Comparison of the Efficacy of Two Novel Antitubercular Agents in Free and Liposome-Encapsulated Formulations

Abstract

Tuberculosis is one of the top ten causes of death worldwide, and due to the appearance of drug-resistant strains, the development of new antituberculotic agents is a pressing challenge. Employing an in silico docking method, two coumaran (2,3-dihydrobenzofuran) derivatives-TB501 and TB515-were determined, with promising in vitro antimycobacterial activity. To enhance their effectiveness and reduce their cytotoxicity, we used liposomal drug carrier systems. Two types of small unilamellar vesicles (SUV) were prepared: multicomponent pH-sensitive stealth liposome (SUV_mixed) and monocomponent conventional liposome. The long-term stability of our vesicles was obtained by the examination of particle size distribution with dynamic light scattering. Encapsulation efficiency (EE) of the two drugs was determined from absorption spectra before and after size exclusion chromatography. Cellular uptake and cytotoxicity were determined on human MonoMac-6 cells by flow cytometry. The antitubercular effect was characterized by the enumeration of colony-forming units on Mycobacterium tuberculosis H₃₇Rv infected MonoMac-6 cultures. We found that SUV_mixed + TB515 has the best long-term stability. TB515 has much higher EE in both types of SUVs. Cellular uptake for native TB501 is extremely low, but if it is encapsulated in SUV_mixed it appreciably increases; in the case of TB515, quasi total uptake is accessible. It is concluded that SUV_mixed + TB501 seems to be the most efficacious antitubercular formulation given the presented experiments; to find the most promising antituberculotic formulation for therapy further in vivo investigations are needed.

Keywords: 2,3-dihydrobenzofuran; absorption spectrometry; colony-forming units; coumaran; dynamic light scattering; flow cytometry; monocomponent liposome; pH-sensitive stealth liposome.

Figure 1

Global trends in the estimated number of incident tuberculosis (TB) cases with the new and relapse occurrences and the estimated number of deaths caused by TB, 2000–2018. (Shaded areas represent uncertainty intervals.)

Figure 2

(A–F) Homogeneity and long-term stability of liposomes are measured by dynamic light scattering (DLS). The size distribution of the two types of small unilamellar vesicles (SUVs) (SUV_mixed and SUV_DPPC) without and with the two antitubercular agent candidates separately (TB501 and TB515): blue curve freshly prepared; red after one week; green after five weeks (in the case of empty SUV_DPPC after five weeks, the DLS autocorrelation function could not be evaluated).

Figure 3

(A–D) Determination of encapsulation efficiency (EE). Absorption spectra of the encapsulated antitubercular agent candidates (TB501 and TB515) in the two different SUV formulation (SUV_mixed and SUV_DPPC), represented with normalized (optical density (OD) before (black curves) and after (red curves) size exclusion chromatography (SEC). Green curves show the signal of the scattered light of liposomes themselves. These curves were used to calibrate the normalization of OD. The inset in part D shows the original spectra without normalization.

Figure 4

Internalization of TB515 antitubercular compound encapsulated in SUV_mixed by MonoMac-6 human monocytic cells captured by fluorescence microscopy. Column (A) displays bright-field images of untreated control cells and treated cells (c = 25 μM; 3 h) Column (B) displays untreated and treated cells (c = 25 μM; 3 h) imaged with WideBlue Filter.

Figure 5

Chemical structure of the two in silico identified small molecular weight trisubstituted coumaran (2,3- dihydrobenzofuran) antitubercular agent candidates: TB501 and TB515.