Long-acting implant-forming formulation of anti-tuberculosis drug rifabutin


Tuberculosis (TB) is a bacterial disease caused by Mycobacterium tuberculosis (Mtb) that usually affects the lung, but it can also affect other parts of the body. It is often thought to be a disease from the past. However, it remains one of the main killer infectious diseases, particularly in developing countries. Moreover, approximately one-fourth of the world’s population has a latent TB infection with the potential for reactivation.1 The Covid-19 pandemic has had devastating effects on tuberculosis testing, prevention and treatment access. As a result, for the first time in more than a decade, TB mortality has increased from 1.4 million in 2019 up to 1.5 million in 2020.

Long acting (LA) parenteral drug formulations that provide sustained drug release over weeks or months, could be sufficient to successfully treat TB with only one or two injections of the drug. Such approach could potentially improve adherence to treatment regimen and reduce treatment failure. LA biodegradable in situ forming implant (ISFI) formulations are liquid drug formulations that solidify after subcutaneous injections and form implants. This allows less invasive and less painful administration than solid implants.  Additionally, the biodegradable nature of the polymer matrix in the formulation eliminates the need for surgical implant removal.2 In our recent article “A long-acting formulation of rifabutin is effective for prevention and treatment of Mycobacterium tuberculosis, 3 we showed that the ISFI technology can be harnessed for anti-TB drugs.

Solidification of the liquid ISFI formulation occurs after injection into the hydrophilic environment of the subcutis. This is a consequence of the unique composition of ISFI formulations: drug, biodegradable polymer and water miscible solvents that dissolve both drug and polymer.2 Upon injection, phase transition occurs by solvent exchange and polymer precipitation, which results in the formation of a solid implant consisting of biodegradable polymer and drug. The drug release from the implant is controlled by polymer degradation4 and can be extensively optimized by changes in the composition of the liquid formulation to meet specific clinical needs. The standard of care for initiating treatment of TB is four-drug therapy. RFB-ISFI alone is thus not appropriate for TB treatment. Nevertheless, one-drug regimens are efficient for highly prevalent latent tuberculosis infection (LTBI). Long-acting RFB-ISFI can therefore change a months-long daily oral treatment of LTBI to a regimen with just several subcutaneous injections.

Figure 1: Long-acting rifabutin formulation solidifies after injection. a The liquid injectable of LA-RFB formulation. b The solidified LA-RFB implant after injection, scale bar is 5 mm. c Microphotographs of the implant surface and the internal structure with and without additives. Scale bar is 2 μm for implant surface and 10 μm for internal structures.

Typical optimization steps include testing of different biodegradable polymers, changes in polymer molecular weight, use of different biocompatible water miscible solvents and addition of various additives. Long-acting formulations require high drug load to maintain sustained drug release for extended periods of time. To accommodate higher rifabutin (RFB) load in the injectable formulation, we manipulated its solubility in biocompatible solvents by amphiphilic additives. Amphiphilic additives improve drug solubility in water by trapping molecules and forming nanoscale aggregates; micelles.5 Two solvents commonly used to prepare ISFI, DMSO and NMP, are polar solvents and thus we expected that amphiphilic additives will increase rifabutin solubility in those solvents. Indeed, amphiphilic additives Kolliphor®HS 15, TPGS, Tween 80, Tween 20, and Pluronic F68 and F127 were able to dramatically increase the saturated solubility of RFB in both DMSO and NMP. This allowed us to significantly increase drug load in long-acting RFB formulations (RFB-ISFI). Interestingly, higher drug load also lowered initial release bursts and extended drug release in vitro and in vivo.

Biodegradable implants degrade by bulk degradation that depends on diffusion of water into the implant.6,7 Degradation can be slowed by suppressing water invasion into the implant by increasing implant hydrophobicity, which results in slower implant erosion8. Indeed, we showed that erosion of implants with higher load of RFB, a hydrophobic drug, is slower compared to implants with lower rifabutin load. The higher drug load of rifabutin also made the microstructure of RFB-ISFI implant more organized.

Figure 2: The efficacy of RFB-ISFI against Mycobacterium tuberculosis infection. a Experimental schema for prevention efficacy testing of RFB-ISFI. BALB/c mice were treated with a single injection of RFB-ISFI or placebo and two weeks later were exposed to Mtb. b The bacterial burden in lung, liver, and spleen of placebo (n=6) and RFB-ISFI (n=6) treated mice four weeks after Mtb exposure, mean +/- SEM are shown, c Experimental schema for post-exposure RFB-ISFI treatment. BALB/c mice were exposed to Mtband one-week later treated with placebo or RFB-ISFI. d The bacterial burden in mice 7 days post Mtb exposure (n=6). e The bacterial burden four weeks post Mtb exposure, mean +/- SEM are shown. f Formalin-fixed whole lung lobes of mice treated with placebo or LA-RFB, white lesions caused by Mtb (white arrows) are visible in lung from mice which received placebo but not in lung from RFB-ISFI treated mice. g H&E staining of lung sections shows pathological changes in mice treated with placebo but not in mice treated with RFB-ISFI.

To test the relevance of the RFB-ISFI formulation we developed, we devised 2 experiments in BALB/c mice. In the first experiment, mice were injected with RFB-ISFI or placebo ISFI two weeks before exposure to aerosol of Mtb. In the second experiment, acutely infected mice with TB were treated with RFB-ISFI or placebo ISFI. Infection, measured as colony-forming units (CFU) in tissues and pathological changes in lung, was assessed 4 weeks post exposure. In both experiments, placebo treated mice had high CFU count in all tissues tested and lung pathology typical for TB infection in mice. However, all RFB-ISFI treated mice had no detectable TB infection. These experiments demonstrated the efficacy of the RFB-ISFI in vivo.


1          WHO. Global tuberculosis report 2021, <https://www.who.int/publications/i/item/9789240037021> (2021).

2          Kempe, S. & Mader, K. In situ forming implants - an attractive formulation principle for parenteral depot formulations. J. Control. Release 161, 668-679, doi:10.1016/j.jconrel.2012.04.016 (2012).

3          Kim, M. et al. A long-acting formulation of rifabutin is effective for prevention and treatment of Mycobacterium tuberculosis. Nature communications 13, 4455, doi:10.1038/s41467-022-32043-3 (2022).

4          Zhang, X. et al. Effect of Polymer Permeability and Solvent Removal Rate on In Situ Forming Implants: Drug Burst Release and Microstructure. Pharmaceutics 11, doi:10.3390/pharmaceutics11100520 (2019).

5          Liu, L. et al. Kolliphor(R) HS 15 Micelles for the Delivery of Coenzyme Q10: Preparation, Characterization, and Stability. AAPS PharmSciTech 17, 757-766, doi:10.1208/s12249-015-0399-5 (2016).

6          Tamada, J. A. & Langer, R. Erosion kinetics of hydrolytically degradable polymers. Proc. Natl. Acad. Sci. U. S. A.90, 552-556, doi:10.1073/pnas.90.2.552 (1993).

7          von Burkersroda, F., Schedl, L. & Gopferich, A. Why degradable polymers undergo surface erosion or bulk erosion. Biomaterials 23, 4221-4231, doi:10.1016/s0142-9612(02)00170-9 (2002).

8          Boimvaser, S., Mariano, R. N., Turino, L. N. & Vega, J. R. In vitro bulk/surface erosion pattern of PLGA implant in physiological conditions: a study based on auxiliary microsphere systems. Polym Bull 73, 209-227, doi:10.1007/s00289-015-1481-6 (2016).

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