, we identified first generation cephalosporins having clinically relevant inhibitory concentrations, both alone and in synergistic drug combinations. Common chemical patterns required for activity against Mycobacterium tuberculosis were identified using structure-activity relationships (SAR) studies. Numerous cephalosporins were synergistic with rifampicin, the cornerstone drug for TB therapy, and ethambutol, a first-line anti-TB drug. Synergy was observed even under intracellular growth conditions where beta-lactams typically have limited activities. Cephalosporins and rifampicin were 4- to 64-fold more active in combination than either drug alone; however, limited synergy was observed with rifapentine or rifabutin. Clavulanate was a key synergistic partner in triple combinations. Cephalosporins (and other beta-lactams) together with clavulanate rescued the activity of rifampicin against a rifampicin resistant strain. Synergy was not due exclusively to increased rifampicin accumulation within the mycobacterial cells. Cephalosporins were also synergistic with new anti-TB drugs such as bedaquiline and delamanid. Studies will be needed to validate their in vivo activities. However, the fact that cephalosporins are orally bioavailable with good safety profiles, together with their anti-mycobacterial activities reported here, suggest that they could be repurposed within new combinatorial TB therapies.

Conflict of interest statement

R.G.d.R., A.S.V., F.C., D.B., L.B., A.M.-L. and S.F.-B. are employees of GlaxoSmithKline, a producer of the generic drug amoxicillin/clavulanate (Augmentin). All other authors declare no conflicts of interest. The funders had no role in the study design, data collection and interpretation, or the decision to submit the work for publication.

Figures

Figure 1. Antimicrobial activity of cephalosporins against…
Figure 1. Antimicrobial activity of cephalosporins against Mycobacterium tuberculosis strains, including multidrug resistant clinical isolates.
BC strains are clinical isolates from British Columbia, Canada. DS, Drug sensitive; MDR, multi-drug resistant. GEN, first, second and third cephalosporin generation; ROA, route of administration; po, oral; iv, intravenous.
Figure 2. Qualitative SAR of cephalosporins against…
Figure 2. Qualitative SAR of cephalosporins against M. tuberculosis H37Rv.
Chemical elements highlighted in red indicate modifications from the parent compounds cephalexin, cefapirin and cefdinir.
Figure 3. Synergistic interactions between rifampicin and…
Figure 3. Synergistic interactions between rifampicin and selected beta-lactams against M. tuberculosis H37Rv cultured in different media.
“RIF FICI” is the Fractional Inhibitory Concentration Index of every compound in combination with rifampicin. An FICI ≤ 0.5 indicates synergy. An FICI > 0.5 indicates no interaction. Nb, 7H9 + GLY + ADC without Tyloxapol is the standard medium used in all other experiments.
Figure 4. Dose response characterization of selected…
Figure 4. Dose response characterization of selected beta-lactam and rifamycin combinations against M. tuberculosis.
(A) Intracellular (THP1) dose response curves of beta-lactams (BLM) alone and in the presence of 1/4xMIC concentrations of rifampicin againstM. tuberculosis H37Rv-Luc. Intracellular MICRIF = 16 ng/mL. (B) Comparison of the synergistic effects of rifampicin and rifabutin with beta-lactams in 7H9 medium. Dose response studies of the beta-lactams in combination with sub-inhibitory concentrations of rifampicin (RIF; 2 ng/mL; 1/8xMIC) and rifabutin (RBT; 0.5 ng/mL; 1/4xMIC). The MIC of rifampicin and rifabutin were 16 and 2 ng/mL, respectively.
Figure 5. Rifamycin accumulation in M. bovis…
Figure 5. Rifamycin accumulation in M. bovis BGC.
Rifampicin and rifabutin at 1 μg/mL were added to cell cultures incubated at 37 °C. Cells were pre-treated overnight in the presence of ethambutol (positive synergistic control; 5 μg/mL) or isoniazid (negative control; 0.25 μg/mL).
Figure 6. Kill kinetics of rifampicin, cephradine…
Figure 6. Kill kinetics of rifampicin, cephradine and ethambutol alone and in combination against M. tuberculosis H37Rv.
(A) Sub-inhibitory concentrations of cephradine (0.5xMIC, 4 μg/mL) enhanced the antibacterial and sterilizing activities of rifampicin and ethambutol (no growth observed after 60 days; upper right panel). (B) Dose dependent sterilizing activity of cephradine. Increased concentrations of cephradine (4xMIC, 32 μg/mL) enhanced the sterilizing activity of rifampicin (no growth observed after 60 days; bottom middle panel). (C) The stabilities of rifampicin and cephradine alone and in combination in 7H9 media were analysed by UPLC-MS/MS. MIC values of the individual drugs are used to express drug concentrations (i.e., 1, 4, 10-fold their MIC concentrations). RIF, rifampicin; CPD, cephradine; EMB, ethambutol. MICRIF = 0.03 μg/mL; MICCPD = 8 μg/mL; MICEMB = 2 μg/mL.
Figure 7. Synergistic triple combinations of rifampicin…
Figure 7. Synergistic triple combinations of rifampicin and beta-lactams.
Dose response curves of rifampicin alone and in the presence of several beta-lactam combinations against M. tuberculosis H37Rv (rifampicin susceptible) and its rifampicin resistant derivative M. tuberculosisH37Rv H526D. Dose response curve of rifabutin were also included for comparison. For the drug susceptible strain, beta-lactam and clavulanate concentrations were 0.06 μg/mL and 8 μg/mL, respectively. For the rifampicin resistant strain, beta-lactam and clavulanate concentrations were 0.125 μg/mL and 5 μg/mL, respectively. AMX, amoxicillin; CFX, cefadroxil; CLV, clavulanate; FAR, faropenem; MER, meropenem; RBT, rifabutin; RIF, rifampicin.
Figure 8. Synergistic interactions of cephradine and…
Figure 8. Synergistic interactions of cephradine and faropenem with a panel of anti-TB drugs against M. tuberculosis H37Rv.
Fractional inhibitory concentrations of every drug within every pairwise combination were calculated and plotted as isobolograms to allow visual inspection of the drug interactions. A straight line indicates a non-interaction profile while curves closer to the axis origins and falling within the box indicate a synergistic profile. Isobologram curves are colour coded according to their synergistic profile. Red, synergistic interaction; Green, additive; and blue, no interaction. Cephradine, solid lines; faropenem, broken lines.

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