Potential antimicrobial agents for the treatment of multidrug-resistant tuberculosis

In vitro studies

Phenothiazine family compounds, including chlorpromazine and thioridazine, are known to possess appreciable levels of antimicrobial activity against M. tuberculosis organisms in vitro. The anti-MDR-TB activity of thioridazine and chlorpromazine was similar [43]. These compounds may be concentrated more than tenfold by macrophages that phagocytise M. tuberculosis [44]. It is likely that the concentration of either phenothiazine required to kill M. tuberculosis cells in vitro is achieved because the macrophage is not only able to concentrate phenothiazines but also to make these compounds available in an active form to the cytoplasmic structure of the macrophage that houses the entrapped phagocytised bacterium [43]. Thioridazine inhibits the growth of clinical isolates of M. tuberculosis that are resistant to streptomycin, rifampicin, isoniazid, ethambutol and pyrazinamide (first-line antituberculous drugs) [16]. The minimum inhibitory concentration of thioridazine required for 50% inhibition of M. tuberculosis H37Rv clinical isolates is 2.5 μg·mL⁻¹ [17]. Because thioridazine does not appear toxic to the macrophage in vitro, it could be used in the treatment of intracellular M. tuberculosis infections [43].

In vivo studies

Groups of five female BALB/c mice were injected intraperitoneally with 1×10⁶ CFU·mL⁻¹ of M. tuberculosis and treated with thioridazine in a dose range of 0.05–0.5 mg·day⁻¹, which is equivalent to that used for psychosis in humans (1200 mg·day⁻¹). There was a >5-log reduction in the number of CFU per millilitre derived from the lungs of infected mice compared with the control group within 1 month [18].

Clinical studies

When thioridazine is administered orally it is rapidly absorbed, with peak plasma concentrations occurring within 2–3 h. Thioridazine is widely distributed in tissues like the liver, blood and kidney [19, 20], yet it is distributed less favourably to the brain [45]. It is not useful for treatment of cavitary pulmonary M. tuberculosis because the concentration of thioridazine required for killing or inhibiting M. tuberculosis outside the macrophage exceeded the concentration that could be achieved in patients receiving standard dosages [43].

Thioridazine undergoes 5-sulfoxidation and N-demethylation by cytochrome P450 (CYP)1A2 and CYP3A4, while CYP2D6 catalyses mono- and di-2-sulfoxidation of thioridazine in the human liver. CYP2D6 and CYP3A4 catalyse thioridazine mono-2-sulfoxidation [62]. Thioridazine is almost entirely (99.85%) bound to serum proteins; its metabolites are bound to serum proteins to a lesser extent. This could be a problem because a small change in the binding capacity of proteins can alter the unbound concentration of thioridazine that has clinical activity. The unbound concentrations of thioridazine metabolites that include thioridazine side chain sulfoxide and thioridazine side chain sulfone are higher (two and nine times, respectively) than the unbound concentration of thioridazine [46].

Thioridazine can be used as adjuvant for regimens already containing several other drugs for treatment of MDR-TB. It is administered in drug-resistant TB until the susceptibility of strains is known [44].

Of 17 extensively drug-resistant (XDR)-TB patients, 14 were treated with thioridazine in combination with linezolid and/or moxifloxacin at a daily dose of only 25 mg for 2 weeks, after which the dose was increased by 25 mg weekly until it reached 200 mg·day⁻¹. The combined therapy of thioridazine, moxifloxacin and linezolid cured 61% of patients, and 22% of patients who were still on follow-up showed a beneficial response. Thioridazine was discontinued in two patients because of pancytopenia in one and allergic dermatitis in the other. In this study, the authors speculate that thioridazine could have contributed to an earlier bacteriological sputum conversion. No prolongation of the QT interval or other cardiac complication was observed in those patients who received thioridazine. Combined therapy including linezolid, moxifloxacin and thioridazine was associated with a relapse-free cure in most cases [58]. The quality of life of the patients was improved shortly after the inclusion of thioridazine in their regimens; most of them benefited from less night sweating, increased appetite and weight gain, and decreased levels of anxiety [58]. In another study, thioridazine cured 10 out of 12 patients; the other two patients also responded, but they dropped out of the programme [63].

The side-effects of thioridazine, as for all phenothiazines, are dose-dependent and include QTc prolongation, thereby increasing the risk of torsade de pointes with subsequent risk of sudden death [64]. To avoid the risk of sudden death in patients whose QTc interval is significantly increased, patients should be screened by ECG before and during treatment with thioridazine [63]. Because chlorpromazine causes frequent and serious side-effects when administered chronically, it is not a good candidate drug for treatment of MDR-TB [65].

In vitro studies

Metronidazole showed no effect when added to bone marrow-derived macrophages infected with M. tuberculosis and did not decrease the bacterial load, although a high concentration of metronidazole was used [22]. Adding metronidazole to a regimen of rifampicin, moxifloxacin and amikacin and/or capreomycin significantly improved killing of dormant (anaerobic and drug-tolerant) M. tuberculosis in an adipocyte model [23].

An in vitro study was conducted to determine the effect of metronidazole against M. tuberculosis incubated under aerobic conditions. Although a high concentration of metronidazole was used (512 μg·mL⁻¹), it had no effect on aerobic cultures of M. tuberculosis. However, under anaerobic conditions, when 32 μg·mL⁻¹ of metronidazole was incubated with M. tuberculosis alone and in combination with either rifampicin or isoniazid, this caused a reduction of 69% and 95% in CFU·mL⁻¹, respectively. Again in this study, 8 days of anaerobic exposure of dormant bacilli to 8 μg·mL⁻¹ of metronidazole caused a 2.7-log reduction in the number of CFU. Rifampicin enhanced the bactericidal activity of metronidazole when used in combination, leading to a 3.68-log unit decline in CFU·mL⁻¹ [21].

In vivo studies

Although metronidazole had no effect on the growth of M. tuberculosis in lungs of aerosol-infected mice at a dose of 15 mg·kg⁻¹, there was a relatively small but statistically significant reduction in bacterial counts during a chronic phase of disease. This chronic phase, in which no marked changes in bacterial load can usually be detected, established after the onset of acquired cellular immunity in mice as a result of the progressively growing M. tuberculosis, resulted in a proportion of bacilli shifting into a dormant state [22, 67]. The possible explanation for the low activity of metronidazole is that M. tuberculosis is not in a state of anaerobic metabolism in which it is susceptible to metronidazole [22].

In another in vivo study using a granuloma model of M. tuberculosis dormancy (mouse hollow-fibre model), metronidazole (100 mg·kg⁻¹) failed to show a beneficial effect against bacilli, although there was immunohistochemical and mutant survival-based evidence of tissue hypoxia. The possible explanations for this result could be a less-than-optimal concentration of metronidazole, poor penetration into granulomatous lesions or lack of hypoxic conditions to permit reductive activation of metronidazole [24].

Metronidazole at 50 and 100 mg·kg⁻¹ in guinea pigs infected with M. tuberculosis combined with standard regimens showed no significant activity, perhaps because of poor penetration of metronidazole into the necrotic core of the hypoxic granulomas or the concentrations of metronidazole effective against M. tuberculosis under completely anaerobic and microaerophilic conditions [25]. These results were comparable to the in vivo study, where treatment of BALB/c and C3HeB/FeJ mice that developed highly organized necrotic lesions following a TB infection, called the Kramnik model, with 200 mg·kg⁻¹ metronidazole for 7–8 weeks had no bactericidal activity against M. tuberculosis, although necrotic lesions in the Kramnik model showed evidence of hypoxia [26].

Clinical studies

The oral bioavailability of metronidazole is almost complete (98.9%) [49]. Peak serum levels are reached within 1–3 h [68]. 10–20% metronidazole is bound to plasma proteins [68]. The half-life of metronidazole is about 8 h [49]. Metronidazole is distributed in different tissues with various percentages of penetration, and it has good penetration into the cerebrospinal fluid (CSF) and central nervous system (CNS) [27]. It is metabolised in the liver, resulting in the formation of two oxidation products: the alcohol metabolite 1-(2-hydroxyethyl)-2-hydroxymethyl-5-nitroimidazole, which has an antimicrobial activity of 30–75% compared with metronidazole; and the acidic metabolite 2-methyl-5-nitroimidazole-1-acetic acid, which has 5% of the activity metronidazole and is only detected in patients with renal dysfunction [69, 70]. Metronidazole and its metabolites are mainly excreted in urine [71].

There are few clinical studies of metronidazole for TB. In a single-blinded study, metronidazole (400 mg three times daily) or a placebo were administered for the first 8 weeks in addition to the standard antituberculous regimen. Addition of metronidazole resulted in a significant improvement in clinical response, with reduction of sputum quantity, enhanced radiographic improvement and improved sensitivity to antituberculousdrugs compared with the placebo group. The results showed a 56% improvement in the sensitivity of TB bacilli to antituberculous drugs on follow-up over 12 weeks in the metronidazole group, compared with 38% in placebo group. This study confirmed that metronidazole has a beneficial adjuvant role in the treatment of TB [50].

In an ongoing clinical trial, metronidazole is being used in MDR-TB patients at a dose of 500 mg three times daily in combination with standard second-line antituberculous drugs. The results of this study are expected soon [72].

Metronidazole is generally well-tolerated. Adverse reactions include reversible neutropenia, minor gastrointestinal side-effects, metallic taste, vaginal and urethral burning, and darkening of the urine. CNS side-effects include ataxia, vertigo, peripheral neuropathy and headache [73]. Metronidazole can produce a reaction similar to that of disulfiram when administered to patients using alcohol because the interaction between metronidazole and ethanol leads to a toxic accumulation of acetaldehyde in the blood [51]. Care should be taken in using this drug in alcohol-abuse patients.

In vitro studies

Doxycycline suppressed MMP1 and MMP3 as well as tumour necrosis factor-α secretion from primary human macrophages infected with M. tuberculosis at 72 h in a dose-dependent manner. This drug is bacteriostatic to M. tuberculosis clinical isolates with an minimum inhibitory concentration (MIC) of 2.5 μg·mL⁻¹ [31]. In 69 MDR-TB isolates obtained from patients in the Samara region of Russia, five (7.4%) isolates were resistant to doxycycline [32]. In vitro MIC values of tigecycline against clinical isolates of M. tuberculosis were as high as 8–64 μg·mL⁻¹ [28].

In vivo studies

Doxycycline decreased mycobacterial replication in infected guinea pigs but showed no effect on MMP activity. At a dose of 5 or 20 mg·kg⁻¹, doxycycline monotherapy suppressed lung CFU at 10 weeks in a dose-dependent manner. The results showed that doxycycline improved the outcome for TB by acting directly on mycobacterial proliferation rather than on MMP activity [31]. No publications mentioned the antimicrobial activity of tigecycline in vivo.

Clinical studies

Doxycycline is available in oral form, unlike tigecycline, which is only available in an injectable form [29]. This drug is absorbed well from the gastrointestinal tract, with bioavailability ranging between 75% and 100% [29]. It is absorbed quickly and reaches its maximum serum concentration within 4 h [29]. Maximum serum concentration and area under the plasma concentration time curve (AUC) of tigecycline are proportional to the dose [29, 52]. In epithelial lining fluid, the AUC of tigecycline was 2.28 μg·h⁻¹·mL⁻¹, which is higher than that of serum (1.73±0.64 μg·h⁻¹·mL⁻¹) [30]. Doxycycline is lipophilic and penetrates well into tissues, especially the brain, eye, prostate and intestinal epithelia [29]. Higher concentrations of doxycycline are also found in the kidney and liver [75]. Tigecycline has a large volume of distribution, 7–10 L·kg⁻¹ [76]. It shows good penetration into tissues like bones, skin, liver and lung [29]. The protein binding of doxycycline is 60–95%, while that of tigecycline ranges between 71% and 89% [29].

Tigecycline has a half-life of 15–36 h, which is greater than the 12-h half-life of doxycycline [29, 30]. No more than 15% of tigecycline is excreted in the urine in an unchanged form [29, 54]. About 30–40% of doxycycline is excreted unchanged in the urine [29].

Doxycycline is safe in humans and may suppress immunopathological MMPs, thereby reducing tissue damage in TB patients using a low dose (20 mg twice daily). It may achieve sufficient concentration in the lung interstitium to decrease the growth of M. tuberculosis and modulate MMP activity and expression [31]. No clinical data are available for the potential use of tigecycline for the treatment of MDR-TB in humans. The most common side-effects of doxycycline are gastrointestinal problems and skin reactions [77]. In a study with healthy subjects, tigecycline had no serious side-effects except for nausea and vomiting, which were dose-related [54].

In vitro studies

Peripheral blood mononuclear cells of healthy and HIV subjects were preincubated with 100–1000 ng·mL⁻¹ of DDC and then infected with M. tuberculosis H37Rv. DDC reduced the CFU number of these intracellular mycobacteria. DDC also enhances the antimycobacterial activity of monocyte-derived macrophages from healthy volunteers injected with 5 mg·kg⁻¹ body weight DDC ex vivo. DDC can enhance macrophage maturation by the induction of 1,25-dihydroxycholecalciferol (vitamin D3) [34]. Vitamin D3 may play an important role in the pathological process of TB by downregulating the levels of MMPs and upregulating the levels of tissue inhibitor of metalloproteinase [79]. Vitamin D3 also mediates the production of cathelicidin, which is a monocyte–macrophage peptide with significant antituberculous activity, in the context of Toll-like receptor 2/1-ligand activation [80]. An in vitro study showed that DDC was highly active against M. tuberculosis with a MIC of 8 μg·mL⁻¹ [33].

DSF and DDC showed antituberculous activity against more than 40 clinical isolates of M. tuberculosis, including MDR/XDR-TB strains. The MIC₉₀ (MIC required to inhibit growth of 90% of organisms) of DSF and DDC against clinical isolates were 1.56 and 3.13 μg·mL⁻¹, respectively. They also showed bactericidal activity against intracellular M. tuberculosis in human monocyte leukaemia cell line (THP-1) at 6–30 μg·mL⁻¹ and 10–30 μg·mL⁻¹, respectively [35]. Since no cross resistance of DSF and DDC with other antituberculous drugs was observed, these compounds may be of potential value for future regimens against MDR/XDR-TB [35].

In vivo studies

DSF kills M. tuberculosis at 80–160 mg·kg⁻¹ in a mouse model with chronic TB [35]. DDC enhances the activity of pyrazinamide and rifampicin twofold when co-administered in mice at 100 mg·kg⁻¹, showing activity against persisters of M. tuberculosis [33].

Clinical studies

DSF is rapidly and completely absorbed following oral administration, and is quickly reduced to DDC. DDC is metabolised to diethylamine, carbon disulfide (CS₂), DDC methyl ester, DDC glucuronide and DDC sulfate; a small amount of DDC is reoxidised to DSF. DSF, DDC and CS₂ are widely distributed throughout the body in lipids of various tissues, and the highest levels of these compounds are found in skeletal muscle [36]. Humans eliminate more than 90% of orally administered DSF within 3 days by renal clearance as DDC, DDC glucuronide and, to a small extent, as DDC sulfate, and via the breath as CS₂ [36]. Few publications showed the activity of DSF or its metabolites against TB. Only one study found that DDC reduces the incidence of infections in HIV co-infected TB patients, probably by stimulating the antimicrobial activity of mononuclear phagocytes [34].

The side-effects of DSF are mainly on the CNS and include psychosis or a confusional state that occurs in the early period of DSF therapy with higher dosages of DSF (500 mg·day⁻¹). Another serious side-effect is peripheral neuropathy. All these side-effects were reversible [81]. Rarely, DSF can cause fatal hepatitis [81].

In vitro studies

M. tuberculosis strains appeared to be susceptible to SXT in 43 (98%) out of 44 isolates tested. These isolates were sensitive to TMP/SMX at MIC ≤1/19 μg·mL⁻¹ [37]. Another study showed that SMX inhibits 80% and 99% growth of all 117 clinical isolates at a MIC of 19 mg·L⁻¹ and 38 mg·L⁻¹, respectively [38]. Drug susceptibility testing in a recent study mentioned that MIC values of SMX for M. tuberculosis ranged between 4.75 and 25 μg·mL⁻¹ [55]. In 7H9 broth, M. tuberculosis was susceptible to SMX (MIC₉₀ 8 μg·mL⁻¹). In that study, SMX achieved excellent activity against M. tuberculosis [39]. One in vitro study showed that M. tuberculosis strain H37Rv was susceptible to SMX and not to TMP at an MIC of 8.5 μg·mL⁻¹. When SXT was added to an isoniazid- or rifampicin-treated M. tuberculosis culture isolate, SMX with and without TMP was efficient at killing and preventing its growth, thereby preventing the emergence of drug resistance [40].

In an in vitro study, M. tuberculosis strains were exposed to either TMP/SMX in combination, SMX or TMP alone, or SMX in combination with first-line TB drugs (isoniazid, rifampicin and ethambutol). TMP had a negligible effect on the growth of M. tuberculosis, while SMX inhibited 80% of the growth at 4.75 mg·L⁻¹. There was no synergistic activity between the TMP and SMX combination, but an additive effect was observed. SMX had a synergistic effect combined with rifampicin, an additive effect combined with ethambutol, and no effect with isoniazid [41].

Clinical studies

The total absorption of SMX is 85–90%. Concentrations of the non-protein-bound fraction of SMX to TMP in plasma varied between subjects, ranging from 1:5 to 1:40 [42]. The explanation could be that SMX has a smaller volume of distribution (10–20 L) than TMP (69–133 L). In the blood, SMX is bound to plasma proteins to an extent of 58–66% (34–42% free) [42, 85]. SMX is well distributed in most body fluids and also in CSF, so it may be a good candidate for treating TB meningitis [42]. Concentrations of SMX in sputum, middle-ear fluid and paranasal sinus fluid are about 20–27% of serum, and in bronchial secretion 60–100% of those in serum [86]. SXT penetrates epithelial lining fluid easily because it is lipophilic and inflammation independent [56, 57]. Following distribution of SMX, it is partially acetylated and glucuronide-conjugated in the liver [87]. SMX excretions are 60–65% acetyl derivative, and 15% appear as glucuronides. These metabolites constitute 70% of total SMX in urine and 20–35% in plasma [42]. The plasma half-life of SMX is 9 h [42].

Limited data are available on the pharmacokinetic parameters of SXT in MDR-TB patients. Only one study described the pharmacokinetic parameters after receiving 480 mg and 960 mg of SXT. Pharmacokinetic parameters of SMX in TB patients including AUC, clearance and volume of distribution are lower than the values observed in patients with other indications. These patients seem to display a consistent pharmacokinetic profile for SMX. SXT was safe and well-tolerated, except for one patient who had gastrointestinal side-effects after receiving 960 mg of SXT [55].

Adherence and tolerance to the drug was good when SXT was used in a daily dose of 960 mg as prophylaxis to reduce the mortality in adults with HIV infection and TB, by preventing opportunistic infections. The rates of occurrence of one or several side-effects that could be due to SXT were similar in the placebo group (n=372) and in the SXT group (n=371) [88]. An SXT dose of 960 mg·day⁻¹ added to routine care improved the survival of HIV-positive TB patients dramatically; SXT prophylaxis should therefore be added to the routine care of HIV-positive TB patients [89].

In five clinical studies summarised in one report [83], SXT was safe, feasible and effective in the prophylactic treatment of HIV patients with TB; it has beneficial effects including lower mortality, fewer hospital visits, weight gain and improved CD4 cell counts, and it reduced plasma HIV in P. jiroveci pneumonia, malaria and other bacterial (pneumococcal) airway infections. The impact on infections with M. tuberculosis was not considered in these studies.

In general, TMP-SMX is a safe medication and is tolerated well. The most common side-effects include gastrointestinal intolerance, nausea, vomiting, anorexia and diarrhoea [87]. Possible side-effects in the blood are hyperkalaemia, a slight increase in serum creatinine levels (not representing loss of glomerular filtration but rather reversible decrease in tubular excretion of the creatinine molecule) and hypernatraemia. These effects occur especially in patients with renal dysfunction [87, 90, 91]. Haematological abnormalities include leukopenia, agranulocytosis and thrombocytopenia, and haemolytic and aplastic anaemia [92].