Rifaquizinone: A Novel Rifamycin-Quinolone Hybrid Antibiotic

Antibiotic-resistant bacteria inside a biofilm, 3D illustration.

Image credit: Adobe Stock

Rifaquizinone is a novel dual-pharmacophore antibiotic that combines rifamycin and a fluoroquinolone-like compound in a single molecule.¹ This combined molecule demonstrates activity primarily on RNA polymerase, much like rifampin, while inhibiting DNA gyrase and DNA topoisomerase IV.² Previously described as CBR-2092 and TNP-2092, rifaquizinone was broadly tested for activity against Staphylococcus aureus biofilm formation,³ methicillin-resistant S aureus (MRSA), streptococcal species, Mycobacterium tuberculosis,⁴ fluoroquinolone-resistant S aureus,⁵ and Helicobacter pylori.⁶ Of these, further evaluation of rifaquizinone as a treatment for prosthetic joint infection (PJI) was pursued.^7,8 This pursuit was influenced by the Infectious Diseases Society of America guidelines for staphylococcal PJI, which recommend rifampin and either ciprofloxacin or levofloxacin.⁹ The bactericidal activity of fluoroquinolones is thought to be potentiated by the biofilm activity of rifampin; however, antagonism has been observed between these drugs.¹⁰

This antagonism, as well as the challenges associated with utilizing multiple medications and the development of resistance genes, has also played a role in directing research into a dual pharmacophore medication like rifaquizinone. In vitro testing of rifaquizinone has illustrated promising results for meeting these challenges. When first presented in 2007, time-kill studies against nongrowing S aureus demonstrated a more significant bactericidal effect than rifampin, multiple fluoroquinolones, and combination therapy.³ Further time-kill studies comparing rifaquizinone with rifampin or ciprofloxacin against S aureus strains demonstrating rifampin resistance, fluoroquinolone resistance, and combined resistance showed similar results.¹ Time- and concentration-dependent cidality was observed at 1 μg/mL and 10 μg/mL, regardless of resistance genes present.

Of significant note in this study, fluoroquinolone efflux pump gene expression had no effect on minimum inhibitory concentration (MIC) for rifaquizinone, illustrating the possibility that larger molecules with multiple pharmacophores can evade such resistance mechanisms. This was also shown in a prior study where upregulated fluoroquinolone efflux had no effect on rifaquizinone MIC or mutant prevention concentration.⁵ Although the fluoroquinolone pharmacophore was studied for cidality and evasion of resistance, the rifampin pharmacophore was examined for simultaneous biofilm penetrance. The initial study found a dose-dependent bactericidal curve against S aureus biofilms, with sterilization observed after 7 days.³ In that same study, S aureus was cultivated in a Centers for Disease Control and Prevention (CDC) biofilm reactor, and viable colony forming units (CFU) were measured after vancomycin or rifaquizinone treatment, with the latter significantly reducing CFU to nearly undetectable amounts. Similar results were seen in colony biofilm studies using methicillin-susceptible and methicillin-resistant S aureus compared with rifampin, gatifloxacin, and rifampin combined with multiple fluoroquinolones.⁵ Further biofilm activity testing was performed against 40 S aureus and 40 S epidermidis strains with visual turbidity assessed to determine minimum biofilm inhibitory concentration (MBIC) and minimum biofilm bactericidal concentration (MBBC).⁷ The results showed a ratio between MBIC/MIC and MBBC/MIC that was much smaller compared with vancomycin, daptomycin, rifampin, ciprofloxacin, and combination rifampin/ciprofloxacin. In vivo studies of murine catheter biofilm formation reproduced the significant log₁₀ reductions in methicillin-susceptible and fluoroquinolone-resistant S aureus.¹¹ From the same study, a rabbit infective endocarditis model with MRSA and fluoroquinolone/methicillin–resistant S aureus found the vegetative density reduction of rifaquizinone was similar to that of vancomycin, levofloxacin/rifampin, and improved upon ciprofloxacin/rifampin.

These findings led to the development of a rat knee prosthesis model.⁸ The knee prostheses were inoculated with MRSA, then they were treated with intra-articular saline, vancomycin, or rifaquizinone at different concentrations. With rifaquizinone, there was a dose-dependent decrease in knee width, serum α1-acid glycoprotein, serum C-reactive protein (CRP), tissue IL-6, and tissue tumor necrosis factor-α. Vancomycin outperformed the highest dosage of rifaquizinone in decreasing serum CRP, observed biofilm formation on scanning electron microscopy, and posttreatment soft tissue bacterial CFUs. However, the highest dosage of rifaquizinone matched or outperformed vancomycin in all histological scoring of osteomyelitis. Only vancomycin and the highest dosage of rifaquizinone managed to prevent loosened prostheses, with similar results found in bone health measurements. This collection of findings is promising for rifaquizinone as a potential treatment for staphylococcal PJI. Coupled with this are the postantibiotic effects and postantibiotic, sub-MIC effects found in earlier work.¹ With poor vascularity in articular spaces, the continued efficacy of an antibiotic after it has been administered and at sub-MIC concentrations could be an important factor for guiding therapy in the future. However, these results may be at odds with changes in the management of orthopedic infections. Literature has shown a potential shift toward oral antibiotic regimens rather than parenteral,¹² whereas rifaquizinone has shown poor bioavailability outside the intestinal lumen.¹³ This localization of antibiotic therapy could limit the application of rifaquizinone for staphylococcal prosthetic joint infections to parenteral treatment; however, similar to that of oral vancomycin, oral rifaquizinone could provide other clinical indications such as treatment of Clostridioides difficile infection and hepatic encephalopathy. The possible oral applications of rifaquizinone evaluated in the aforementioned studies were primarily aimed at efficacy against H pylori,⁶ C difficile, and in a similar manner to that of rifaximin for hyperammonemia in hepatic encephalopathy.¹³ Activity against H pylori had a dose- and time-dependent effect, similar to current regimens with fewer medications involved, but still required lowered pH to be effective.

The results were promising enough to lead to a human safety and tolerability trial with a proton pump inhibitor. The impact on gut microbiota family and genus matched that of rifaximin, prompting a pharmacokinetic profile and efficacy trial in patients with cirrhosis and hyperammonemia. Curiously, studies were performed to assess the ability of rifaquizinone to impact the survivability of S aureus strains cultivated for or directly injected intracellularly. Based on the sources provided by the literature, this was done due to the presence of intracellular S aureus found in recurrent infectious rhinosinusitis.¹⁴ For the purposes of studying rifaquizinone, intracellular studies were performed using murine reticulum cell sarcoma⁴ and macrophages.¹ Although this may provide some insight into the ability of rifaquizinone to decrease a potential reservoir in PJI, there may be more benefit in assessing the intracellular activity in other cell lines. In so doing, the efficacy of the rifampin pharmacophore could be further assessed in the treatment of certain mycobacteria, especially in the context of fluoroquinolone use in tuberculosis as well as resistant strains of Mycobacterium avium-intracellulare and Mycobacterium kansasii. As illustrated in prior work, despite the fluoroquinolone-like pharmacophore, the gram-negative activity of rifaquizinone seems limited to Shigella sonnei, Yersinia enterocolitica, Salmonella spp, and Neisseria gonorrhoeae.¹³ The broad use of fluoroquinolones in the empiric treatment of cystitis and pneumonia has been identified as a precipitating factor in the development of resistance,¹⁵ with fewer options as multidrug-resistant organisms continue to proliferate. Although rifaquizinone demonstrates continued efficacy despite resistance and potential durability based on in vitro mutation prevention concentration/ mutation selection window testing, the lack of efficacy against gram negatives most likely to demonstrate fluoroquinolone resistance may be an example of the limit- ations of multipharmacophore antibiotics. Ma and Lynch discussed that prior dualacting molecules struggled with several challenges in order to provide activity for each pharmacophore.¹¹ Cleavage was required to activate the second active site, larger molecule sizes limited bioavailability, and some pharmacophores were unable to provide any additional activity. Rifaquizinone appears to have been designed with the intent of using a known combination to target a specific problem, which could have inherently limited its ability to be used outside of that indication. This raises questions about future medications made in this fashion. Will future dual-pharmacophore medications be limited to niche uses? Nonetheless, rifaquizinone appears to be a promising new drug in an era of increasing antimicrobial resistance.

References

1.Robertson GT, Bonventre EJ, Doyle TB, et al. In vitro evaluation of CBR-2092, a novel rifamycin-quinolone hybrid antibiotic: microbiology profiling studies with staphylococci and streptococci. Antimicrob Agents Chemother. 2008;52(7):2324-2334. doi:10.1128/AAC.01651-07

2.Robertson GT, Bonventre EJ, Doyle TB, et al. In vitro evaluation of CBR-2092, a novel rifamycin-quinolone hybrid antibiotic: studies of the mode of action in Staphylococcus aureus. Antimicrob Agents Chemother. 2008;52(7):2313-2323. doi:10.1128/AAC.01649-07

3.Doyle TB, Bonventre EJ, Robertson GT, Roche ED, Lynch AS. In vitro studies of the efficacy of CBR-2092, a novel rifamycin-quinolone hybrid antibiotic, in killing staphylococcal cells in biofilms. Presented at: 47th Interscience Conference on Antimicrobial Agents and Chemotherapy; September 17-20, 2007; Chicago, IL. Available at: https://www.researchgate.net/publication/266880997_In_Vitro_Studies_of_the_Efficacy_of_CBR-2092_a_Novel_Rifamycin-Quinolone_Hybrid_Antibiotic_in_Killing_Staphylococcal_Cells_in_Biofilms_Summary_and_Conclusion (Date Accessed: 02/07/2025)

4. Du Q, Doyle TB, Duncan L, Robertson GT, Lynch AS. In vitro microbiology profiling of CBR-2092, a novel rifamycin-quinolone hybrid antibiotic. Presented at: 47th Interscience Conference on Antimicrobial Agents and Chemotherapy; September 17-20, 2007; Chicago, IL. Available at: https://www.researchgate.net/publication/265825720_In_Vitro_Microbiology_Profiling_of_CBR-2092_a_Novel_Rifamycin-Quinolone_Hybrid_Antibiotic_Summary_and_Conclusion (Date Accessed: 02/07/2025)

5.Robertson GT, Bonventre EJ, Doyle TB, et al. Comparative in vitro activity of CBR-2092, a novel rifamycin-quinolone hybrid antibiotic, and rifampin+quinolone combinations. Presented at: 47th Interscience Conference on Antimicrobial Agents and Chemotherapy; September 17-20, 2007; Chicago, IL. Available at: https://www.researchgate.net/publication/267194420_Comparative_In_Vitro_Activity_of_CBR-2092_a_Novel_Rifamycin-Quinolone_Hybrid_Antibiotic_and_RifampinQuinolone_Combinations_Summary_and_Conclusion (Date Accessed: 02/07/2025)

6.Wang B, Zhao Q, Yin W, et al. In-vitro characterisation of a novel antimicrobial agent, TNP-2092, against Helicobacter pylori clinical isolates. Swiss Med Wkly. 2018;148:w14630. doi:10.4414/smw.2018.14630

7.Fisher C, Schmidt-Malan S, Yuan Y, et al. 691. Activity of TNP-2092 against biofilms formed by prosthetic joint infection–associated staphylococci. Open Forum Infect Dis. 2019;6(suppl 2):S313. doi:10.1093/ofid/ofz360.759

8.Dai T, Ma C, Zhang F, et al. The efficacy and safety of an intra-articular dual-acting antibacterial agent (TNP-2092) for implant infection–associated methicillin-resistant Staphylococcus aureus. J Infect Dis. 2024;229(6):1658-1668. doi:10.1093/infdis/jiad588

9.Osmon DR, Berbari EF, Berendt AR, et al; Infectious Diseases Society of America. Diagnosis and management of prosthetic joint infection: clinical practice guidelines by the Infectious Diseases Society of America. Clin Infect Dis. 2013;56(1):e1-e25. doi:10.1093/cid/cis803

10. Neu HC. Synergy and antagonism of combinations with quinolones. Eur J Clin Microbiol Infect Dis. 1991;10(4):255-261. doi:10.1007/BF01966998

11.Ma Z, Lynch AS. Development of a dual-acting antibacterial agent (TNP-2092) for the treatment of persistent bacterial infections. J Med Chem. 2016;59(14):6645-6657. doi:10.1021/acs.jmedchem.6b00485

12.Li HK, Rombach I, Zambellas R, et al; OVIVA Trial Collaborators. Oral versus intravenous antibiotics for bone and joint infection. N Engl J Med. 2019;380(5):425-436. doi:10.1056/NEJMoa1710926

13.Yuan Y, Wang X, Xu X, et al. Evaluation of a dual-acting antibacterial agent, TNP-2092, on gut microbiota and potential application in the treatment of gastrointestinal and liver disorders. ACS Infect Dis. 2020;6(5):820-831. doi:10.1021/acsinfecdis.9b00374

14.Plouin-Gaudon I, Clement S, Huggler E, et al. Intracellular residency is frequently associated with recurrent Staphylococcus aureus rhinosinusitis. Rhinology. 2006;44(4):249-254.

15.Piddock LJV. Fluoroquinolone resistance. BMJ. 1998;317(7165):1029-1030. doi:10.1136/bmj.317.7165.1029