Combination Chemo- and Immunotherapy Against Latent Pulmonary Tuberculosis

Annually, two million people die from active tuberculosis (TB). A significant fraction of the deaths is from the reactivation of a dormant form of tuberculosis called latent tuberculosis (LTB). LTB is more difficult to treat because LTB patients are asymptomatic and thus challenging to diagnose. Within the lungs of LTB patients, Mycobacterium tuberculosis (Mtb) is encapsulated by structures called granulomas which are impenetrable to anti-TB drugs when delivered by the conventional oral or injectable route. The goal of this study is to formulate a spray dried powder (SDP) that incorporates a live bacterial immunotherapeutic agent, bacillus Calmette–Guérin (BCG), with an anti-TB drug, isoniazid, loaded in nanoparticles (INH NP). We hypothesize that when the SDP is delivered by the pulmonary route it will disrupt the lung granulomas leading to the elimination of Mtb from the LTB patient.

The INH containing poly (lactic-co-glycolic acid) (PLGA) NPs were synthesized by an emulsification/solvent evaporation method. Afterwards, the INH NPs were characterized for size, zeta-potential (Malvern Zetasizer), and INH drug loading in the NPs. INH loading in the NPs was assessed by UV spectrometry at an absorbance of 262 nm, measuring INH presence in the supernatant to indirectly calculate drug loading. The INH NPs were resuspended with BCG in a L-leucine solution at 1:1.5/ NPs: leucine ratio. Leucine was chosen as an excipient for its excellent flow properties. The suspension was spray-dried using a Büchi mini spray dryer at a flow rate of 3 mL/min, inlet temperature of 130±2 °C, and an outlet temperature of 48±2 °C. The final mass of the SDP was used to calculate the final product yield. The particle size of the SDP was obtained using a Malvern Aerosizer.

The size of the INH NPs were 282.11±3.97 nm with a zeta-potential of -5.02±0.13 mV, making them ideal for inducing an immune response and maximizing the uptake by macrophages in the lungs. In addition, INH NPs were optimally loaded with 38.6±2.7 mg INH per 100 mg NPs, resulting in a SDP loaded with adequate INH to potentially eliminate the LTB in the lungs. The BCG loading in the SDP was 3.3 x 10⁷ CFU/mg. We predict this BCG loading to be sufficient to break open the granulomas, induce an immune response, and target Mtb. A spray drying yield of 36.9% was obtained. In addition, the size of the SDP was 2.47 ± 0.05 μm, which is within the appropriate size range of 1-3 μm for deep lung delivery. Our data shows that the BCG and INH loaded NPs, the combination of immuno- and chemotherapy, can successfully be incorporated in an inhalable dry powder and used as a potential delivery system in LTB patients. Further studies are needed to evaluate the immunogenicity of BCG and the toxicity of INH NPs in animal models. This inhalable dry powder may serve as the next step in controlling and eradicating LTB infections caused by one of the most virulent bacterial pathogens.