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Formulation and Delivery - Chemical

(T1430-06-37) An Inhalable Spray Dried Powder of Linezolid and Antimicrobial Peptide for Tuberculosis Treatment

Tuesday, November 11, 2025
2:30 PM - 3:30 PM CT
Purpose: Tuberculosis (M. tb) is a deadly disease primarily targeting the lungs that has plagued the lives of millions of people around the world and remains a constant threat as the world’s leading cause of death from a single infectious agent after COVID-19. Current treatment regimens for tuberculosis were developed many decades ago and suffer from limitations such as drug resistance, systemic side effects, and lengthy and complicated therapy, limiting patient compliance. Further, the disease’s resistance to most oral first-line drugs indicates a need for a more reliable and successful therapy, one that exerts a localized effect directly to the lungs in the form of an inhalable powder. Linezolid (LZD), an antibiotic approved by the FDA in 2000, has proven effective against  M. tb and is used in treating Multi-Drug Resistant TB. Its bacteriostatic activity makes it a promising choice for a new therapy. Nisin (NP), an antimicrobial peptide, has recently gained interest as it showed efficacy against bacteria. A combination of these two drugs was optimized and spray dried into a dry powder inhalation to offer a novel, synergistic approach for multidrug-resistant tuberculosis treatment. Success of this approach will decrease the likelihood of drug resistance as well as reduce systemic toxicity and allow a localized effect.   

Methods: For bacterial studies, a M. tb avirulent strain H37Ra was used. The minimum inhibitory concentration (MIC) of individual drugs was obtained through the resazurin-based microtiter assay, a colorimetric assay that determines the susceptibility of bacteria to antibiotics. In addition, MIC assay with drugs combination was performed to determine the ratio of the drugs needed to exert a therapeutic effect. Upon completion of the combination study, the Fractional Inhibitory Concentration Index (FICI) was used, a technique for determining whether the combination of the two drugs resulted in an antagonistic, additive, or synergistic effect. After, the drugs in the optimized ratio were spray dried to obtain a dry powder formulation. The spray drying parameters involved the aspirator at 100%, the inlet temperature of 90°C, and a pump rate of 3 mL/min. The excipients Mannitol, L-Leucine, and Trehalose were used in a 75:15:10 ratio. To quantify the amount of drug present in the spray-dried powder, a gradient HPLC method was developed. The melting endotherm, crystallinity, and moisture content of the powder were evaluated by Differential Scanning Calorimetry (DSC), Powder X-ray Diffraction (PXRD), and Thermogravimetric Analysis (TGA), respectively.  

Results: The experimental values from the MIC for Linezolid and Nisin peptide were 1 µg/mL and 1.5 µg/mL, respectively as seen in Figures 1A and 1B. These values are in alignment with published literature that reported MIC for LZD ~0.5-1 µg/mL and Nisin peptide ~1.5 µg/mL. The drug combination showed a synergistic effect with FICI value of 0.375 (Table I). The range for two drugs to be considered synergistic is below 0.5, additive is 0.5-4.0, and antagonistic is anything greater than 4. FICI value < 0.5 suggests enhanced antibacterial efficacy when used in combination. The drug combination of 1:1.3 (LZD: NP) was spray-dried and the formulations averaged a yield of 58 ± 2% w/w. The DSC graph shows that the spray-dried formulation maintained characteristic endothermic peaks of mannitol and trehalose. Linezolid underwent a melt–recrystallization–melt process, exhibiting a polymorphic transition at approximately 155°C followed by melting at around 180°C, as shown in Figure 2. This shift in p</span>olymorphs was confirmed through literature search and was consistent with its findings. The PXRD results showed Linezolid’s crystalline peaks by itself were consistent with published literature whereas the spray dried formulation displayed a shift from its crystalline nature to an amorphous one. The amorphous nature of the spray dried powder is desirable for its stability, solubility, and bioavailability. In addition, the spray dried powder contained approximately 1 % w/w moisture as determined by TGA. Low moisture is favored for the success of dry powder formulations as it minimizes the risk of agglomeration, improving flow properties and ensuring effective lung deposition after consistent powder release aerosolization. 

Conclusion: The combinatory formulation of Linezolid and Nisin antimicrobial peptide offers promising potential as an inhalable formulation displaying a synergistic effect against M. tb bacteria. The successful development of this inhalable combination therapy could significantly enhance treatment outcomes for pulmonary tuberculosis. Our ongoing studies involve testing the spray dried powder against the bacterial strain, determining the aerosolization and deposition of the powder, as well as evaluating its stability.  

References: Maccaroni, E, et al. “Polymorphism of linezolid: A combined single-crystal, powder diffraction and NMR study.” International Journal of Pharmaceutics, vol. 351, no. 1–2, 3 Mar. 2008, pp. 144–151, https://doi.org/10.1016/j.ijpharm.2007.09.028.


Table I. Minimum inhibitory concentration values of Linezolid and Nisin individually and in combination. The calculated FICI value shows a synergistic effect. (synergy ( <0.5), additive (0.5-4.0), and antagonism (>4.0) effect.)


Figure 1. Minimum Inhibitory Concentration of Linezolid (A) and Nisin (B) represented as a heat map. The MIC is indicated in the red box. (C) Checkerboard assay visualized by bacterial viability heatmap and as an image (D).


Figure 2: (A) Differential scanning calorimetry thermograms for Linezolid, Spray Dried Blank, Linezolid-Nisin Spray Dried Powder, Trehalose, L-Leucine, and Mannitol. (B) Powder X-Ray Diffractogram of Linezolid, Spray Dried Formulation, Spray Dried Blank, L-Leucine, Trehalose, Mannitol