Review

The early bactericidal activity of antituberculosis drugs Expert Review of Anti-infective Therapy Downloaded from informahealthcare.com by Washington University Library on 12/26/14 For personal use only.

Expert Rev. Anti Infect. Ther. 12(2), 223–237 (2014)

Andreas H Diacon*1 and Peter R Donald2 1 Department of Medical Biochemistry, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa 2 Department of Paediatrics and Child Health, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa *Author for correspondence: Tel.: +27 824 201 677 Fax: +27 219 389 476 [email protected]

Early bactericidal activity studies measure the ability of antituberculosis treatments to reduce the burden of Mycobacterium tuberculosis in sputum specimens collected overnight from smear-positive pulmonary tuberculosis patients during the first 14 days of therapy. This confirms the efficacy of novel agents or drug combinations in human patients, allows comparison of different drug dosages and a preliminary assessment of the drugs’ pharmacokinetics and toxicity in closely observed patients. In the past few years several novel antituberculosis agents have demonstrated significant early bactericidal activity and progressed to studies of longer duration. Most recently the early bactericidal activity of drug combinations was found to be similar to results predicted by murine studies. This may contribute to expediting the future progress of drug evaluation. KEYWORDS: bedaquiline • delamanid • early bactericidal activity • PA-824 • rifampicin • sutezolid • treatment • tuberculosis

Since the first antituberculosis agents became available in the mid-1940s, quantification of tuberculosis bacilli in sputum and the fall, or rise, in their numbers in response to the initiation of treatment has been used to follow the progress of patients and evaluate the efficacy of drugs and regimens. In recent years, this methodology has become established as the first clinical assessment of the efficacy of proposed antituberculosis agents in a relatively small number of sputum smear-positive pulmonary tuberculosis patients. Studies of this early bactericidal activity (EBA) have been traditionally carried out over 2 days but have more recently been conducted for up to 14 days. EBA studies demonstrate efficacy in conjunction with pharmacokinetic analysis, allow a preliminary assessment of toxicity and, when a range of doses is assessed, permit determination of the optimal dose to carry forward to more comprehensive clinical trials in larger patient numbers. When pharmacokinetics are studied in association with doseranging studies, drug exposure can be linked to bactericidal activity. The last decade has seen the application of EBA studies in the first clinical evaluation of a number of new antituberculosis agents and also in novel combinations and regimens. In this paper, we review experience with the evaluation of the EBA of antituberculosis agents and regimens and www.expert-reviews.com

10.1586/14787210.2014.870884

current developments regarding the technique; EBA refers to the fall in log10CFU of Mycobacterium tuberculosis per ml sputum per day; the period in days follows EBA, thus EBA 0– 14 refers to the fall in log10CFU over the first 14 treatment days. Alternatively, the viable bacterial load in sputum can be measured with time to culture positivity in liquid culture media, expressed in hours, which is inversely related to the number of viable bacteria in the sputum sample inoculated. Similar to EBA measured with log10CFU, bactericidal activity is determined by the daily prolongation of time to a positive (TTP) from baseline. Development of the methodology

In early reports of the treatment response of pulmonary tuberculosis patients, acid-fast bacilli were often quantified by sputum microscopy, but following studies of Fenner et al. published in 1949, the quantification of viable CFU of M. tuberculosis became a repeatable and reliable investigation [1]. It should be noted that these investigators emphasized at the outset that what was being counted were not single bacilli, but CFU of bacilli that might hypothetically consist of any number of bacilli from 1 to 100. Several groups soon used this technology as a means of evaluating the treatment response of individual patients [2,3]. In 1957, the group around Crofton and


2014 Informa UK Ltd

ISSN 1478-7210

223

Expert Review of Anti-infective Therapy Downloaded from informahealthcare.com by Washington University Library on 12/26/14 For personal use only.

Review

Diacon & Donald

Stewart in Edinburgh noted that serial quantitative studies were a useful technique for a preliminary pilot trial of a new drug. Wishing to evaluate the efficacy of pyrazinamide (PZA) and finding few suitable patients they stated that “…a careful serial assessment of the number of bacilli in sputum would be the most sensitive way of estimating the value of the drug combination” [4]. At the conclusion of their studies they established beyond doubt the efficacy of PZA and concluded: “It is possible to obtain valuable information about new drugs by a trial on a relatively small number of patients provided these have chronic disease, extensive cavitation and sputum consistently positive on direct smear” (FIGURE 1). Thereafter, several other groups used quantification of the numbers of both acid-fast bacilli and viable CFU counts, and their fall or rise, to evaluate the progress of patients [5,6], and also to formally evaluate specific drugs such as ethionamide [7], para-aminosalicylic acid (PAS) and thiocarlide [8], rifampin (RMP) [9], ethambutol (EMB) and PAS [10]. However, it was the study of Jindani et al. (published in 1980) carried out in Nairobi among African patients that set new standards for EBA studies that, broadly, still apply today; this is probably also the first paper to use the term ‘early bactericidal activity’ [11]. For the first time, a wide range of drugs and drug combinations were studied intensively with sputum specimens collected overnight every second day for the first 14 days of treatment. Twenty-seven different drugs and regimens were evaluated in 124 patients. Noteworthy details included the collection of 12-h overnight sputum samples, collection of two ‘baseline’ pretreatment sputum specimens and the use of selective media to prevent overgrowth of contaminants rather than the use of an alkali, such as sodium hydroxide, that destroys a significant number of the bacilli present in sputum [12]. Over the first two treatment days, isoniazid (INH) was the most active drug with a mean daily log10 fall in CFU of 0.722. Any drug combination containing INH was also influenced by the activity of INH and also tended to have a high EBA 0– 2 days. RMP at the usually recommended dose of 10 mg/kg had a modest 0–2 day value (0.187), but this rose to 0.41 at a dose of 20 mg/kg; during the subsequent 2–14 days, the daily fall in counts following RMP treatment was 0.096 following a dose of 10 mg/kg, but 0.15 at a dosage of 20 mg/kg. It is also noteworthy that EMB at 15 mg/kg dose, as currently recommended, had a low EBA 0–2 days (0.05) and an equally low EBA 2–14 (0.03), but at a dose of 25 mg/kg, this improved to 0.37 for days 0–2, but was still poor, in fact negative, for days 2–14; for a dose of 50 mg/kg, however, the EBA 0–2 days was 0.38, and for days 2–14 a remarkable 0.45. This is the highest value ever documented for any single drug or combination of drugs during this period, but the drug cannot be used at this dose due to the great risk of ocular toxicity. One further study evaluated an EMB dosage of approximately 25 mg/kg and found an EBA 0–2 of 0.245 (standard deviation [SD]: 0.152) [13]. The response to PZA was particularly interesting as this drug had already made a considerable contribution to treatment 224

shortening. Despite a negligible 0–2 day fall in counts (0.044/ day), the drug eventually killed at least 95% of the mycobacterial population by day 14 (0.110/day). The PZA results are not given in detail, but appear in a figure and it is relevant, particularly in the light of more recent experience with newer, hopefully equally sterilizing drugs, that in four of the nine patients evaluated there appears to be a fairly rapid fall in counts, while in the remaining five patients the counts actually increase or remain static until day 4 before falling. PZA has now acquired a new lease of life and has been shown in both murine [14] and EBA studies [15] to improve the action of some of the newer agents. Given the importance of PAS in regimens for multidrug-resistant and extensively drug-resistant tuberculosis, it is also notable that, although usually considered a bacteriostatic drug, its activity during treatment days 0–2 at a dose of 15 g was 0.259, the second highest recorded in this study. Importantly, it was only during the first two treatment days that significant differences emerged among the drugs and regimens evaluated and this contributed to a decision to limit later studies to this period. Between 1990 and 2005, most EBA studies were carried out over the first two treatment days and evaluated the majority of available antituberculosis agents contributing to increased understanding of their actions and the relationship between efficacy and pharmacokinetics. These and later studies will be reviewed in two groups, those with monotherapy and those evaluating drug combinations. EBA studies of monotherapy 1980–2005 Isoniazid

INH has consistently been the most bactericidal drug over the first two treatment days with a fall in CFU of close to 0.5 log10CFU/ml sputum/day following the usual dose of 300 mg daily. In a dose-ranging EBA study it was also established that increasing the dose of INH above approximately 300 mg will not improve bactericidal activity over treatment days 0–2 in patients with fully drug-sensitive bacilli, and conversely, that even at a total daily dose of as low as 18 mg, INH still has a detectable bactericidal effect [16]. This finding of a ceiling to the dose-related response of INH in EBA studies is in accordance with the findings of long-term clinical studies [17]. Further studies including pharmacokinetics and N-acetyl-transferase genotyping showed that the EBA90 (that EBA that is 90% of the mean maximum EBA) was associated with a 2 h INH concentration of 2.19 mg/ml and an AUC(0–¥) of 10.52 mg/ml/h [18] and that the N-acetyl-transferase 2 genotype also had a significant effect on EBA [19]. Rifamycins

A large study by the US Public Health Service published in 1979 evaluated three RMP-INH regimens for treatment of pulmonary tuberculosis and found a daily RMP dose of 600 mg to be superior to 450 mg but not inferior to 750 mg for bacteriologic conversion and prevention of relapse [20], and that doses below 9 mg/kg body weight were probably inadequate Expert Rev. Anti Infect. Ther. 12(2), (2014)

Early bactericidal activity of antituberculosis drugs

Case 2

Expert Review of Anti-infective Therapy Downloaded from informahealthcare.com by Washington University Library on 12/26/14 For personal use only.

Viable count per ml. concentrate

Case 4 Resistance first detected

Resistance first detected

106 105 104 103 102

Viable count per ml. concentrate 0

106 105 104 103 102 0

10 20 30 40 50 60 70 80 90 100

10 20

Culture positivity *

+++ ++ + -VE 0

Direct film positivity **

Direct film positivity ** 0

+++ ++ + -VE

10 20 30 40 50 60 70 80 90 100 Days

+++ ++ + -VE

0

10 20

10 20 30 40 50 60 70 80 90 100

4 2 0 0

10 20 30 40 50 60 70 80 90 100 Days

30 40 50 60 70 80 90 100 Days

0

10 20 30 40 50 60 70 80 90 100

+++ ++ + -VE Days

Days No. bacilli per concentrate field **

30 40 50 60 70 80 90 100 Days

Days Culture positivity *

Review

No. bacilli per concentrate field **

20 10 0 0

10 20 30 40 50 60 70 80 90 100 Days

Figure 1. Early use of Mycobacterium tuberculosis CFU counting to evaluate the efficacy of pyrazinamide and thiocarlide in a patient with pulmonary tuberculosis. Graphs of serial readings of the degree of positivity of the sputum of two cases during the first 100 days of treatment with pyrazinamide and oxytetracycline. The intermittent vertical line indicates the time at which resistant organisms were first isolated. *Culture positivity: +++: Confluent growth over whole slope; ++: Innumerable discrete colonies; +: 20–100 colonies. **Direct film positivity: +++: More positive than standard smear 1; ++: Less positive than standard smear 1, but more positive than smear 2; +: Less positive than standard smear 2. Reprinted with permission from [4]
Elsevier (1957).

for treatment of pulmonary tuberculosis. Several rifamycins, RMP, rifapentine and rifabutin were later the subject of dose ranging EBA studies. A dose-related increase in activity was found for these drugs and is illustrated in FIGURE 2 [11,21–24]. The simultaneous determination of pharmacokinetics of these drugs showed an increase in peak plasma concentration (Cmax) and area under the curve (AUC) that was associated with an increase in EBA 0–2 days, and in the case of RMP and rifapentine, also over 0–5 days (TABLE 1) [23]. At the currently recommended RMP dosage (8–12 mg/kg bodyweight), these studies confirmed earlier opinions that a RMP Cmax serum concentration of approximately 10 mg/ml was desirable for optimal activity [25]. These EBA studies suggested that, provided unexpected toxicity does not occur, exploration of higher RMP doses given for a longer period might reveal not only greater bactericidal activity, but also greater sterilizing activity. More recently, an as yet unpublished study administered even higher RMP doses www.expert-reviews.com

and increased drug exposure and bactericidal activity were found up to a dose of 35 mg/kg [26]. A fourth rifamycin, rifalazil, with several promising characteristics, such as a low MIC, failure to induce hepatic cytochrome P450 and low protein binding, was also evaluated at two doses, 10 and 25 mg/kg, in a 14-day study in combination with INH and compared with INH alone [27]. Unfortunately, an unexpectedly high variance in the results of the INH and INH combined with RMP groups made the interpretation of the data difficult and the effect of the individual rifamycins was probably overwhelmed by that of INH during this early treatment phase. Aminoglycosides

Streptomycin (SM), in common with other aminoglycosides, was identified during in vitro studies as highly bactericidal [28], but this was not confirmed in EBA studies [11,29,79]. The relatively 225

Review

0.5

0.4

EBA CFU

Expert Review of Anti-infective Therapy Downloaded from informahealthcare.com by Washington University Library on 12/26/14 For personal use only.

0.3

0.2

0.1

Diacon & Donald

Rifabutin Rifampicin Rifapentine

administration. Despite maximum concentrations in the 15 mg/kg dosage group reaching a mean of 39.2 (SD ± 9.0) mg/ml, well above the MIC of 2–4 mg/ml, a very low EBA days 0–2 of only 0.052 was found. Regarding aminosidine, it is curious that, although relatively weakly bactericidal, it has not found a place in the management of therapeutically destitute, extremely drug-resistant patients with few remaining therapeutic options. Pyrazinamide

PZA was introduced into antituberculosis chemotherapy in 1952 and its action in promoting sterilization of tuberculosis 0.0 lesions noted in murine studies [31]. Clinical studies confirmed its attributes, but were also marked by a high incidence of -0.1 hepatotoxicity [32]. As noted above, early 1200 0 200 400 600 800 1000 therapeutic studies by Crofton and Stewart RMP/RFP/RFB dosage (Mg) made use of the enumeration of CFU in sputum to demonstrate the action of the Figure 2. The relationship between early bacterial activity 0–2 and dosage of drug [4]. The demonstration of its sterilizthe rifamycins rifampin, rifabutin and rifapentine summarized across several ing capacity in association with RMP studies. revolutionized tuberculosis treatment Data taken from [11,21–24,39–41]. introducing ‘short-course’ regimens of low dosage-related EBA of SM, amikacin and aminosidine is 6 months’ duration [33]. EBA studies of PZA have been confusing summarized in TABLE 2 and illustrated in FIGURE 3. The relatively poor in that, despite its excellent sterilizing capacity, it has almost no EBA 0–2 of SM and other aminoglycosides may be a consequence detectable activity during the first 2–4 treatment days [11,13], of the acid surroundings in lung cavities as the activity of the ami- although by the end of 14 days observation its activity matched noglycosides is adversely affected by a low pH. It should also be that of other drugs with an EBA 2–14 of 0.113 compared with noted that the EBA of SM at a dose of 15 mg/kg, as currently rec- those of RMP and INH, 0.096 and 0.113, respectively [11]. More ommended because of fear for toxicity such as irreversible hearing recently, EBA studies of drug combinations have shown an addiimpairment, is very low (0.043; SD: 0.10). In early clinical studies tive effect when PZA has been combined with new drugs bedaquiwhere SM was coupled to other relatively weak drugs, such as thi- line (BDQ) and PA-824 leading to a much more rapid onset of oacetazone or PAS, the long-term clinical response to treatment improved bactericidal activity [15]. was unsatisfactory [30]. Thus, SM is neither a good sterilizing drug nor very bactericidal at the dosages currently used. In the case of Fluoroquinolones amikacin, the serum concentrations were determined during the The fluoroquinolones entered antituberculosis therapy by EBA study at 1, 2, 3 and 4 h after amikacin intramuscular default as agents for the treatment of drug-resistant tuberculosis [34], and are now an essential compoTable 1. Early bactericidal activity and pharmacokinetics of rifampin nent of regimens for the management of in relation to dosage. drug-resistant tuberculosis. Studies of the EBA of ofloxacin found a high EBA secRMP Serum Cmax Ref. AUCI EBA fall in log10CFU/ml ond only to that of INH [38,39] and sevdosage sputum/day (mg/ml) (mg·h/ml) eral subsequent studies have confirmed (mg) Day 1 Day 5 Day 1 Day 5 Days 0–2 Days 2–5 the high EBA of the newer fluoroquino150 2.53 1.49 13.1 7.8 0.028 (0.187) 0.002 (0.059) [23] lones [36,37]. However, surprisingly, with the exception of ciprofloxacin (CFX) [35], 300 3.19 2.89 24.5 15.1 0.121 (0.130) 0.111 (0.072) [23] no dose ranging studies have been car600 13.00 9.53 100.1 65.2 0.221 (0.247) 0.226 (0.144) [23] ried out to establish the therapeutic margin of the fluoroquinolones. The EBA 1000 14.00 171.0 0.440 (0.240) 0.300 (0.110) [24] studies that have been done are summarAUCI: Area under the time-concentration curve extrapolated to infinity; EBA: Early bactericidal activity. Data taken from [23]. ized in TABLE 3 [35–40]. In the study of 226

Expert Rev. Anti Infect. Ther. 12(2), (2014)

Review

Johnson et al. [37], the EBA days 0–7 of moxifloxacin (MFX), levofloxacin and gatifloxacin, together with their pharmacokinetics, was evaluated in comparison with those of INH. The AUC/MIC was calculated and was correlated with the EBA days 0–2 for gatifloxacin but not for levofloxacin or MFX, but no correlation with the EBA days 2–7 was found for any of the three fluoroquinolones.

Table 2. The early bactericidal activity of the aminoglycosides streptomycin, amikacin and aminosidine (paromomycin).

EBA studies of drug combinations 1980–2005

Aminosidine

Study (year)

n

Dose

0–2 days (SD)

2–14 days

Jindani et al. (1980)

4

1g

0.094

0.096

Donald et al. (2001)

8

7.5 mg/kg

-0.025 (0.10)

9

15 mg/kg

0.043 (0.10)

10

30 mg/kg

0.133 (0.09)

7

7.5 mg/kg

0.066 (0.216)

15

15 mg/kg

0.092 (0.140)

12

5 mg/kg

0.0405 (0.10)

13

10 mg/kg

0.0453 (0.14)

15

15 mg/kg

0.0518 (0.10)

Donald et al. (2000)

During the landmark study of Jindani et al., 12 drug combinations were evaluated and are summarized in TABLE 4 and Amikacin Donald et al. (2001) the findings of certain of the combinations reassessed in 2003 [11,41]. During treatment days 0–2, the rapid early kill of INH did not appear to increase nor decrease following the addition of any SD: Standard deviation. other drugs and therefore additional activity from another drug added to INH in this period would be difficult to measure. Only three of the two-drug combinations did not contain INH and the results during the first two treatment days of the combination of RMP with EMB (EBA days 0–2: 0.558) and RMP with SM (0.325) were similar to the those of two-drug combinations containing INH; the combination of SM and PZA, however had a lower EBA days 0–2: (0.118). During the subsequent 12-day period, RMP appeared to be the dominant drug and when accompanied by INH their combined activity appeared to accelerate from day 6 onward and during days 2–14 the combination of INH and RMP had the highest value (0.227) of all the groups evaluated. In two interesting studies, the activity of CFX accompanying INH and RMP and MFX accompanying INH were studied early in treatment. In the first study [42], INH 300 mg was compared with CFX 750 mg during the first 7 days of treatment and the percentage fall in CFU counts/ ml sputum was slightly greater for the INH group (97%) than for the CFX group (88.7%), while the daily rate of fall derived by linear regression was 0.25 log10CFU/ml/day for INH compared with 0.30 log10CFU/ml/day for CFX; these differences did not reach statistical significance. As part of this study, a CFX regimen of INH, RMP and CFX (9 patients) was compared with one of INH, RMP and PZA (11 patients) with sputum counts being carried out on days 0, 3, 7, 10 and 14 and then weekly for the rest of the first 2 months therapy. No difference between the two regimens was apparent for the first 2 weeks, but thereafter a significant difference emerged and by 8 weeks all 11 patients receiving INH, RMP and PZA were cultured negative but only 6 of 9 patients receiving the CFX regimen. In a later study, the combination of INH and MFX was evaluated and a daily fall in counts during the first 2 days treatment of www.expert-reviews.com

Ref.

Streptomycin [11] [29]

[78]

[29]

0.60 log10CFU/ml/day found; this did not differ significantly from the values for INH in previous studies and it was concluded that MFX did not inhibit the action of INH [11,43]. New antituberculosis agents & drug combinations assessed in EBA studies 2005–2013

In 2005, a new era in antituberculosis drug evaluation began with the clinical evaluation of the first of a series of new Aminosidine (Donald et al. 2000) Amikacin (Donald et al. 2001) Streptomycin (Donald et al. 2002) Streptomycin (Jindani et al. 1980)

0.3

0.2 EBA CFU

Expert Review of Anti-infective Therapy Downloaded from informahealthcare.com by Washington University Library on 12/26/14 For personal use only.

Early bactericidal activity of antituberculosis drugs

0.1

0.0

-0.1 0

5

10

15

20

25

30

35

Dosage, mg/kg

Figure 3. The dosage-related early bacterial activity days 0–2 of the aminoglycosides streptomycin, amikacin and aminosidine. Data taken from [11,29,78,79].

227

Review

Diacon & Donald

Table 3. The early bactericidal activity of fluoroquinolones determined by the fall in CFU of Mycobacterium tuberculosis. Study (year)

n

Dose (mg)

Baseline

0–2 days (SD)

2–5 days (SD)

Ref.

9

250

6.822

0.046 (0.10)

9

500

7.054

0.092 (0.09)

10

1000

6.750

0.121 (0.14)

11

1500

7.035

0.205 (0.17)

Chambers et al. (1998)

10

600

0.41 (0.59)

Sirgel et al. (2000)

11

800

0.391 (0.19)

Gosling et al. (2003)

11

400

0.53 (0.31)

Pletz et al. (2004)

8

400

Johnson et al. (2006)

10

400

6.19 (0.77)

0.35 (0.33)

[37]

10

1000

6.53 (0.66)

0.45 (0.35)

[37]

10

400

6.65 (0.60)

0.35 (0.27)

[37]

Ciprofloxacin

Expert Review of Anti-infective Therapy Downloaded from informahealthcare.com by Washington University Library on 12/26/14 For personal use only.

Sirgel et al. (1997)

[35]

Ofloxacin [38]

0.165 (0.20)

[39]

Moxifloxacin [40]

0.273 (CI: 0–0.546)

[36]

Levofloxacin Johnson et al. (2006)

Gatifloxacin Johnson et al. (2006) SD: Standard deviation.

antituberculosis agents, BDQ, previously known as TMC207. These studies were characterized by the period of evaluation being extended to 7 or 14 days after treatment commencement and by simultaneous pharmacokinetic evaluation and close observation for toxicity. BDQ was first assessed in a doseranging EBA study carried out over treatment days 0–7. In addition, delamanid (DLM) previously known as OPC-67683, PA-824, SQ-109 and sutezolid have been assessed. The newer fluoroquinolones that have been used for the management of drug-resistant tuberculosis and are still identified by WHO as ‘second-line’ drugs, have also been incorporated in newer regimens tested in EBA studies, along with clofazimine, a leprosy drug suggested for repurposing for treatment of tuberculosis. During this period, the evaluation of TTP in liquid culture medium as a measure of bactericidal activity has become standard practice and the evaluation of drug combinations as a prelude to longer-term combination studies commenced [44,45]. EBA studies of monotherapy 2005–2013 Bedaquiline

BDQ was initially studied over the first 7 days of treatment at 3 dosages, 25, 100 and 400 mg and in comparison with INH 300 mg and RMP 600 mg [46]. INH and RMP had the expected rapid onset of bactericidal activity, while BDQ 400 mg displayed significant activity only from day 4 onward; no significant activity was detected following the lower BDQ dosages. Although its activity over the 7-day period was inferior to that of INH and RMP, the 400 mg dosage matched their activity 228

during the period days 4–7; this slow onset of BDQ activity was not unexpected based on experience in previous in vitro and in vivo animal studies and subsequent experiments studying the mechanism of action of BDQ in more detail [47,48]. TTP was also determined in a subset of patients enrolled in this study; while TTP prolongation ranked the regimens similarly to those based on the fall in CFU counts, they did demonstrate significant activity also for the BDQ 200 mg dose and found the BDQ effect at 400 mg equivalent to that of RMP (TABLE 5) [44]. BDQ monotherapy was also evaluated in two further EBA studies over the first 14 days of treatment and the results are summarized in TABLE 6. A daily BDQ dosage of 400 mg, but preceded by loading dosages 700 and 500 mg on treatment days 1 and 2, respectively, was studied alone (and together with PA-824 and PZA, see below); following the expected slow onset of BDQ activity, there was a trend toward accelerating activity measured by the daily fall in CFU counts of -0.022, 0.043 and 0.123 over days 0–2, 0–7 and 7–14, respectively and also prolongation of TTP (EBATTP 2.043, 4.625 and 7.466, respectively) [15]. In a further dose-ranging study over 14 days, BDQ doses of 100, 200, 300 and 400 mg were evaluated, also following loading doses during the first two treatment days; all BDQ groups showed significant bactericidal activity that was continued to the end of the 14-day evaluation period with a trend over the period 0–2 days, but also 0– 14 days for greater activity at the higher dosages in association with rising exposure to BDQ shown during simultaneous pharmacokinetic studies, as illustrated in FIGURE 4 [49]. Expert Rev. Anti Infect. Ther. 12(2), (2014)

Early bactericidal activity of antituberculosis drugs

Expert Review of Anti-infective Therapy Downloaded from informahealthcare.com by Washington University Library on 12/26/14 For personal use only.

Delamanid

During the first EBA evaluation of this new nitro-dihydroimidazooxazole, four dosages (100, 200, 300 and 400 mg) were evaluated and pharmacokinetics studied simultaneously [50]. The data from 42 patients who completed treatment were analyzed and the results of the trial appear in TABLE 7 [50]. All dosages were associated with a significant decline in CFU that was monophasic; the mean decline in all groups combined was 0.04 ± 0.056 log10CFU/ml sputum/day and the decline was significant from day 2 onward; the decline was less than dose-proportional and plateaued at the 300 mg dosage, possibly due to dose-limited absorption of the formulation used. Although the response did not differ between dosages, a fall of >0.9 log10 CFU/ml sputum over the 14 days was experienced in 70 and 80% of patients receiving doses of 200 and 300 mg, respectively but only 45 and 27% of patients receiving 100 and 400 mg, respectively. A moderate, but significant correlation was found between the fall in CFU counts and Cmax and AUC0–24h indicating exposure dependence and this is illustrated in FIGURE 5 [50]. PA-824

Review

Table 4. The early bactericidal activity of drug combinations during the study of Jindani et al. (1980). Early bactericidal activity Combinations

n

0–2 days

2–14 days

HS

4

0.510

0.066

HM

4

0.702

0.051

HZ

4

0.494

0.098

RM

4

0.558

0.124

SZ

4

0.118

0.176

SR

4

0.325

0.210

HR

4

0.714

0.227

SHR

4

0.321

0.146

HRM

4

0.487

0.121

SHZ

4

0.799

0.153

SHRZ

4

0.685

0.161

SHRZM

4

0.686

0.200

H: Isoniazid; M: Ethambutol; R: Rifampin; S: Streptomycin; Z: Pyrazinamide. Data taken from [11].

PA-824, a small nitro-imidazooxazine molecule showed promise during in vitro and in vivo studies of both bactericidal and sterilizing activity [14,51,52]. PA-824 has been the subject of two binding licensed by the US FDA for treatment of Gramdose-ranging EBA studies summarized in TABLE 8 [53,54]. Preclini- positive bacterial infections such as pneumonia or bacteremia. cal evaluation suggested that evaluation of dosages from 200 to It is also active against M. tuberculosis and has been used with 1200 mg would suffice to establish the highest safe dosage and anecdotal success for the management of multidrug-resistant also the lowest efficacious dosage permitting determination of and extensively drug-resistant tuberculosis [55]. In a single EBA the therapeutic margin. In the event, the results of the first study, two LZD dosages were evaluated, 1200 mg daily in two EBA study found that the effect for four dosages ranging from 600 mg doses and a single daily dosage of 600 mg [56]. The 200 to 1200 mg was similar. This was also the first study dur- mean EBA 0–2 days was 0.26 (SD 0.42) following 600 mg ing which the preplanned evaluation of EBA by determination two-times a day (b.i.d.) and 0.18 (SD 0.27) after a single daily of TTP was undertaken [54]. This result had not been predicted dose of 600 mg; during the period 2–7 days the EBA was by murine studies, while Phase I studies had found sub-dose weaker with a mean of 0.04 (SD 0.11) and 0.09 (SD 0.17), proportional increases in drug levels across the evaluated dosages. It was Table 5. The early bactericidal activity days 0–7 of a range of doses speculated that as the serum PA-824 concentrations were above MIC throughout of bedaquiline, isoniazid and rifampin measured by A, the fall in CFUs of Mycobacterium tuberculosis, and B, the prolongation in the dosing interval the main driver of PA-824 activity might be time above time to a positive signal in liquid culture in hours. MIC rather than any ratio between Cmax A. Daily fall in log10/ml B. Daily prolongation of sputum of CFU of time to a positivity in or AUC and MIC. Consequently, a secM. tuberculosis liquid culture (h) ond study evaluated dosages of 50, 100, 150 and 200 mg [53]. The results indiDosage (mg) n EBA 0–7 days (SD) n EBA 0–7 days (SD) cated a trend toward lower activity at the BDQ 25 8 -0.004 (0.078) 7 -0.408 (4.176) 50 mg dosage. In both studies, no serious BDQ 100 6 0.097 (0.102) 6 6.131 (5.872) adverse events were encountered and dosages of 100–200 mg appeared appropriBDQ 400 6 0.107 (0.095) 7 12.296 (8.763) ate for further studies. INH 300

5

0.269 (0.061)

4

14.23 (6.360)

Linezolid & sutezolid

RMP 600

7

0.219 (0.124)

8

12.491 (9.188)

Linezolid (LZD) is an oxazolidinone with high bioavailability and low protein

BDQ: Bedaquiline; EBA: Early bactericidal activity; INH: Isoniazid: RMP: Rifampin; SD: Standard deviation. Data taken from [44].

www.expert-reviews.com

229

Review

Diacon & Donald

significant role in the prevention of resistance in companion drugs that would be one of the main functions of LZD in the management of MDR or Dosage (mg) Baseline n 0–2 days n 2–14 days Ref. extensively drug-resistant tuberculosis. Daily fall in CFU counts following BDQ monotherapy preceded by loading LZD pharmacokinetics were also doses studied and although drug exposure was lower with q.d. dosing, time above 100 6.302 (0.697) 14 0.004 (0.168) 14 0.047 (0.074) [49] MIC appeared adequate for manage200 6.001 (0.903) 15 -0.033 (0.128) 15 0.071 (0.047) [49] ment of other resistant bacterial 300 6.071 (1.087) 13 0.015 (0.149) 13 0.088 (0.071) [49] infections. Sutezolid (PNU-100480) is a LZD ana400 6.625 (0.756) 14 0.093 (0.136) 14 0.106 (0.078) [49] log with bactericidal activity against 400 5.956 (1.060) 15 -0.022 (0.121) 14 0.076 (0.069) [15] M. tuberculosis in the hollow fiber, whole Daily TTP prolongation following BDQ monotherapy preceded by loading blood bactericidal activity and mouse moddoses els. The EBA of sutezolid was recently assessed in groups of 25 patients receiving 100 104.8 (23.5) 13 1.5 (2.3) 13 4.4 (5.9) [49] 600 mg b.i.d. or 1200 mg q.d. for the first 200 111.1 (30.6) 13 3.7 (4.3) 13 4.3 (3.1) [49] 14 days of treatment. The mean EBA days 300 115.7 (40.8) 13 4.1 (4.7) 13 5.1 (5.3) [49] 0–2 days was 0.08 (SE 0.091) following 600 mg b.i.d. and 0.05 (SE 0.088) after a 400 88.5 (10.8) 12 6.2 (3.3) 12 5.3 (3.5) [49] single daily dose of 1200 mg; during the 400 110.9 (21.8) 15 2.0 (6.0) 14 5.8 (3.6) [15] period 2–14 days the mean EBA was BDQ was administered in doses of 100, 200, 300 or 400 mg on days 3–14, preceded by single daily loading 0.09 (SE 0.014) and 0.07 (SE 0.017), doses of 200, 400, 500 and 700 mg, respectively on treatment day 1 and 100, 300, 400 and 500 mg, respecrespectively. Compared with LZD, which tively on treatment day 2. BDQ: Bedaquiline; TTP: Time to culture positivity. produced effects approaching statistical sigData taken from [15,49]. nificance during the first 2 days of treatrespectively. The differences between once-daily (q.d.) and ment, but not subsequently, sutezolid showed slightly weaker b.i.d. dosing were not significant. The EBA days 0–2 was activity over the first 2 days but sustained and stronger activity comparable with that of RMP and suggests a modest, but during the later phases of the study [57]. 8

0.20 EBA (0–2) EBA (0–14)

0.15

EBA (0–2) EBA (0–14)

7 6 EBA_TTP

0.10 EBA_CFU

Expert Review of Anti-infective Therapy Downloaded from informahealthcare.com by Washington University Library on 12/26/14 For personal use only.

Table 6. Daily fall in CFU of Mycobacterium tuberculosis (CFU) and prolongation in time to a positive signal in liquid culture in hours following bedaquiline monotherapy preceded by loading doses.

0.05 0.00

5 4 3 2

-0.05

1

-0.10

0 4.0

4.2

4.4

4.6

LogAUC (ng.h/ml)

4.8

5.0

4.0

4.2

4.4

4.6

4.8

5.0

LogAUC (ng.h/ml)

Figure 4. Relationship between EBACFU 0–2 and 0–14 and EBATTP 0–2 and 0–14 and bedaquiline AUC0–24 at different dosages. Relationship of the early bactericidal activity (EBA) of bedaquiline (BDQ) related to the area under the time concentration curve of BDQ for the different dosage groups on treatment days 1 and 14. BDQ was administered in doses of 100, 200, 300 or 400 mg on days 3–14, preceded by single daily loading doses of 200, 400, 500 and 700 mg, respectively on treatment day 1 and 100, 300, 400 and 500 mg, respectively on treatment day 2. EBA over treatment days 0–2 and days 0–14 was measured by the daily fall in CFU of Mycobacterium tuberculosis (left panel) or the daily prolongation of time (h) to positive signal (right panel) in patients with sputum smear-positive pulmonary tuberculosis. Error bars represent standard error. AUC: Area under the curve; EBA: Early bactericidal activity. Reprinted with permission from [49]
American Society for Microbiology (2013).

230

Expert Rev. Anti Infect. Ther. 12(2), (2014)

Early bactericidal activity of antituberculosis drugs

Table 7. The early bactericidal activity days 0–14 of a range of dosages of delamanid measured by the fall in CFU of Mycobacterium tuberculosis.

Recently in pursuit of the goal of the more rapid advancement of new regimens, Daily fall in CFU counts following delamanid monotherapy four combinations of drugs were studied Dosage (mg) Baseline n 0–2 days n 2–14 days and the results of these EBA studies are summarized in TABLE 9 [15]. It is notable that 100 7.06 (0.40) 11 0.066 (0.165) 11 0.026 (0.044) PZA added to BDQ and PA-824 appeared 200 6.75 (0.71) 10 0.138 (0.271) 10 0.038 (0.060) to increase their bactericidal activity and bring about its more rapid onset [15]. This 300 6.72 (0.49) 10 0.023 (0.193) 10 0.063 (0.096) is in keeping with the effect predicted by 400 6.82 (0.65) 11 0.049 (0.205) 11 0.018 (0.033). murine studies [58]. Also noteworthy is the Data taken from [50]. putative new regimen of PA-824, MFX and PZA that had efficacy equivalent to the existing ‘first-line’ for the period of study. Sputum should be collected over regimen of INH, RMP, PZA and EMB and that, in this small 12–16 h overnight including at least two samples for the calcugroup of patients, no signs were encountered of unexpected or lation of the baseline bacterial sputum burden [59]. The processundue toxicity. It is likely that future antituberculosis drug- ing of specimens is an important aspect that has been fully regimen development will follow this pathway of evaluating drug described elsewhere [60–62]. combinations first in 14-day EBA studies before proceeding to While reasonably consistent results have been obtained during serial colony counting studies over the first 8 weeks of treatment EBA studies of different drugs and drug classes, anomalous results have occurred, the most obvious example being the findand then Phase III full-scale randomized controlled studies. ing of significant activity for the combination of amoxicillin/ clavulanic acid by a group recruiting patients in the USA and Methodological issues Since the study of Jindani et al., the methodology of EBA stud- Turkey [38], but a lack of activity in a study conducted in Cape ies has changed little [11]. Attention should be paid to recruiting Town, South Africa [63]. Three papers have considered possible patients who have not been treated for the current episode of sources of variation during EBA studies [61,62,64] and these tuberculosis and have a bacterial sputum burden high enough include the nature and extent of disease among the patients, the to provide positive cultures throughout the envisaged period of collection and processing of specimens, laboratory procedures study. This has been traditionally achieved by recruiting micro- and the experience of the relevant laboratory and the pharmacoscopy sputum smear-positive patients. The study subjects kinetics of the drugs under investigation. In an analysis of should be producing sufficient sputum to remain productive results of eight EBA studies from Cape Town, the main source Delamanid 100 mg Delamanid 200 mg Delamanid 300 mg Delamanid 400 mg All doses regression line All doses 95% Cl

1 Maximum change in log cfu

1 Maximum change in log cfu

Expert Review of Anti-infective Therapy Downloaded from informahealthcare.com by Washington University Library on 12/26/14 For personal use only.

EBA studies of drug combinations 2005–2013

Review

0

-1

-2

-3

-4

0

-1

-2

-3

-4 0

100

200 300 Cmax, ng/ml

400

500

0

100

200

300

400

500

AUC0-24h, ng*h/ml

Figure 5. Relationship between DLM Cmax and AUC0–24h and the decline in CFU counts by treatment day 14. Relationship between Cmax and AUC0–24h and maximum fall in log10 CFU/ml sputum at day 14. A moderate correlation was observed between higher drug exposure and increasing fall of log10 CFU/ml on day 14 for Cmax (R = 0.298, p = 0.044) and for AUC0–24h (R = 0.219, p = 0.144). Cmax: Peak plasma concentration; AUC: Area under the curve. Reprinted with permission from [50] of the International Union Against Tuberculosis and Lung Disease
The Union (2011).

www.expert-reviews.com

231

Review

Diacon & Donald

Table 8. The early bactericidal activity days 0–14 of a range of dosages of PA-824 measured by the fall in CFU of Mycobacterium tuberculosis and the prolongation in time to a positive signal in liquid culture. Dosage (mg)

Baseline

n

0–2 days (SD)

n

2–14 days (SD)

Ref.

Expert Review of Anti-infective Therapy Downloaded from informahealthcare.com by Washington University Library on 12/26/14 For personal use only.

PA-824 daily fall in CFU counts 50

6.1

14

0.093 (0.211)

12

0.059 (0.060)

[53]

100

5.8

15

0.111 (0.332)

15

0.088 (0.085)

[53]

150

6.0

15

-0.009 (0.290)

14

0.096 (0.098)

[53]

200

6.1

14

0.160 (0.255)

14

0.104 (0.083)

[53]

200

6.5

15

0.109 (0.487)

12

0.106 (0.063)

[54]

600

6.3

13

0.096 (0.226)

12

0.113 (0.079)

[54]

prevent drug resistance in companion drugs, this important characteristic might be obscured by recruitment of patients with lower CFU counts, if the effect on the EBA days 0–2 is sufficiently compromised. The clarity of a dose-related response might also be compromised; it is also uncertain to what degree the use of prolongation of TTP might also be affected by this factor. This aspect should be evaluated in future studies. Determination of EBA by TTP in liquid culture media

While the technique of counting CFU in sputum specimens as the basis for EBA 1200 6.1 15 -0.035 (0.420) 11 0.113 (0.099) [54] determination is now well established, the technique requires adequately trained labPA-824 daily TTP prolongation oratory personnel, attention to detail and 50 100 15 1.483 (8.153) 13 2.958 (2.652) [53] the availability of appropriate laboratory 100 115 14 -1.345 (8.586) 15 5.744 (3.973) [53] facilities certified for good laboratory practice located in close proximity to the 150 103 15 4.867 (12.755) 15 4.594 (5.035) [53] site of patient recruitment. TTP has now 200 104 13 3.096 (8.202) 13 5.391 (3.608) [53] been shown to deliver results similar to 200 102 13 1.115 (15.256) 11 3.833 (2.954) [54] those of CFU counting in the ranking of EBA results [45]; this is also shown by the 600 112 13 5.788 (12.173) 13 5.090 (2.768) [54] results of studies completed since 1000 113 11 2.795 (9.230) 12 4.069 (1.916) [54] 2005 that are summarized above. Automated read out of liquid culture results 1200 96 11 1.400 (7.659) 12 4.868 (3.224) [54] can replace the labor-intensive counting SD: Standard deviation; TTP: Time to a positive signal in liquid culture. Data taken from [53,54]. of CFU. Liquid media culture systems are now widely available and their use of variation appeared to be between patient variation in disease does not require the same degree of laboratory sophistication as features and sputum sampling [61]. In order to minimize varia- CFU counting. Despite the similarity of the results of studies tion, attention to detail is essential at every step of the process. using CFU counting and TTP to rank agents and regimens, there are hints of subtle differences in some studies, which Initial mycobacterial load in sputum might indicate that different aspects of EBA are indeed being In early EBA studies with established antituberculosis agents, measured by these techniques. the patients recruited were usually heavily sputum smearThus, as noted above, in the first study of BDQ, when the positive and baseline CFU counts were frequently 7 or results of TTP were also available for comparison over the first 8 log10CFU/ml sputum. More recently, patients with baseline 7 days treatment in a smaller subset of patients, prolongation CFU counts in the region of 5–6 log10CFU/ml sputum are in TTP found significant activity for the mid-dose of 100 mg more frequently encountered. This is probably due to a limited BDQ that was not shown by the fall in CFU counts; similarly number of patients able to fulfill the rigorous selection criteria the activity of BDQ 400 mg that was somewhat inferior to for proof-of-concept studies of novel compounds, and exacer- that of RMP regarding the fall in CFU counts was shown to bated by the recent advent of the GeneXpert MTB/RIF tech- be identical to that of RMP by TTP. Larger studies of serial nology, which enables the establishment of a diagnosis of colony counting and TTP throughout the first 2 months of pulmonary tuberculosis at lower bacterial load level than spu- therapy will assist in clarifying whether these subtle differences tum smears. Several early studies showed a relationship between in findings have any greater therapeutic significance [44]. the baseline CFU counts and the EBA days 0–2, higher initial counts being associated with a greater bactericidal activity over Culture contamination treatment days 0–2 in some studies [16,21,38,65]. Contamination by bacteria and fungi of the solid media plates As it seems likely that greater activity during the first used for CFU counting is a recurring problem in mycobacteri2 days of therapy is associated with the ability of an agent to ology and can cause the loss of significant amounts of data in 1000

232

6.3

15

0.025 (0.340)

14

0.095 (0.062)

[54]

Expert Rev. Anti Infect. Ther. 12(2), (2014)

Early bactericidal activity of antituberculosis drugs

Review

Table 9. The early bactericidal activity days 0–14 of different combinations of bedaquiline, PA-824, pyrazinamide and moxifloxacin measured by the fall in CFU of Mycobacterium tuberculosis and the prolongation in time to a positive signal in liquid culture. Combinations

Dosage

Baseline

n

0–2 days

n

0–7 days

n

2–14 days

Expert Review of Anti-infective Therapy Downloaded from informahealthcare.com by Washington University Library on 12/26/14 For personal use only.

Daily fall in CFU counts following combinations of drugs BDQ and PZA

400 mg and 20–30 mg/kg

5.911 (1.060)

15

0.079 (0.167)

15

0.106 (0.119)

15

0.143 (0.109)

BDQ and PA-824

400 mg and 200 mg

6.680 (0.719)

14

0.114 (0.149)

14

0.114 (0.089)

14

0.114 (0.047)

Pa-824 and PZA

200 mg and 20–30 mg/kg

5.910 (1.045)

15

0.170 (0.082)

14

0.155 (0.040)

14

0.148 (0.043)

Pa-824, MFX and PZA

200 mg, 400 mg and 20–30 mg/kg

5.835 (1.101)

15

0.315 (0.133)

12

0.225 (0.091)

13

0.222 (0.130)

Daily prolongation in TTP following combinations of drugs BDQ and PZA

400 mg and 20–30 mg/kg

99.067 (13.376)

15

12.393 (10.475)

15

11.115 (8.299)

15

9.970 (6.987)

BDQ and PA-824

400 mg and 200 mg

100.300 (37.864)

15

3.941 (9.156)

15

3.948 (3.968)

14

5.855 (2.785)

Pa-824 and PZA

200 mg and 20–30 mg/kg

103.050 (30.852)

15

10.243 (5.982)

15

10.243 (5.982)

14

8.805 (3.468)

Pa-824, MFX and PZA

200 mg, 400 mg and 20–30 mg/kg

96.883 (20.779)

15

21.018 (11.506)

14

19.396 (13.024)

13

18.482 (22.582)

The BDQ dosage of 400 mg daily was preceded by a dosage of 700 mg on treatment day 1 and 500 mg on treatment day 2. BDQ: Bedaquiline; MFX: Moxifloxacin; PZA: Pyrazinamide. Data taken from [15].

EBA studies, but also in serial colony counting studies during the first 2 months of therapy; rates of contamination varying from 15 to 27% are recorded, the risk being greater later in treatment. This problem is most often solved by the use of NaOH treatment of sputum prior to culture [66]. This, however, has been shown to substantially reduce the number of viable CFU cultured during investigations quantifying viable bacilli [67]. Notably, NaOH is used in processing sputum samples for TTP but not for CFU counting. The most likely consequence of this decontamination might be to obscure the effect of more highly bactericidal drugs such as INH and thus their potential to protect companion drugs from the emergence of resistant mutant bacilli [16]. As decontamination is a standard procedure prior to culture in liquid media there may also be an effect in regard to TTP determinations early in therapy, but this has not been evident in studies combining the two evaluations. Optimization of the antifungal component of the selective media has been shown to reduce the rate of contamination, although with the disadvantage of a slight reduction in the M. tuberculosis colony counts. This may be particularly relevant in tropical countries with a high humidity [66]. Transport & storage of sputum before analysis

A further problem in this regard is that the increased number of new drugs entering clinical evaluation has necessitated the www.expert-reviews.com

enrollment of increasing numbers of patients at multiple sites, often at some distance from a central laboratory. This might lead to an interval of several days before sputum specimens are delivered to the laboratory with a likely loss of viability of a proportion of bacilli and an increased risk of contamination. This was recently evaluated during series of EBA studies during which 817 TTP and 794 CFU results were processed [68]. Time elapsed before processing varied from 0 to 3 days and specimens were maintained on ice at 2–8˚C when any delay occurred in processing of the specimen. The CFU and TTP results did not differ taking into account time to processing; there was, however, a trend toward a slightly longer TTP over time elapsed. A similar tendency was also noted among sputum specimens of less than 5 ml, but this also did not appear to influence the CFU counts. The authors concluded that central laboratories could be used for EBA and similar studies provided no more than 3 days elapsed before processing of sputum specimens. Alternative means of determining EBA

Given the difficulties inherent in the determination and interpretation of EBA by CFU counting, a number of other methodologies have been explored in addition to the prolongation of TTP discussed above. These include the PCR [12,42], antigen 85 induction [69,70], whole blood bactericidal assay [71,72], the 233

Expert Review of Anti-infective Therapy Downloaded from informahealthcare.com by Washington University Library on 12/26/14 For personal use only.

Review

Diacon & Donald

measurement of mRNA [73,74], molecular bacterial load assay [75] and most recently the use of GeneXpert MTB/RIF to quantitate mycobacteria in sputum [76,77]. Each of these techniques could potentially either add value to the current determination of the fall in sputum CFU counts or replace it. PCR may remain positive after sputum becomes culture-negative and may thus not necessarily reflect bactericidal activity of a new drug. Determination of mRNA is a marker of cell viability and its decline has been noted to parallel the decline in viable CFU during tuberculosis treatment. The whole blood bactericidal activity assay has the potential advantage of reflecting drug activity against non-replicating persisting organisms. The true value of each of these methodologies will become clear only on completion of long-term clinical studies followed by the evaluation of relapse rates. GeneXpert MTB/RIF has the advantage of the availability of rapid results and ease of handling, however, like the use of enumeration of acid-fast bacilli [59], it is not able to distinguish viable and non-viable bacilli, an essential element in the assessment of new drugs and its discriminative power between different drugs and combinations of drugs is limited [77]. Conclusion

Determination of EBA is now the first clinical investigation undertaken in the development of new antituberculosis drugs and regimens; the technique provides objective repeatable results regarding bactericidal efficacy of a drug or regimen and its relationship to pharmacokinetics and can aid the selection of an appropriate drug dosage to take forward to further clinical studies. These studies also provide an opportunity for a first intensive evaluation of toxicity in a small number of patients. The technique does not yet provide information regarding sterilization of tuberculosis lesions, although it is probable that sterilization commences soon after the start of treatment. Clear identification of the different populations of mycobacteria that are probably present in sputum at the start of treatment, and measurement of their rise or fall, might contribute to the identification of early signals of sterilizing activity. This area will start to be resolved only when the results of long-term studies of new regimens regarding relapse rates become available. In the interim, any reference to EBA findings, 2-month culture negativity, serial sputum colony counting studies or murine studies regarding sterilization remains speculative. It is likely that studies making use of TTP will assist in increasing the number of sites where EBA studies can be done and thus make possible the more rapid evaluation of new drugs and regimens in a wider spectrum of patients. Expert commentary

Determination of EBA is now established as the first clinical evaluation of a new antituberculosis agent to be undertaken in pulmonary tuberculosis patients. A significant fall in CFU of M. tuberculosis in the sputum of such patients following

234

administration of the relevant drug confirms the antituberculosis activity of the agent and allows its progression to further, longer trials, enrolling considerably more patients. Recently, the determination of EBA by measuring CFU of M. tuberculosis has been augmented by determination of the prolongation of TTP in liquid media. The results of this procedure have ranked the efficacy of antituberculosis agents very similarly, albeit with subtle differences in some cases. It is likely that TTP and culture conversion in liquid media will be used on a larger scale in future studies because of their greater ease of execution and the need to rapidly enroll larger numbers of patients from different communities and countries. Importantly, studies of EBA also allow a first evaluation of toxicity in closely observed hospitalized patients and the study of the pharmacokinetics and pharmacodynamics of agents in relation to a range of drug dosages. For the foreseeable future, studies of the EBA will likely retain their place as the initial clinical evaluation to be carried out on a new antituberculosis agent or regimen. Five-year view

The next 5 years seem likely to bring an ever-increasing tempo of antituberculosis drug development and with it the necessity for accelerated drug evaluation. The tuberculosis situation in the world also remains desperate in many countries so that there is growing urgency regarding the development and assessment of new antituberculosis agents and regimens. Although a fairly clear, logical pathway for antituberculosis drug assessment has now been developed from the laboratory to the tuberculosis patient, the growing body of experience may well bring new insights and new surrogate markers of drug efficacy to the fore. Ideally, an early marker of antituberculosis drug efficacy should be able to distinguish sterilizing and bactericidal activity and do this at an early stage of clinical assessment. At present, early bactericidal activities do not do this, but no more accurate methodology is yet apparent to supplant the value of EBA studies in the early assessment of a new drug. As the current new drugs and regimens enter longer-term evaluation, the role of EBA studies and other surrogate markers of possible sterilizing activity in assessing a new drug should become clearer and newer techniques may augment, or replace, EBA studies in early antituberculosis drug assessment. Acknowledgement

We thank Dr L van der Merwe for drawing the original figures. Financial & competing interests disclosure

PR Donald is supported by the South African National Research Foundation. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Expert Rev. Anti Infect. Ther. 12(2), (2014)

Early bactericidal activity of antituberculosis drugs

Review

Key issues • For the first time in four decades, new antituberculosis drugs have entered clinical evaluation and there is every prospect of new regimens being registered for clinical use in the near future. • There is now an established pathway of evaluation for new antituberculosis agents and regimens commencing in the laboratory, leading to evaluation in animal models, most often using mice, and then to studies in healthy humans and finally pulmonary tuberculosis patients.

Expert Review of Anti-infective Therapy Downloaded from informahealthcare.com by Washington University Library on 12/26/14 For personal use only.

• Encouragement comes from the recent finding that certain drug combinations have similar patterns of synergistic and antagonistic activity in mouse models and in human early bactericidal activity (EBA) studies. This promises to reduce the time needed to develop new treatment regimens. • EBA studies provide the first opportunity to demonstrate antituberculosis efficacy in patients; there are no currently used antituberculosis agents that have not shown efficacy in 14-day studies of EBA. • Several single drugs and combinations have now progressed to Phase III clinical trials and in due course it will become easier to retrospectively confirm (or refute) the value of early bactericidal studies and interpret their findings more accurately. • Studies of EBA in pulmonary tuberculosis patients provide an opportunity for the evaluation of toxicity in small groups of hospitalized patients under careful observation and for the conduct of pharmacokinetic studies allowing study of efficacy in relation to drug exposure in tuberculosis patients. From these results, a drug dosage can be selected to take forward to later Phase II and III studies. • Together with later stage evaluations, EBA studies also allow the evaluation of the potential value for drug assessment of other biomarkers based on sputum or other body fluids.

Papers of special note have been highlighted as • of interest •• of considerable interest 1.

Fenner F, Martin SP, Pierce CH. The enumeration of viable tubercle bacilli in cultures and infected tissues. Ann NY Acad Sci 1949;52(5):751-64

•

A key classic study providing the underpinning for all subsequent studies of mycobacterial enumeration.

2.

Jensen KA, Kjaer I, Torning K, et al. Chemotherapy in tuberculosis. Acta Tuberc Scand 1954;29(2):95-105

3.

4.

Joiner CL, Maclean KS, Chalmers DG, et al. Chemotherapy of pulmonary tuberculosis. Lancet 1953;265(6778): 152-5 Stewart SM, Murdoch JM, Crofton JW, et al. Pyrazinamide together with oxytetracycline in patients with tubercle bacilli resistant to streptomycin, PAS and isoniazid. Br J Tuberc Dis Chest 1957; 51(2):158-67

•

One of the first known quantitative assessments of the activity of antituberculosis drugs in sputum.

5.

Hobby GL, Holman AP, Iseman MD, et al. Enumeration of tubercle bacilli in sputum of patients with pulmonary tuberculosis. Antimicrob Agents Chemother 1973;4(2): 94-104

6.

Yeager H Jr, Lacy J, Smith LR, et al. Quantitative studies of mycobacterial

www.expert-reviews.com

the Amplicor PCR assay. J Clin Microbiol 1995;33(7):1944-7

populations in sputum and saliva. Am Rev Respir Dis 1967;95(6):998-1004

References 7.

Brouet G, Chevallier J, Meeus Bith L, et al. Supplementary Clinical Study of Alpha– Propyl-Isonicotinic Acid Thioamide (1321 Th). Rev Tuberc Pneumol (Paris) 1965;29:133-58

8.

Cooperative controlled trial of thiocarlide (DATC), PAS and bed rest alone in short-term single-drug treatment in retreated cavitary pulmonary tuberculosis. Beitr Klin Erforsch Tuberk Lungenkr 1969;139(2): 115-39

9.

10.

11.

••

12.

Gyselen A. Rifampicin in the retreatment and original treatment of advanced pulmonary tuberculosis. Bull Int Union Tuberc 1970;43:60-3 Schutz I. A comparison of rifampicin, ethambutol and PAS in short-term monotherapy. Preliminary results of the third cooperative study of the WATL. Acta Tuberc Pneumol Belg 1969;60(3): 437-41 Jindani A, Aber VR, Edwards EA, et al. The early bactericidal activity of drugs in patients with pulmonary tuberculosis. Am Rev Respir Dis 1980;121(6):939-49 First formal early bactericidal activity study with single agents and combinations carried out over 14 days. Yajko DM, Wagner C, Tevere VJ, et al. Quantitative culture of Mycobacterium tuberculosis from clinical sputum specimens and dilution endpoint of its detection by

13.

Botha FJ, Sirgel FA, Parkin DP, et al. Early bactericidal activity of ethambutol, pyrazinamide and the fixed combination of isoniazid, rifampicin and pyrazinamide (Rifater) in patients with pulmonary tuberculosis. S Afr Med J 1996;86(2):155-8

14.

Nuermberger E, Tyagi S, Tasneen R, et al. Powerful bactericidal and sterilizing activity of a regimen containing PA-824, moxifloxacin, and pyrazinamide in a murine model of tuberculosis. Antimicrob Agents Chemother 2008; 52(4):1522-4

•

Demonstration of combinatorial effects of antituberculosis agents in a murine model.

15.

Diacon AH, Dawson R, von Groote-Bidlingmaier F, et al. 14-day bactericidal activity of PA-824, bedaquiline, pyrazinamide, and moxifloxacin combinations: a randomised trial. Lancet 2012;380(9846):986-93

•

Confirmation that murine estimates of the efficacy of drug combinations [14] can be reproduced in 14-day early bactericidal activity studies in humans.

16.

Donald PR, Sirgel FA, Botha FJ, et al. The early bactericidal activity of isoniazid related to its dose size in pulmonary tuberculosis. Am J Respir Crit Care Med 1997; 156(3 Pt 1):895-900

17.

East African/British Medical Research Council. Isoniazid with thiacetazone (thioacetazone) in

235

Review

Diacon & Donald

the treatment of pulmonary tuberculosis in East Africa – third investigation: the effect of an initial streptomycin supplement. Tubercle 1966;47(1):1-32

Expert Review of Anti-infective Therapy Downloaded from informahealthcare.com by Washington University Library on 12/26/14 For personal use only.

18.

Donald PR, Parkin DP, Seifart HI, et al. The influence of dose and N-acetyltransferase-2 (NAT2) genotype and phenotype on the pharmacokinetics and pharmacodynamics of isoniazid. Eur J Clin Pharmacol 2007;63(7):633-9

••

Pharmacogenetic analysis confirming that a large proportion of homozygous fast acetylators does not reach exposures needed to achieve 90% of the optimum 2-day early bactericidal activity of isoniazid (INH).

19.

Donald PR, Sirgel FA, Venter A, et al. The influence of human N-acetyltransferase genotype on the early bactericidal activity of isoniazid. Clin Infect Dis 2004;39(10): 1425-30

20.

Long MW, Snider DE Jr, Farer LS. U.S. Public Health Service Cooperative trial of three rifampin-isoniazid regimens in treatment of pulmonary tuberculosis. Am Rev Respir Dis 1979;119(6):879-94

21.

Sirgel FA, Botha FJ, Parkin DP, et al. The early bactericidal activity of rifabutin in patients with pulmonary tuberculosis measured by sputum viable counts: a new method of drug assessment. J Antimicrob Chemother 1993;32(6):867-75

22.

Chan SL, Yew WW, Ma WK, et al. The early bactericidal activity of rifabutin measured by sputum viable counts in Hong Kong patients with pulmonary tuberculosis. Tuber Lung Dis 1992;73(1):33-8

23.

Sirgel FA, Fourie PB, Donald PR, et al. The early bactericidal activities of rifampin and rifapentine in pulmonary tuberculosis. Am J Respir Crit Care Med 2005;172(1): 128-35

24.

Diacon AH, Patientia RF, Venter A, et al. Early bactericidal activity of high-dose rifampin in patients with pulmonary tuberculosis evidenced by positive sputum smears. Antimicrob Agents Chemother 2007;51(8):2994-6

25.

Acocella G. Human bioavailability studies. Bull Int Union Tuberc Lung Dis 1989; 64(1):38-40.discussion 40-32

26.

Boeree M, Diacon AH, Dawson R, et al. What is the “right” dose of rifampin? 20th Conference on Retroviruses and Opportunistic Infections, Paper 148LB; Atlanta, GA, USA; 2013. Abstract 128

27.

Dietze R, Teixeira L, Rocha LM, et al. Safety and bactericidal activity of rifalazil in patients

236

with pulmonary tuberculosis. Antimicrob Agents Chemother 2001;45(7):1972-6 28.

Dickinson L. Effect of streptomycin on experimental tuberculosis in guinea pigs. Br J Pharmacol Chemother 1947;2(1):23-6

29.

Donald PR, Sirgel FA, Venter A, et al. The early bactericidal activity of amikacin in pulmonary tuberculosis. Int J Tuberc Lung Dis 2001;5(6):533-8

30.

31.

32.

33.

34.

35.

36.

Tucker WB, Livings DG. Comparative efficacy of three streptomycin and para-aminosalicylic acid regimens of prolonged duration in patients with previously untreated pulmonary tuberculosis. Am Rev Tuberc 1955;72(6):756-82 McCune RM Jr, McDermott W, Tompsett R. The fate of Mycobacterium tuberculosis in mouse tissues as determined by the microbial enumeration technique. II. The conversion of tuberculous infection to the latent state by the administration of pyrazinamide and a companion drug. J Exp Med 1956;104(5):763-802 McDermott W, Ormond L, Muschenheim C, et al. Pyrazinamide-isoniazid in tuberculosis. Am Rev Tuberc 1954;69(3):319-33 Hong Kong Chest Service/British Medical Research Council. Controlled trial of 2, 4, and 6 months of pyrazinamide in 6-month, three-times-weekly regimens for smear-positive pulmonary tuberculosis, including an assessment of a combined preparation of isoniazid, rifampin, and pyrazinamide. Results at 30 months. Am Rev Respir Dis 1991;143(4 Pt 1):700-6 Tsukamura M, Nakamura E, Yoshii S, Amano H. Therapeutic effect of a new antibacterial substance ofloxacin (DL8280) on pulmonary tuberculosis. Am Rev Respir Dis 1985;131(3):352-6 Sirgel FA, Botha FJ, Parkin DP, et al. The early bactericidal activity of ciprofloxacin in patients with pulmonary tuberculosis. Am J Respir Crit Care Med 1997;156(3 Pt 1): 901-5 Pletz MW, De Roux A, Roth A, et al. Early bactericidal activity of moxifloxacin in treatment of pulmonary tuberculosis: a prospective, randomized study. Antimicrob Agents Chemother 2004;48(3): 780-2

37.

Johnson JL, Hadad DJ, Boom WH, et al. Early and extended early bactericidal activity of levofloxacin, gatifloxacin and moxifloxacin in pulmonary tuberculosis. Int J Tuberc Lung Dis 2006;10(6):605-12

38.

Chambers HF, Kocagoz T, Sipit T, et al. Activity of amoxicillin/clavulanate in

patients with tuberculosis. Clin Infect Dis 1998;26(4):874-7 39.

Sirgel FA, Donald PR, Odhiambo J, et al. A multicentre study of the early bactericidal activity of anti-tuberculosis drugs. J Antimicrob Chemother 2000;45(6):859-70

40.

Gosling RD, Uiso LO, Sam NE, et al. The bactericidal activity of moxifloxacin in patients with pulmonary tuberculosis. Am J Respir Crit Care Med 2003;168(11):1342-5

41.

Jindani A, Dore CJ, Mitchison DA. Bactericidal and sterilizing activities of antituberculosis drugs during the first 14 days. Am J Respir Crit Care Med 2003; 167(10):1348-54

42.

Kennedy N, Gillespie SH, Saruni AO, et al. Polymerase chain reaction for assessing treatment response in patients with pulmonary tuberculosis. J Infect Dis 1994; 170(3):713-16

43.

Gillespie SH, Gosling RD, Uiso L, et al. Early bactericidal activity of a moxifloxacin and isoniazid combination in smear-positive pulmonary tuberculosis. J Antimicrob Chemother 2005;56(6):1169-71

44.

Diacon AH, Maritz JS, Venter A, et al. Time to detection of the growth of Mycobacterium tuberculosis in MGIT 960 for determining the early bactericidal activity of antituberculosis agents. Eur J Clin Microbiol Infect Dis 2010;29(12): 1561-5

45.

Diacon AH, Maritz JS, Venter A, et al. Time to liquid culture positivity can substitute for colony counting on agar plates in early bactericidal activity studies of antituberculosis agents. Clin Microbiol Infect 2012;18(7):711-17

46.

Rustomjee R, Diacon AH, Allen J, et al. Early bactericidal activity and pharmacokinetics of the diarylquinoline TMC207 in treatment of pulmonary tuberculosis. Antimicrob Agents Chemother 2008;52(8):2831-5

47.

Andries K, Verhasselt P, Guillemont J, et al. A diarylquinoline drug active on the ATP synthase of Mycobacterium tuberculosis. Science 2005;307(5707):223-7

48.

Koul A, Dendouga N, Vergauwen K, et al. Diarylquinolines target subunit c of mycobacterial ATP synthase. Nat Chem Biol 2007;3(6):323-4

49.

Diacon AH, Dawson R, Von Groote-Bidlingmaier F, et al. Randomized dose-ranging study of the 14-day early bactericidal activity of bedaquiline (TMC207) in patients with sputum microscopy smear-positive

Expert Rev. Anti Infect. Ther. 12(2), (2014)

Early bactericidal activity of antituberculosis drugs

pulmonary tuberculosis. Antimicrob Agents Chemother 2013;57(5):2199-203 50.

Expert Review of Anti-infective Therapy Downloaded from informahealthcare.com by Washington University Library on 12/26/14 For personal use only.

51.

52.

53.

54.

Diacon AH, Dawson R, Hanekom M, et al. Early bactericidal activity of delamanid (OPC-67683) in smear-positive pulmonary tuberculosis patients. Int J Tuberc Lung Dis 2011;15(7):949-54 Hu Y, Coates AR, Mitchison DA. Comparison of the sterilising activities of the nitroimidazopyran PA-824 and moxifloxacin against persisting Mycobacterium tuberculosis. Int J Tuberc Lung Dis 2008;12(1):69-73 Lenaerts AJ, Hoff D, Aly S, et al. Location of persisting mycobacteria in a Guinea pig model of tuberculosis revealed by. r207910. Antimicrob Agents Chemother 2007;51(9): 3338-45 Diacon AH, Dawson R, du Bois J, et al. Phase. II dose-ranging trial of the early bactericidal activity of PA–824. Antimicrob Agents Chemother 2012;56(6):3027-31 Diacon AH, Dawson R, Hanekom M, et al. Early bactericidal activity and pharmacokinetics of PA-824 in smear-positive tuberculosis patients. Antimicrob Agents Chemother 2010;54(8): 3402-7

55.

Lee M, Lee J, Carroll MW, et al. Linezolid for treatment of chronic extensively drug-resistant tuberculosis. N Engl J Med 2012;367(16):1508-18

56.

Dietze R, Hadad DJ, McGee B, et al. Early and extended early bactericidal activity of linezolid in pulmonary tuberculosis. Am J Respir Crit Care Med 2008;178(11):1180-5

57.

58.

59.

Wallis RS, Diacon AH, Dawson R, et al. Safety, tolerability and early bactericidal activity in sputum of PNU-100480 (sutezolid) in patients with pulmonary tuberculosis. XIX International AIDS Conference 2012. Abstract A-452-008620755. Nuermberger E, Rosenthal I, Tyagi S, et al. Combination chemotherapy with the nitroimidazopyran PA-824 and first-line drugs in a murine model of tuberculosis. Antimicrob Agents Chemother 2006;50(8): 2621-5 Hafner R, Cohn JA, Wright DJ, et al. Early bactericidal activity of isoniazid in pulmonary tuberculosis. Optimization of methodology. The DATRI 008 Study Group. Am J Respir Crit Care Med 1997; 156(3 Pt 1):918-23

www.expert-reviews.com

60.

Donald PR, Diacon AH. The early bactericidal activity of anti-tuberculosis drugs: a literature review. Tuberculosis (Edinb) 2008;88(Suppl 1):S75-83

61.

Donald PR, Sirgel FA, Venter A, et al. Early bactericidal activity of antituberculosis agents. Expert Rev Anti Infect Ther 2003; 1(1):141-55

62.

63.

64.

65.

66.

67.

68.

69.

Mitchison DA, Sturm WA. The measurement of early bactericidal activity. In: Malin A, McAdam K, editors. Bailliere’s clinical infectious diseases: mycobacterial diseases (part II). Bailliere Tindall; London, UK: 1997. p. 185-206 Donald PR, Sirgel FA, Venter A, et al. Early bactericidal activity of amoxicillin in combination with clavulanic acid in patients with sputum smear-positive pulmonary tuberculosis. Scand J Infect Dis 2001;33(6): 466-9 Sirgel F, Venter A, Mitchison D. Sources of variation in studies of the early bactericidal activity of antituberculosis drugs. J Antimicrob Chemother 2001;47(2): 177-82 Brindle R, Odhiambo J, Mitchison D. Serial counts of Mycobacterium tuberculosis in sputum as surrogate markers of the sterilising activity of rifampicin and pyrazinamide in treating pulmonary tuberculosis. BMC Pulm Med 2001;1:2 Sloan DJ, Corbett EL, Butterworth AE, et al. Optimizing outpatient serial sputum colony counting for studies of tuberculosis treatment in resource-poor settings. J Clin Microbiol 2012;50(7):2315-20 Peres RL, Maciel EL, Morais CG, et al. Comparison of two concentrations of NALC-NaOH for decontamination of sputum for mycobacterial culture. Int J Tuberc Lung Dis 2009;13(12):1572-5 Kolwijck E, Mitchell M, Venter A, et al. Short-term storage does not affect the quantitative yield of Mycobacterium tuberculosis in sputum in early-bactericidal-activity studies. J Clin Microbiol 2013;51(4):1094-8 Wallis RS, Phillips M, Johnson JL, et al. Inhibition of isoniazid-induced expression of Mycobacterium tuberculosis antigen 85 in sputum: potential surrogate marker in tuberculosis chemotherapy trials. Antimicrob Agents Chemother 2001;45(4):1302-4

Review

70.

Wallis RS, Perkins M, Phillips M, et al. Induction of the antigen 85 complex of Mycobacterium tuberculosis in sputum: a determinant of outcome in pulmonary tuberculosis treatment. J Infect Dis 1998; 178(4):1115-21

71.

Wallis RS, Doherty TM, Onyebujoh P, et al. Biomarkers for tuberculosis disease activity, cure, and relapse. Lancet Infect Dis 2009;9(3):162-72

72.

Wallis RS, Jakubiec W, Mitton-Fry M, et al. Rapid evaluation in whole blood culture of regimens for XDR-TB containing PNU-100480 (sutezolid), TMC207, PA-824, SQ109, and pyrazinamide. PLoS ONE 2012;7(1):e30479

73.

Desjardin LE, Perkins MD, Wolski K, et al. Measurement of sputum Mycobacterium tuberculosis messenger RNA as a surrogate for response to chemotherapy. Am J Respir Crit Care Med 1999;160(1):203-10

74.

Li L, Mahan CS, Palaci M, et al. Sputum Mycobacterium tuberculosis mRNA as a marker of bacteriologic clearance in response to antituberculosis therapy. J Clin Microbiol 2001;48(1):46-51

75.

Honeyborne I, McHugh TD, Phillips PP, et al. Molecular bacterial load assay, a culture-free biomarker for rapid and accurate quantification of sputum Mycobacterium tuberculosis bacillary load during treatment. J Clin Microbiol 2011; 49(11):3905-11

76.

Blakemore R, Nabeta P, Davidow AL, et al. A multisite assessment of the quantitative capabilities of the Xpert MTB/RIF assay. Am J Respir Crit Care Med 2011;184(9): 1076-84

77.

Kayigire XA, Friedrich SO, Venter A, et al. Direct comparison of Xpert MTB/RIF assay with liquid and solid mycobacterial culture for quantification of early bactericidal activity. J Clin Microbiol 2013;51(6): 1894-8

78.

Donald PR, Sirgel FA, Kanyok TP, et al. Early bactericidal activity of paromomycin (aminosidine) in patients with smear-positive pulmonary tuberculosis. Antimicrob Agents Chemother 2000;44(12): 3285-7

79.

Donald PR, Sirgel FA, Venter A, et al. The early bactericidal activity of streptomycin. Int J Tuberc Lung Dis 2002;6:693-8

237