Antituberculosis Activities of Lapachol and β-Lapachone in Combination with Other Drugs in Acidic pH

Abstract

Background:

The treatment of multidrug-resistant tuberculosis (MDR-TB) is a challenge to be overcome. The increase of resistant isolates associated with serious side effects during therapy leads to the search for substances that have anti-TB activity, which make treatment less toxic, and also act in the macrophage acidic environment promoted by the infection.

Objective:

The aim of this study was to investigate lapachol and β-lapachone activities in combination with other drugs against Mycobacterium tuberculosis at neutral and acidic pH and its cytotoxicity.

Design:

Inhibitory and bactericidal activities against M. tuberculosis and clinical isolates were determined. Drug combination and cytotoxicity assay were carried out using standard TB drugs and/or N-acetylcysteine (NAC).

Results:

Both naphthoquinones presented activity against MDR clinical isolates. The combinations with the first-line TB drugs demonstrated an additive effect and β-lapachone+NAC were synergic against H₃₇Rv. Lapachol activity at acidic pH and its association with NAC improved the selectivity index. Lapachol and β-lapachone produced cell morphological changes in bacilli at pH 6.0 and 6.8, respectively.

Conclusion:

Lapachol revealed promising anti-TB activity, especially associated with NAC.

Introduction

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis that affects about 10 million people per year.¹ Despite the adoption of several strategies, TB control and treatment remain a challenge.² The standard treatment is well established and consists in the use of isoniazid (INH), rifampicin (RIF), ethambutol (EMB), and pyrazinamide (PZA), but it presents serious problems in patient adhesion, mainly due to its side effects and extended duration period.^3,4

In addition, there are many cases of resistant TB, which are difficult to treat with the available drugs.^1,4 Multidrug-resistant (MDR)-TB is defined by resistance to the two most potent first-line drugs (INH and RIF), whereas extensively drug-resistant (XDR)-TB, in addition to these, shows resistance to a fluoroquinolone and a second-line injectable drug. In both cases, the treatment becomes more complicated because it requires the use of second-line drugs, which makes the treatment last longer and have more severe side effects than in cases of susceptible TB, which can be treated with first-line drugs.¹ Approximately 18% of TB cases are classified as MDR-TB, of which 6.2% were XDR-TB. In the world, only 56% of patients with MDR-TB and 39% with XDR-TB achieve success in treatment.¹

PZA plays a key role in the treatment of TB and its in vitro activity against M. tuberculosis has to be determined in acid environment.⁵ Early in TB infection, the bacillus is phagocytosed by alveolar macrophages and come into contact with the acid environment. This host immune defense mechanism represents an important step to fight the disease. However, some bacilli are resistant and can survive even under these conditions.⁶ The search for new drugs with anti-M. tuberculosis activity in acidic pH conditions becomes interesting since PZA is the only drug in TB therapeutic regimen able to eliminate the bacillus in the intramacrophagic environment.⁷

Lapachol and β-lapachone are naphthoquinones extracted mainly from the Tabebuia species and already showed potential activity against M. tuberculosis.^8,9 Studies with these compounds suggest that the cytotoxicity mechanism of both is related to the generation of reactive oxygen species (ROS).^10,11 In this view, the association with an antioxidant drug, as N-acetylcysteine (NAC), could reduce the cytotoxicity of these compounds.¹² Moreover, considering that the TB therapeutic regimen consists in the association of drugs, it becomes necessary to perform tests that determine the antimycobacterial activity when two or more agents are combined.¹³

The search for new drugs from natural products and the improvement of the anti-TB therapy using association with already used drugs are of paramount importance.¹⁴ In this sense, the aim of this study was to evaluate (1) the anti-M. tuberculosis activity of lapachol and β-lapachone in neutral (6.8) and acidic (6.0) pH; (2) the effect of these two compounds combined with INH, RIF, EMB, PZA, and NAC; (3) the in vitro cytotoxicity of lapachol and β-lapachone alone and combined with NAC; and (4) the effect of these compounds on bacillus morphology.

Materials and Methods

Extraction of lapachol and synthesis of β-lapachone

Lapachol was extracted from Tabebuia sp. wood, according to Ferreira,¹⁵ and β-lapachone was obtained from lapachol.¹⁶ In brief, sawdust (200 g) was macerated with 600 mL of 10% sodium carbonate solution for 1 hr. The suspension was filtered and carefully acidified with hydrochloric acid (6 mol/L). The resulting suspension was agitated and filtered in a Büchner funnel, under vacuum. The yellow solid was washed with cold distilled water. After drying, it was recrystallized with ethanol. The yield of lapachol obtained from sawdust was 0.8%.

Ninety-eight milligrams of lapachol under stirring and cooled externally with ice was added to 0.3 mL of sulfuric acid and maintained under stirring during 10 min. The solution was stirred for 20 min more at room temperature and then poured into 25 mL of ice-cold water. The orange solid formed was filtered and washed with ice-cold water. The crude β-lapachone obtained was recrystallized with ethanol and the yield of this compound was 76%.

General

The structures of lapachol and β-lapachone were characterized by the analysis of the ¹H and ¹³C NMR spectra. Nuclear magnetic resonance (NMR) ¹H (300.06 MHz) and ¹³C NMR (75.45 MHz) spectra were recorded in deuterated chloroform (CDCl₃) solution in a Mercury-300BB spectrometer, with δ (ppm) and spectra referred to CDCl₃ (δ 7.27 for ¹H and 77.00 for ¹³C) as internal standard (Supplementary Data).

Bacterial strain and drugs

M. tuberculosis H₃₇Rv (ATCC 27294) and nine clinical isolates (one pan-susceptible, six MDR, one INH monoresistant, and one streptomycin monoresistant) were cultured in Middlebrook 7H9 broth supplemented with 10% oleic acid, bovine albumin, dextrose, and catalase (OADC) (7H9-OADC) Enrichment (BBL/Becton-Dickinson, Sparks, MD) at 35°C ± 2°C for 15–21 days. INH, RIF, EMB, and PZA (Sigma, St. Louis, MO) and NAC (Fluimucil^® injectable 100 mg/mL; Zambon, Vicenza, Italy) were obtained commercially.

All clinical isolates used in this research are from the mycobacterial collection of the Laboratory of Medical Bacteriology of the State University of Maringa, Parana, Brazil. This research is registered in the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SisGen) (number AB486FE).

Minimum inhibitory concentration

Minimum inhibitory concentration (MIC) was determined using resazurin microtiter assay plate (REMA) method as described by Palomino et al.¹⁷ with modifications at pH. The pH of the medium was adjusted to 6.0 with HCl 1 N. The compounds were diluted in 7H9-OADC with adjusted pH and transferred to 96-well microplates and, then, 1:2 serial dilutions were performed to obtain dilutions from 250 to 0.48 μg/mL for lapachol and from 250 to 0.0019 μg/mL for β-lapachone. INH (1.0–0.007 μg/mL for susceptible and 32–0.12 μg/mL for resistant isolates) and PZA (800–3.12 μg/mL) were used as reference drugs in neutral (6.8) and acidic (6.0) pHs, respectively. The bacterial suspension was adjusted to OD₆₂₅ using a spectrophotometer and then diluted to obtain final concentration of 7.5 × 10⁶ colony forming units (CFU)/mL. After 7 days under incubation at 35°C ± 2°C, 30 μL of 0.02% resazurin (Acros, Morris Plains, NJ) solution were added to each well and the plates were incubated for 24–48 hr at the same conditions. The reading was performed visually through the resazurin (blue) to resorufin (pink) change by the bacilli metabolism. MIC was determined as the lowest compound concentration that was able to inhibit mycobacterial growth. Medium sterility, DMSO 2.5% (v/v), pure drug/compounds controls and bacterial growth controls were added in all tests. The MIC of EMB, RIF, and NAC also were determined to establish the concentration range for the combination assays.

Minimum bactericidal concentration

The minimum bactericidal concentration (MBC) method was performed from REMA. At 24-hr reading day of MIC, 100 μL from wells that showed no bacilli growth were transferred to Middlebrook 7H11 supplemented with OADC (7H11-OADC). The MBC control was carried out by a 1:500 dilution (100–400 CFU) from the first day of REMA inoculum and 100 μL was transferred to 7H11-OADC. After 28 days of 35°C ± 2°C incubation, CFU counting was performed and the concentration of the compound that killed 99.99% of the bacteria was considered to be bactericidal. The experiments were performed in triplicate.¹⁸

Drug combination assay

The two-dimensional drug combinations technique was performed by resazurin drugs combination microtiter assay (REDCA) according to Caleffi-Ferracioli et al.¹⁹ Lapachol and β-lapachone were tested with INH, EMB, RIF, PZA, and NAC against M. tuberculosis H₃₇Rv. The ranging concentrations of drugs were INH (0.00093–0.25 μg/mL), EMB (0.03–8 μg/mL), RIF (0.00093–0.25 μg/mL), PZA (3.125–800 μg/mL), and NAC (24.4–6,250 μg/mL). The fractional inhibitory concentration index (FICI) was calculated by the formula: FICI = (MIC A + B/MIC A) + (MIC B + A/MIC B), where MIC A + B represents the MIC of compound A when combined with B; MIC B + A, the MIC of compound B when combined with A; and MIC A and MIC B, the MIC of compound A and B tested alone, respectively. The combination effects were classified as synergistic (FICI ≤0.5), additive or indifferent (FICI >0.5–4) and antagonistic (FICI >4).²⁰

Cell cytotoxicity assay

The cytotoxicity assay was performed according to Pires et al.²¹ using VERO cells (ATCC CCL81), kidney epithelial cells extracted from an African green monkey (Chlorocebus sp.), macrophages J774.A1 (reticular sarcoma), and HeLa cells (cervix adenocarcinoma). VERO and HeLa cells were cultured with Dulbecco's modified eagle's medium (Sigma-Aldrich Co., St. Louis, MO) and J774.A1 in RPMI-1640 medium with l-glutamine (Sigma-Aldrich Co.) at 37°C under 5% CO₂ atmosphere. Both media were supplemented with 10% fetal bovine serum (Gibco BRL, Life Technologies, NY). The cells concentration was adjusted to 5.0 × 10⁵ cells/mL, plated in 96-well microplates and incubated for 24 hr at 37°C under 5% CO₂. Then the media were removed and different concentrations of lapachol (6.25–500 μg/mL), β-lapachone (0.00225–250 μg/mL), and NAC (625–50,000 μg/mL), alone and in combination, were added to the wells containing drugs concentrations, except in the controls. Lapachol or β-lapachone was combined with NAC at fixed concentrations (1,000 and 10,000 μg/mL). After 24 hr of incubation under the same conditions, the wells were washed with phosphate-buffered saline (PBS) and 50 μL of 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) dissolved in PBS at 2 mg/mL were added. The microplate was incubated in the dark under 5% CO₂ at 37°C for 4 hr and, then, 150 μL of DMSO were added to the wells. The absorbance was determined at 550 nm using automatic spectrophotometer (Asys Expert Plus; Asys HITECH GMBH, Eugendorf, Austria). The 50% cytotoxicity concentration (IC₅₀) was determined by logarithm regression analysis, using Microsoft Excel^® (2013). The selectivity index (SI) was calculated by the IC₅₀/MIC ratio using the MIC obtained in both pHs.

Scanning electron microscopy

We exposed M. tuberculosis H₃₇Rv with 15–21 days of growing to 0.5 × MIC of lapachol (at acid and neutral pH conditions) and β-lapachone (only at neutral conditions) under stirring (110 rpm) for 12, 24, and 48 hr at 35°C ± 2°C. Samples were washed with PBS and fixed with 2.5% glutaraldehyde (Sigma-Aldrich Co.) in 0.1 M cacodylate buffer (Electron microscopy science, Hatfield, PA) for at least 2 hr at 4°C. Then the samples were washed with fixation solution by centrifugation at 10,000 rpm for 3 min and distributed on glass laminates with poly-l-lysine (Sigma-Aldrich Co.). After the samples were dried, they were gradually dehydrated with 70% to 100% alcohol. Then, a critical point dried in CO₂ was carried out and, finally, metallization with gold was performed. The images were captured by random scanning of microscopic of 20–30 micro-fields in a Quanta 250 scanning electron microscope (Fei, OR). The experiment was performed in duplicate.

Results

Minimum inhibitory concentration and minimum bactericidal concentration

The anti-TB drugs, NAC, lapachol, and β-lapachone MICs and MBCs determined against M. tuberculosis H₃₇Rv reference strain and clinical isolates carried out in neutral (6.8) and acidic (6.0) pHs are described in Tables 1 and 2.

Table 1.

Lapachol and β-Lapachone Minimum Inhibitory Concentration and Minimum Bactericidal Concentration in Neutral (6.8) and Acidic (6.0) pH Against Mycobacterium tuberculosis H₃₇Rv Reference Strain and Clinical Isolates

Mtb strain/isolates	Resistance profile	MIC/MBC (μg/mL)
		Lapachol		β-lapachone
		pH 6.8	pH 6.0	pH 6.8	pH 6.0
H₃₇Rv	Susceptible	31.25/250	7.8/>250	1.95/7.8	3.9/15.6
49	Susceptible	7.8/>250	3.9/>62.5	0.49/31.25	1.95/31.25
64A	INH^R/RIF^R/PZA^R	3.9/>250	7.8/>250	0.49/31.25	3.9/31.25
71A	INH^R/EMB^R/RIF^R/PZA^R	7.8/>250	15.6/>250	0.98/15.6	3.9/31.25
73A	INH^R/RIF^R/PZA^R	15.6/>250	15.6/>250	0.98/3.9	3.9/62.5
109	INH^R/RIF^R/PZA^R	7.8/>250	7.8/250	1.95/15.6	1.95/15.6
309	INH^R/RIF^R	7.8/>250	7.8/>250	0.49/31.25	3.9/31.25
3614	INH^R/RIF^R/EMB^R/SM^R/ETH^R/PZA^R	3.9/250	3.9/>250	0.98/15.6	3.9/31.25
BRF04	INH^R	3.9/>250	7.8/>250	0.98/15.6	3.9/31.25
BRF07	SM^R	3.9/>250	7.8/250	0.49/15.6	3.9/31.25

EMB, ethambutol; ETH, etionamide; INH, isoniazid; MBC, minimum bactericidal concentration; MIC, minimum inhibitory concentration; PZA, pyrazinamide; ^R, resistant; RIF, rifampicin; SM, streptomycin.

Table 2.

Lapachol (L) and β-Lapachone (β) Minimum Inhibitory Concentrations (μg/mL) and Fractional Inhibitory Concentration Index, Obtained by Resazurin Drugs Combination Microtiter Assay, in Combination with Isoniazid, Ethambutol, Rifampicin, Pyrazinamide, and N-acetylcysteine in Mycobacterium tuberculosis H₃₇Rv

Drugs (D)	MIC	MICs combination (REDCA)
Drugs (D)	MIC	MICs D/L	FICI	MICs D/β	FICI
INH	0.06	0.06/31.25	2	0.03/0.48	0.75
EMB	1	1/31.25	2	0.5/1.95	2
RIF	0.06	0.075/15.6	0.625	0.015/0.98	0.75
PZA^a	100	50/3.9	1	50/1.95	0.75
NAC	3,125	1,562/15.6	1	781.25/0.488	0.375
NAC^a	3,125	3,125/1.95	1	781.25/0.12	0.313

REDCA at pH 6.0.

D, drugs; FICI, fractional inhibitory concentration index; NAC, N-acetylcysteine; REDCA, resazurin drugs combination microtiter assay; in bold is synergism.

Lapachol showed great activity against M. tuberculosis H₃₇Rv with MICs ranging from 31.25 to 7.8 μg/mL at pH 6.8 and 6.0, respectively. Moreover, β-lapachone activity was better than lapachol (MIC 1.95–3.9 μg/mL); however, no considerable variation at both pHs against the reference strain was observed. INH and PZA were tested, in the recommended pH (6.8 and 6.0, respectively), to test their in vitro activities and the MICs were 0.06 and 100 μg/mL against M. tuberculosis H₃₇Rv, respectively.

When comparing the results between the two tested pH (6 and 6.8), MIC values of lapachol against susceptible and resistant M. tuberculosis clinical isolates did not differ and, when there was such difference, it was in a dilution. Concomitantly, β-lapachone also showed higher anti-M. tuberculosis activity, evidenced by MIC values, when compared with lapachol. MIC values ranged from 0.49 to 3.9 μg/mL, for all clinical isolates, except for the isolate 109.

MBC of lapachol against the reference strain and clinical isolates ranged from 250 to >250 μg/mL (pH 6.8) and >62.5 to >250 μg/mL (pH 6.0), whereas for β-lapachone it ranged from 3.9 to 31.25 μg/mL (pH 6.8) and 15.6 to 62.5 μg/mL (pH 6.0). No marked difference was observed in the MBC of lapachol and β-lapachone, in both pH, for M. tuberculosis H₃₇Rv and clinical isolates. For β-lapachone MBC, a difference of four dilutions (3.9–62.5 μg/mL) was observed in only one clinical isolate (73A) at pH 6.8 and 6.0, respectively (Table 1).

Drug combinations assay

Table 2 shows the results of the associations of four anti-TB drugs and NAC with lapachol and β-lapachone against M. tuberculosis H₃₇Rv. PZA was tested at pH 6.0 and NAC at both pH. As the activity of PZA depends on acidic pH, tests with this drug were carried out exclusively at pH 6.0. β-lapachone plus NAC combination showed synergic effect in both pH (FICI = 0.375 and 0.313). The β-lapachone or lapachol plus anti-TB drugs (INH, RIF, EMB, and PZA), as well as lapachol plus NAC combinations showed additive effects (FICI >0.5–4) (Table 2).

Cytotoxicity assay

The SI was calculated by using three different cells and lapachol and β-lapachone MICs values in neutral and acidic pHs (Table 3).

Table 3.

Lapachol, β-Lapachone, and N-Acetylcysteine Alone and in Combination Cytotoxicity in J774.A1 Macrophages, VERO, and HeLa Cells

Compounds	J774.A1			VERO			HeLa
	IC₅₀ (μg/mL)	SI		IC₅₀ (μg/mL)	SI		IC₅₀ (μg/mL)	SI
		pH	pH		pH	pH		pH	pH
		6.8	6.0		6.8	6.0		6.8	6.0
L	34	1.08	4.36	158.33	5.07	20.29	125	4.0	16.03
L+NAC 1,000	85	2.72	10.89	243.33	7.79	31.19	62.33	1.99	7.99
L+NAC 10,000	126.7	4.05	16.24	352.5	11.28	45.19	<2.8	<0.09	<0.36
β	3.17	1.63	0.81	3.63	1.86	0.93	0.38	0.19	0.09
β+NAC 1,000	1.01	0.52	0.26	0.06	0.03	0.02	2.4	1.23	0.62
β+NAC 10,000	1.09	0.56	0.28	7	3.59	1.79	<0.02	<0.005	<0.01
NAC	40,000	25.6	12.8	20,000	12.8	25.6	6,000	1.92	1.92

β, β-lapachone; IC₅₀, 50% cytotoxicity concentration; L, lapachol; SI, selectivity index.

Lapachol SI was 1.08, 5.07, 4.0 and 4.36, 20.29, 16.03 for macrophage J774.A1, VERO, and HeLa cells, in neutral and acidic pH, respectively. The NAC combination improved the SI of lapachol in J774.A1 and VERO cells, reaching 16.24 and 45.19 for pH 6.0, respectively. For HeLa cells, the addition of NAC decreased the SI values from 4.0 to 1.99 and 16.03 to 7.99, considering MIC obtained in neutral and acidic pH, respectively.

β-lapachone showed low SI for all tested cells (0.09–1.86) and the combination of NAC did not improve the SI. VERO cells showed the best SI value (1.86) at neutral pH.

Scanning electron microscopy

M. tuberculosis H₃₇Rv were exposed to lapachol (both pH) and β-lapachone (pH 6.8) for 12, 24, and 48 hr. We observed morphological alterations, especially at 24 and 48 hr, when the mycobacteria were exposed to lapachol at acid environment and to β-lapachone under normal pH conditions. Under normal pH, lapachol showed no visible changes in the bacilli at any of the exposure times. The cells treated with lapachol and β-lapachone that presented morphological alterations appeared as rounded and pear shaped, with a greater intensity at the time of 48 hr. The control of nontreated cells revealed typical elongated bacilli cells with shape of variable size arranged singly, in pairs or in irregular clusters. Some cells were dividing by binary fission (Fig. 1).

FIG. 1.

Scanning electron microscopy of Mycobacterium tuberculosis H₃₇Rv after 12 hr (2a–4a), 24 hr (2b–4b), and 48 hr (2c–4c) of exposure to β-lapachone (2) at neutral pH and lapachol at neutral (3) and acidic pH (4). Images 1a and 1b refer to control at 6.8 pH after 12 and 48 hr; 1c refer to control at 6.0 pH. Thin arrow showed pear shaped.

Discussion

The current TB treatment is centered in the administration of polychemotherapy for a long period of time, with a variety of side effects. These facts motivated the search for new options, such as lapachol and β-lapachone, which, through the study of key points that generally are related to the difficulty of TB treatment, could improve and increase the success of treatment. We evaluated the activity in different environments, targeting the discovery of new drugs that are able to eliminate the bacillus in the acid environment, since PZA is the only drug of the treatment regimen with this ability.²² Considering the administration of several drugs together, we also evaluated the effects of the combination with drugs already used in the treatment and with NAC, searching for alternatives to reduce the toxic effects of the therapy.^1,3 Furthermore, the bactericidal action against MDR clinical isolates was verified, since resistance is one of the most important challenges in TB treatment.¹

Minimum inhibitory concentration and minimum bactericidal concentration

In this study, we showed that lapachol and β-lapachone have promising activity in acidic and neutral environments, showing MICs values lower than PZA.²³ We observed that lapachol MICs, determined in two pH (6.0 and 6.8) against M. tuberculosis H₃₇Rv, showed a marked difference. In contrast, no considerable differences were observed between MICs of acid and neutral pH for clinical isolates. Some studies have already shown lapachol and β-lapachone MICs against H₃₇Rv in normal pH conditions, but none in acidic pH.^1,24,25

Quinones have different reduction potentials, and possibly the acidic pH may affect the protonation status of both substances differently. Thus, it can interfere with the amount of ROS formation or even intermediate products.^16,26 The improvement of lapachol action at pH 6.0 could be justified by the possible cyclization catalyzed by the acidic environment in α- and β-lapachone.^16,26,27 Barbosa and Neto¹⁶ reported that amino acids such as cysteine nucleophilically attack the α,β-unsaturated system of β-lapachone leading to heterocyclic ring open. As β-lapachone has a more basic characteristic, this could partially favor protonation in a more diluted acid medium, forming a hydronium ion, which could lead to a loss of its activity in an acid medium.

It is important to highlight that both compounds showed activity against clinical isolates resistant to PZA, with MIC values similar or better than those for M. tuberculosis H₃₇Rv, showing that the activity is maintained independently of the tested clinical isolates resistance mechanism or pH conditions. Considering the difficulty in the treatment of MDR and XDR-TB, the discovery of new drugs able to combat this problem is very important, especially if they have the capacity to eliminate the bacillus in different environments.

Although β-lapachone MIC did not decrease at acidic pH, it showed activity at low concentration values against the reference strain and all MDR clinical isolates. Our neutral pH findings corroborate to those found by Coelho et al.,²⁸ who described MIC = 1.56 μg/mL to M. tuberculosis H₃₇Rv and to RIF-resistant strain. Eyong et al.⁸ reported MBC = 19.53 μg/mL for M. tuberculosis at normal pH conditions. This result is in accordance to the one found in our research, for H₃₇Rv strain and clinical isolates, but a very similar value was observed in both pHs tested.

Lapachol presented MBC = 250 μg/mL (pH 6.8) to M. tuberculosis H₃₇Rv, but it was not bactericidal for the majority of clinical isolates at the tested concentrations. Usually, there is a preference in the discovery of bactericidal drugs because of the action of these in effectively killing and eliminating bacteria, whereas bacteriostatic drugs only inhibit growth.²⁹ However, bactericidal action is generally associated with severe adverse effects, due to the rapid lytic action of the drug against the target microorganism.³⁰ This statement may be related to our results, which showed that, although lapachol did not exhibit bactericidal activity at the tested concentrations, cytotoxicity results were more promising when compared with β-lapachone, which presented low bactericidal concentrations against all clinical isolates.

Furthermore, clinical relevance that bactericidal drugs are better than bacteriostatic drugs is rarely found.³⁰ A meta-analysis sought to evaluate some parameters associated with the clinical relevance and found no significant difference of clinical cure rates between bactericidal and bacteriostatic drugs, and in some cases bacteriostatic drugs presented higher cure rates.³¹ Considering that the treatment of TB consists in the use of multiple drugs, the inclusion of drugs, even bacteriostatic, may contribute to a successful outcome.

Drug combination assay

The associations of β-lapachone and lapachol with first-line TB drugs did not show antagonism, neither synergism, but we observed a decrease in MIC of RIF and PZA when associated with lapachol, and of INH, EMB, RIF, and PZA when associated with β-lapachone, demonstrating that these drugs have a better activity in combination with the naphthoquinones than alone.³² Besides that, the effects of combinations could represent other advantages for the treatment. da Silva et al.⁹ showed that the combination of INH with β-lapachone becomes bactericidal to Mycobacterium fortuitum and Mycobacterium smegmatis. It is worth remembering that our results refer only to the standard strain H₃₇Rv; thus, more studies should be carried out on the drug combination effects against susceptible and resistant clinical isolates.

The association β-lapachone+NAC revealed synergism against H₃₇Rv, but it did not improve the cytotoxicity against the tested cells. Despite this, the use of NAC as adjuvant in the treatment of TB represents a relevant and reliable alternative, due to its low toxicity, and other several advantages already discussed by other authors.^33,34 NAC is a drug commercially available without prescription and administered at high doses (>100 mg/mL).³⁵ Although it is safe and a known nontoxic drug, our cytotoxicity results showed that the IC₅₀ for NAC was lower than the administered dose. However, it should be noted that in vitro cytotoxicity studies may not accurately represent in vivo effects.³⁶

Cytotoxicity

Lapachol was less toxic than β-lapachone for all tested cells and this is in agreement with the results of some studies, which show that β-lapachone presents lower values of IC₅₀ against a varied group of cells, compared with lapachol.^37,38 In contrast, Costa et al.³⁹ found IC₅₀ >500 μg/mL to lapachol in THP-1 cells.

Candidates to continue the screening for a new TB drug must have the SI >10.⁴⁰ We found promising SI for lapachol in VERO and HeLa cells, when considering its activity in acid environment. The IC₅₀ for lapachol in macrophages was very similar to the one found by Rocha et al. (32.4 μg/mL).⁴¹ In our study, the IC₅₀ for HeLa cells was higher (125 μg/mL) than the findings of recent studies with HeLa and other human leukemic cell lines (K562, Lucena-1, Daudi, and MCF-7); however, the cells were incubated with the drugs during 72 hr, unlike the time adopted by our group (24 hr).^42,43 The SI of lapachol alone was <10 in neutral pH, but this index was increased in VERO and macrophages by the association of NAC. This may be related to the action of NAC in the oxidative stress control against the ROS generation by lapachol.^43,44 We observed that the highest concentration of NAC (10,000 μg/mL) showed better SI than the lowest (1,000 μg/mL) when associated to lapachol, indicating that the concentration of NAC is relevant to reach less toxicity. It is important to emphasize that this association did not hamper the anti-M. tuberculosis activity of lapachol, as shown by REDCA.

Fluimucil consists of acetylcysteine, which acts as a captor of ROS and provides cysteine, a precursor of glutathione that also has antioxidant action.⁴⁴ NAC already showed action against susceptible and resistant M. tuberculosis and the coadministration in MDR-TB treatment is considered useful due to its advantage in preventing some adverse effects associated with the therapy, such as hepatotoxicity and ototoxicity.^33,34 Kranzer et al. (2015)³⁴ showed that NAC inhibits intra- and extracellular growth of M. tuberculosis in acid and neutral conditions and decreases the oxidative stress associated with TB.

β-lapachone is known to present low levels of IC₅₀, especially against tumor cells.^37,45,46 This fact is explained by its particular action mechanism dependent on the activity of the enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1), which is overexpressed in human cancer cells and plays a protective role against semiquinone-generated ROS cytotoxic effects.^11,46,47 Da Silva Junior et al.⁴⁸ found very low IC₅₀ results to tumor cells (<1.8 μM) compared with peripheral blood mononuclear cells (>20 μM). Sun et al.⁴⁵ showed that β-lapachone induces necrotic cell death in several human cancer cell lines, but not in normal cells. Based on this, it is suggested that β-lapachone activity is more selective against cancer cells.^46,49 Our results also showed selectivity of β-lapachone to HeLa cells, which is derived from a human cervix adenocarcinoma cell line (Table 3). Although macrophages belong to a reticular sarcoma cell line, it is derived from murine cells and do not express the enzyme NQO1, showing similar results to VERO.

IC₅₀ of β-lapachone to VERO cells was similar to the one found by Pereira et al. to BSC40 cells (2 μg/mL), which also come from African green monkey's kidney.³⁸ A recent study showed that most of the metabolites found in human blood have IC₅₀ >1,000 μM for normal and carcinogenic cell lines, and a similar study was performed with lapachol, in which lapachol is biotransformed by Bifidobacterium sp. and Lactobacillus acidophilus in less toxic metabolites against fibroblasts cell line.^50,51 Although our results did not demonstrate an ideal SI value for β-lapachone, these data represent different alternatives to other studies that aimed to improve cytotoxicity.

In general, the association of NAC with β-lapachone did not decrease cytotoxic effects, probably because this naphthoquinone is capable of acting through several alternative pathways, independent of ROS generation, to promote cell death.^52,53 Reinicke et al.⁵⁴ associated β-lapachone with NAC and did not find differences in IC₅₀ in MDA-MB-231 breast cancer cells. Other study showed that the treatment of cells with NAC did not interfere in the formation of ROS by β-lapachone exposure.⁵⁵ The association of β-lapachone with NAC 10,000 μg/mL in HeLa cells showed low IC₅₀, probably because NAC's IC₅₀ is smaller than this value.

Scanning electron microscopy

The effect of lapachol was evaluated at acid and neutral pH conditions, since we observed that its action against M. tuberculosis differs according to the pH. We observed that the exposure to lapachol at acidic pH and β-lapachone at neutral pH induced loss of bacillary morphological characteristic, with the appearance of rounded and pear shapes (Fig. 1). Very similar alterations were found by recent studies with INH and eupomatenoid-5.^56,57 Our results help to clarify the death dynamics of the TB bacillus when in contact with these naphthoquinones. Scanning electron microscopy findings are consistent to the MIC of lapachol at different pH conditions, demonstrating that this compound is able to produce changes in the cell wall only at acidic environment.

Conclusion

Considering our in vitro findings, lapachol and β-lapachone showed promising activity against M. tuberculosis H₃₇Rv and MDR clinical isolates in acid and normal environment and presented additive effect in combination with TB drugs. The use of NAC associated with lapachol improved the cytotoxicity without hampering the anti-M. tuberculosis activity and could be a possible strategy to reduce the side effects in the treatment of TB. β-lapachone had bactericidal effect at low concentrations against M. tuberculosis H₃₇Rv and MDR clinical isolates and presented synergic effect in M. tuberculosis H₃₇Rv, but its association with NAC did not improve its toxicity. The improvement of cytotoxicity can be sought through in further studies using micro- or nanoparticles, as well as liposomes.

Footnotes

Acknowledgments

We thank all collaborators, especially of Chemistry and Bacteriology Medical Laboratory of the State University of Maringa.

Disclosure Statement

No competing financial interests exist.

Funding Information

No funding was received for this article.

Supplementary Material

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